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Diploma Thesis - Fachhochschule Jena : Maschinenbau
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1. T AVA AVAVA Fachhochschule Jena EADS nl University of Applied Sciences Jena ase rium Fachbereich Maschinenbau Fachgebiet Mechatronik Diploma Thesis Theme System tests and optimisation of an automated biotechnology facility for tissue engineering Submitted by Born on Matrikel Nr University Supervisor Mentor Date of Theme lssue Due Date Daniel Redlich 07 10 1982 in Schleiz 270487 Herr Professor Dr Grabow Herr Dr Ing Peter Kern 10 03 2009 12 08 2009 1 a A AVATA Fachhochschule Jena Page 2 EAD a University of Applied Sciences Jena SErium Abstract The modern biotechnology has evolved rapidly during the last decades Whereas by now the cultivating and implantation of fully functional tissue to diseased areas of the patient became possible This method of tissue engineering is called regenerative medicine and the creation of these tissues is called tissue engineering As the tissue cultivation is a recent field of research a lot of corresponding phenomena are still unknown For this reason the European Space Agency ESA decided to develop a Tissue Cultivation Unit to study the influence of zero gravity on different kinds of tissues Thus the establishment of the Biotechnology Mammalian Tissue Culturing Facility BMTC came into being It is common practice to develop a ground model to verify the technical feasibility and get reproducible results on earth before the
2. Number Part Ball shaped External Handler F Function The robot can grip this part and handle the ECM 2 ECM Housing Carrier of all other parts of the ECM and connector to the Peristaltic Pump see chapter 3 1 3 3 ISMATEC Tube Track Provides the proper guidance of the tube anda safe pump function 4 Tube adapter Connection of tube and Fluid Storage Cassette FSC 5 Quad Ring Is used to tighten the I F between FSC and Spray Pump 6 Fluid Storage Cassette FSC Storage of the cultivation medium Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena University of Applied Sciences Jena Page 19 1 EADS pose aj SSErium Gas exchanger The Spray pump and the fluorescence sensors are mounted on it 7 Cover plate Closes the ECM mechanically Allows to pass gas through it to aerate the 8 Bio foil cultivating liquid 9 Atomiser of the Spray Pump Injects a small amount of medium into the FSC Insert for the fluorescence Provides the mounting of the fluorescence 10 sensors sensors onto FSC Fluorescence Sensors with Measures the O and the pH value of the 11 counter part for the Optical cultivation liquid Multiplexer 12 Spray pump housing Provides the storage for the cultivation liquid Ensures that all of the medium in the Spray 13 Exhaust foil tube Pump can
3. Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 63 EADS ae University of Applied Sciences Jena SSErium Start Priming Connection of the Cleaning Unit Setup Cock in flow direction Waste Storage Setup Cock in flow direction Filling Channel N 1 Port Aspiration from Channel N 1 channel N by Cavro Pump Expiration to channel 9 Ext Waste by Cavro Pump Aspiration from channel N by Cavro Pump N N 1 Expiration to channel 9 Ext Waste by Cavro Pump End Priming Figure 3 42 Flow chart of the Cleaning Procedure Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AYATA Fachhochschule Jena Page 64 EADS et University of Applied Sciences Jena SSErium 3 3 Modular Support System Robot The Modular Support System in the following sections also called Robot is the handling system for the ECMs and the LHA Figure 3 43 shows the Katana 6M180 which has been chosen for the automation of the BMTC The term 180 refers to the configuration of the gripper which spans an angle of 180 to the arm it is connected to Katana 6M180 Technical Specifications 11 e Weight 4 3 kg e Lift able Weight 0 5 kg e Power Consumption 60 W e Precision 0 1 mm e Operational range 60 cm e Degrees of freedom 5 1 rotating b
4. Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 107 EAD z University of Applied Sciences Jena SSErium gt Set variable Parameters Q Command Queuing 0 Disable 1 Enable B Oscillatory Pump Frequency in s Period From 8 up to 99 s C Oscillatory Pump Stagnation Time in s Can be set if the frequency is at least 8 S D Oscillatory Pump Offset in Can be set 0 100 0 8 mm Examples gt Q0 gt B50 Internal Parameters P Save current position in position number two digits from 01 to 20 C Checksum O Disable 1 Enable V Verbose mode Sends verbal messages through the serial port for debugging 0 Disable 1 Enable 2 Enable for Trinamic Board R Release position offset O Optical sensor position offset Y Spray pump position offset SLr Set lower limit for the Release Servo in OCR value between 115 for 1 ms and 230 for 2 ms SLR Set upper limit for the Release Servo in OCR value between 115 for 1 ms and 230 for 2 ms SLo Set lower limit for the Optical Servo in OCR value between 115 for 1 ms and 230 for 2 ms SLO Set upper limit for the Optical Servo in OCR value between 115 for 1 ms and 230 for 2 ms SLy Set lower limit for the Spray Servo in OCR value between 115 for 1 ms and 230 for 2 ms Daniel Redlich System tests and op
5. Figure 4 3 BMTC Control tab Actuator Bridge Servo Teach in and Testing C Cultivation Unit Cultivation Unit 2 per RC SetMin SetMax Optics jaan nal E Seba Spray per E SetMin Set Max Release Pe eee eee eee eee eee eee Important notice the values shown in this window are not updated from the values in the pC Please be careful that you receive a response for all the teach in moves you make If one is ignored the value shown here will not be the real one until a new move is received Figure 4 4 BMTC Control window to teach the positions of the servos The Table 4 3 explains all buttons that are used in the tab Actuator Bridge and additionally the button of the window to the servos Table 4 3 Overview of the buttons in the tab Actuator Bridge Button Function Start Starting the movement to the above selected position Stop Stopping the movement Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a 4 AVATA Fachhochschule Jena Page 85 EAD a University of Applied Sciences Jena SSErium Reference Starting the reference run to find the zero position Debug modus 1 Debug modus 2 Activating the debug modus 1 The Actuator Bridge sends commands for any action that is realised Activating the debug modus 2 The Actuator Bridge sends all commands to the computer that are send to and received from the Stepper Moto
6. sssssesnssssssesereesnssssesereesrsssererennesssseorreenesse 82 Figure 4 3 BMTC Control tab Actuator Bridge ccsssccccssssececeesseeeesseaeceeseseeecesseneeeeesenes 84 Figure 4 4 BMTC Control window to teach the positions of the servos ccccssesssseeeeeeeees 84 Figure 4 5 BMTC Control tab Liquid Handling eessccccsssceceessecececseaececsseeecssseeeeeeessnes 88 Figure 4 6 BMTC Control tab Robot sceccisacsccsascuiovacaeadenstaenenivnannivarsancecandelogedactemigueiandage 90 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 adaa Fehinsactlg dena iad EADS on Figure 9 1 Flow chart of the Cleaning Procedure ccssscccsssscececsssececsssseeeceeseneeceessaaeeeees 110 Figure 9 2 Flow chart of the Priming Procedure cccssccccessseecesssnececesseeeecessseeeeessaaeeeeess 111 Figure 9 3 Flow chart of the Filling Procedure cceccsssscecesseceeecssaeeecesseeecesseeeeeessaaeeeeess 112 Figure 9 4 Flow chart of the Seeding Procedure ccsssccccessseecesssntececssseeeeceesseeeesseaeeeeess 113 Figure 9 5 Flow chart of the Washing ProC CUure ccccsscccsssssececesseeeceeseeeecessneeeseeeaaeeeenes 114 Figure 9 6 Schematic sketch of the a 9 pin D Sub male plug cssccecessssceceesssteeeessseeeeees 115 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineeri
7. Cultivation Cultivation 9 Unit 1 s Unit 2 s Start Read Parameter Pump1 Inner Tubing Diameter Peristaltic Pump Actuator Bridge Liquid Handling Cultivation Unit 1 and Cultivation Unit 2 Pump 2 Inner Tubing Diameter r Rotation Speed in Percent Rotation Speed in Percent Calibrated Flow Rat Calibrated Flow Rat Read Parameter Choose revolution Clockwise C Counter Clockwise Send Parmeter Choose the Pump which become new parameters Pump 1 C Pump 2 Pumpi and Pump 2 Tubing Diamet Inner T bing Diameter Approximate Flow Rate min max 0 044 4 4 fe Reactor Type 083mm x Rotating Speed Send Parameter Received Figure 4 2 BMTC Control tab Peristaltic Pump To get an overview of the buttons in this tab see Table 4 2 Table 4 2 Overview of the buttons in the tab Peristaltic Pump Button Function Start Starting rotation of the chosen Peristaltic Pump to the chosen direction Stop Stopping the rotation of the Peristaltic Pump Read Parameter Reading the parameters of the pump that are adjusted Send Parameter Sending the new parameters to the pump Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 83 EAD a University of Applied Sciences Jena SErium 4 3 Actuator Bridge The Actuator Bridge can be controlled by th
8. Figure 3 16 3D CAD Model of the Optical MUX The Figure 3 17 shows how the Light Fibre from the instrument is connected to the Fluorescence Sensor in the FSC Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 35 EADS a University of Applied Sciences Jena SSErium Section view of the Connector Port Counterpart of the Connector Port Flow direction of the cultivation liquid Fluorescence Sensor with a fluorescence ink spot at the bottom side Figure 3 17 Section view of connection between the Fluorescence Sensors and the Light Fibres 3 1 3 Peristaltic Pump For the transport of the cultivation liquid through the ECMs a Peristaltic Pump is used Therefore the Peristaltic Pump IPC from ISMATEC was chosen and connected to a computer by using the serial interface of the pump to enable its controlling The communication protocol is documented in the manual of the ISMATEC pump 4 This pump had to be modified by another student The Figure 3 18 shows the original unmodified ISMATEC Pump Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AYATA Fachhochschule Jena Page 36 EADS ae University of Applied Sciences Jena SSErium Figure 3 18 ISMATEC Peristaltic Pump in the original state To use the pump as part of the Cultivation Un
9. Modular Support System Manual Exchange Interface Figure 3 27 Manual Exchange Interface with ECM 3 2 4 Liquid Handler Adapter LHA When the Liquid Handler Adapter LHA is connected with the Modular Support System the ECMs can be supplied with Cultivation Liquid To fulfil this task the LHA is connected with the Cavro Pump see chapter3 2 7 over a spiral tube and an I F When the LHA is not in use the Modular Support System is placed into the Storage Position As a waste bag is attached to the Storage Position it is possible to pump a cleaning solution through the LHA and clean it The I F in Figure 3 28 has on the one side a Canula and on the other side a septum These both are stored in the interface box The box fixates the position of these two parts and so they can not change their position This construction enables to sterilise these parts of the Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AMA AVATA Fachhochschule Jena Page 46 EADS et University of Applied Sciences Jena interface from the outside and reunite them in a contamination free state This became necessary because the LHA and the Cavro Pump are placed at vertically opposite sides When the Medium Storage has to be refilled it can be taken out of the BMTC and the interface has to be detached to avoid the burst of the tubes The Figure 3 28 shows the LHA with the spiral tube and the I F Se
10. These alignment restrictions are not necessary for the ECMs because they are using these degrees of freedom to get suitably handled Whereas the Figure 3 44 shows this alignment its connection to the LHA can be seen in Figure 3 29 The following changes of the robot had to be realised during this diploma thesis e Enlargement of the gripper hole e Fit advancement of the alignment for the LHA e Improvement of the robot nut holder for the LHA connection e Aluminium material of the LHA body was exchanged by PEEK material The enlargement of the hole was necessary to improve the fit of the sphere in the gripper Through this modification the dimensions of the gripper had to be adapted as well to ensure the stability of the gripper Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVAVA Fachhochschule Jena Page 66 EADS za University of Applied Sciences Jena SErium During the first tests of the robot with the LHA it exposed that the I F of these both have too much backlash That is why the cup nut see Figure 3 28 was measured and the inner dimensions of the alignment were redefined After the fitting of the alignment was improved a changing of the position of the LHA was identified This problem existed due to the bad connection of the aluminium sheet to the gripper based by a single screw This aluminium sheet connects the robot with the Nut holder for the LHA
11. 35 Sensor board to measure the pressure on port 5 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a 4 AVATA Fachhochschule Jena Page 54 EADS et University of Applied Sciences Jena SErium During the tests of this diploma thesis it turned out that an additional Contamination Protection Cover around the syringe of the Cavro Pump became necessary The Figure 3 36 shows an additional adapter that makes it possible to attach a tubular foil around the syringe and the pump piston This guarantees that the Cavro Pump can work contamination free The space between the adapter and the plunger was sealed with silicone d S 9 a Sant Contamination Protection Cover Adapter of the Contamination Protection Cover Figure 3 36 Cavro Pump with adapter and tubular film to make the syringe contamination free The Contamination Protection Cover of the Cavro Pump is made of plastic and had to be tested carefully to ensure that the foil does not get clamped by the moving piston and it does not get cracks The program was written on the basis of the long term test program described in section 3 1 2 1 Only the sequence of commands was adapted for this application The Figure 3 37 shows the MMI and a set of typical operational parameters of the long term test of the Cavro Pump Daniel Redlich System tests and optimisation of an automated biotechnology facility
12. EADS ea University of Applied Sciences Jena SErium 6 Bibliography 1 2 3 4 5 EADS Space Transportation GmbH Simplified BMTC Ground Demonstrator TN 1 Scientific and Technical Background Issue 1 p 25 Precision Sensing GmbH Instruction Manual OXY 4 4 Channel Fibre Optic Oxygen Meter p 51 f http www presens de fileadmin user_upload downloads manuals OXY 4 UM3 pdf Last viewed 10 08 2009 Precision Sensing GmbH PH 4 mini 4 Channel Fibre Optic pH Meter p 43 f http www presens de fileadmin user_upload downloads manuals pH 4 mini UM1 1 pdf Last viewed 10 08 2009 ISMATEC Operational Manual Tubing Pump with planetary drive IPC IPC N 23 03 07 Tecan Systems Operating Manual Cavro XLP 6000 Modular Syringe Pump p 1 2 f October 2005 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 94 EADS a University of Applied Sciences Jena SSErium 6 7 8 9 10 11 Oxygen absorber RP K http www cwaller de deutsch htm teil6 3 sauerstoffarmelagerung htm information Last viewed 10 08 2009 Prof Dr Ing habil Dr h c Gottfried W Ehrenstein Dr Ing habil Sonja Pongratz Best ndigkeit von Kunststoffen 1 Auflage Carl hanser Verlag M nchen 2007 p 530 Prof Dr Ing habil Dr h c Gottfried W Ehrenstein Dr Ing habil Sonja Pongratz Best ndigkei
13. F VED gt 5C0 2 gt COMMAND RECEIVED gt gt Q0BF 22 04 2009 16 21 03 22 04 2009 16 21 05 22 04 2009 16 21 05 22 04 2009 16 21 09 23 2 Example of successfully Statement which signalizes that the 5 completed command command was accurately received 2 gt MOTOR HAS RY POSITION Indicates what operation was executed by the Actuator Bride 22 04 2009 16 22 37 22 04 2009 16 22 37 22 04 2009 16 22 46 22 04 2009 16 22 52 22 04 2009 16 22 57 22 04 2009 16 23 03 22 04 2009 16 23 28 22 04 2009 16 23 33 22 04 2009 16 23 39 MOVED TO POSITION A FRISA ECM UNLOCKES ECM L gt says that the command was fulfilled correctly I says that a problem occurred during the 22 04 2009 16 24 18 i i 55 04 3009 16 24 18 execution of the belonging command 22 04 2009 16 24 18 MOTO z TOV aS _ ACOMMAND RECEIVED ne COMMAND RECEIVED gt MO3BO ALL SERVOS RESETED COMMAND RECEIVED R183 NOT READY COMMAND IGNORED COMMAND RECEIVED RO82 NOT READY COMMAND IGNORED COMMAND RECEIVED 38C NOT READY COMMAND IGNORED Example of an occurring error Figure 3 12 Cut out of the log file of the long term test program for the Actuator Bridge Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 31 EAD a University of Applied Sciences Jena SErium 3 1 2 2 Solving
14. University of Applied Sciences Jena SErium Status codes There is several status data that can be requested see Command Specification The response comes attached to the Command accepted response in the same line after the echo see Bridge Response General Status Requested with the command S or SG Possible responses O Not_yet_ready 1 Ready 2 Waiting System was reseted 4 Stepper motor board was reseted Reference search needed 5 The Stepper motor board is not responding 6 The values of the Oscillatory Pump make it exceed the limits of the mechanism Configuration Status Requested with the command SC Possible responses 00 Queuing mode disabled Checksum disabled 01 Queuing mode disabled Checksum enabled 10 Queuing mode enabled Checksum disabled Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 103 EAD za University of Applied Sciences Jena aserium 11 Queuing mode enabled Checksum enabled Light Laser sensor Status Requested with the command SL This status request returns 0 if the TTL signal coming from the sensor is low and 1 if it is high This information tells if all the release mechanisms are locked or one of them is loose At the time of this version of the document the sensor was not yet chosen so the meaning
15. alignment of the Canula with the septum I Fs of the ECMs The interface consists of e The sphere e The short threaded bold e The cap nut The sphere will be used to grab the LHA with the Modular Support System The cap nut and the threaded bold ensures that the LHA can not change its adjustment The LHA has finally the same vectorial axial orientation as the end effector of the robot At the start of this diploma thesis the LHA was available only as a functional model which can be seen in Figure 3 29 It was built to test the interaction of the Modular Support System with the LHA Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 48 EADS ee University of Applied Sciences Jena SErium Modular Support System I F between LHA and Modular Support System Figure 3 29 Old Storage Position with LHA and Modular Support System The following changes have been made on the LHA e Fitting accuracy of the front Luer Lock Canula I F has been improved o To ensure a Save interaction with the Canula Rack e The liquid transport through the LHA has been implemented and tested o The liquid transport through the LHA is essential for the liquid support of the ECMs e The diameter holes responsible for the liquid transport of the LHA had been minimised o The dead volume of the LHA had been as minimal as it is possible old dea
16. be exhausted Protect the exhaust tube against penetration of 14 Exhaust foil tube protection a Canula 15 Septum I F of the Spray Pump penetration by the Canula 16 Septum cover Fixes the septum on the Spray Pump In the reactor the tissue will be cultivated the 17 Reactor with mounting plate reactor specific mounting plate attaches the reactor to the ECM Housing Actually there should be a tube in Figure 3 4 to connect the both tube adapters But to keep the expressiveness of the construction this part had to be left out This mentioned tube is shown in Figure 3 7 and Figure 3 8 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 20 EAD a University of Applied Sciences Jena SErium 3 1 1 1 The ECM housing The ECM housing is responsible for the mechanical accommodation of all parts of the ECM and provides the connection I F between the ECM and the Peristaltic Pump This is realised by a Locking Mechanism on the one side and a clamp on the other side The I F is pictured in Figure 3 21 The housing is designed and produced by IVSS but the entire concept and its outer dimensions are defined by Astrium Due to its reusability only a small amount of them is needed So it is sufficient to produce it by milling This causes another advantage The inner dimensions can be adapted depending on the not yet defined final shape of
17. biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 62 EAD paea University of Applied Sciences Jena SSErium All tubes have to be cleaned with the following liquids in this order 1 Solution of isotonic Sodium Chloride as washing medium 2 Sterilisation solution H202 3 Double distilled water or isotonic NaCl as washing medium Samples of the Solution of Sodium Chloride and the Double distilled water have to be extracted in a test tube to check by a specialised lab if the sterilisation was successful Each test tube should be filled with liquid 0 5 ml of every channel to get an information if all tubes are sterile But when the BMTC is switched on the first time or after a long break a sample of every single channel has to be taken in its own test tube 6 ml This procedure is recommended to ensure clean channels and in case to know the location of a contaminated one The liquid that was used to clean the tubes of the LH should be expired trough port five because this port is connected to the stored LHA who is connected to a waste bag to collect the used liquids Furthermore is it possible to transport the LHA with the Modular Transport System to the test tubes and inject the samples into them and it is feasible to remove the used liquids over port five to clean the LHA and all tubes too To properly clean all parts of the LH the following procedure is performed for every above mentioned liquid
18. biotechnology facility for tissue engineering L Ma 4 AVATA Fachhochschule Jena Page 10 EADS eh University of Applied Sciences Jena SErium weekend scenario during which the whole BMTC system is supposed to operate autonomously without any human intervention for the time of one experimental run Furthermore the BMTC has to interact with the outer environment Those external services include manual servicing reloading of media consumables disposables sample vial exchange collection of used medium as samples and other minor tasks The Waste Disposal is for the remove of disposal materials like the Canulas from the Liquid Handling and used media To ensure a constant temperature of 37 C in the inner of the BMTC a hull covers the entire facility So not only the temperature but also the atmosphere inside the BMTC can be controlled Legend Environment Monitoring Control Physical interaction Aw or A transport of reactors a xperiment Chamb Aodules Transport of liquids te Transport of solid waste and resupply ne Power and Control External Analysis ft Periodic manual Sampling vials servicing media agents Microscope slides etc Sampling vials etc Figure 2 1 System concept of the BMTC 1 The Figure 2 2 below shows an initial concept of the BMTC It demonstrates a schematic overview and the set up of the components Daniel Redlich System tests and optimisation of an automated biotechnology fac
19. conditions a cultivation liquid is required to supply the tissue with nutrients In the reactor the tissue will be cultivated and in the supply part the liquid will be circulated and analysed Another important device is the Liquid Handling its supplies the ECMs with new cultivation liquid and removes the old one as waste sample All operational necessary liquids are stored in the Medium Storage The Sample Storage instead is used to store and freeze the removed cultivation liquid from the ECM This makes it possible for the scientist to perform later a detailed analyse of the cultivation liquid The tissue needs stress to grow properly This is provided by Mechanical Stimulation through the following methods e Flow induced shear stress e Uni axial compression There are two possibilities to analyse the tissue inside or outside of the BMTC If an external analysis of the tissue shall be performed then the ECMs have to be unloaded from the BMTC by the Mechanical Exchange Interface and the tissue has to be analysed by a scientist For the internal analyse a diagnostic and an imaging system has been installed into the BMTC Additionally the BMTC features a Modular Support System which functions as the key element in providing all needed mechanical transport and positioning services to the ECMs by using a handling robot This is especially required for proper operation of the so called Daniel Redlich System tests and optimisation of an automated
20. facility is launched to space So the BMTC has firstly been developing on this purpose When took over the project the design concept of the BMTC ground model was already finalized and a lot of parts were already constructed So the main objective of my thesis was to finish the project and improve its functional and operational robustness This process and the achieved results are documented within this diploma thesis whereas contributed work is explicitly mentioned Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 3 EAD a University of Applied Sciences Jena SErium Table of Contents 1 2 2 1 2 2 3 1 3 1 1 3 1 2 3 1 3 3 2 3 2 1 3 2 2 3 2 3 3 2 4 3 2 5 3 2 6 3 2 7 3 2 8 3 2 9 3 3 3 3 1 3 3 2 3 3 3 3 3 4 3 4 4 1 4 2 4 3 Objective of the Diploma Thesis cccccsessssecececeeeesesseeeeeeeeseesssseaeeeeeeseeeees 7 The Simplified Biotechnology Mammalian Tissue Culturing Facility Ground Demonstrator S BMTC GD sssssessessrrrrressssrrrerrressesrrirrrnssesreerrrersseserereneseeseere 8 General Information ssssssesssesesesssrerssensssssressstesstessreseressstessrenstensesenereseseenseens 8 System COMCEDLE ssciss viisesieisedediexinsiavseaielsseisad dane a AA A AN seen 9 Test Analysis and Improvement of the BMTC s ssssssssssssssseseneseressssrrsssrsesee 13 Cultivation UIE iesise
21. following steps with the test setup in displayed in Figure 3 40 LHA Interface LY a Spiral tube 0000 0 g Balance O O Cavro Pump DIGITAL IN 1 DIGITAL IN 2 GND 24V Power Supply Unit RS232 interface eu IEJ Figure 3 40 Sketch of the test setup for the measurement of the dead volume Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a 4 AVATA Fachhochschule Jena Page 59 EAD a University of Applied Sciences Jena SErium Steps 1 Ensuring that no liquid is in the tubes on port 5 2 Aspiration and expiration of water into port 4 to ensure this tube is filled with water Noting the weight of the tank filled with water Aspiration of 10 ml through port 4 into the syringe of the Cavro Pump Expiration of the 10 ml water through port 5 Se a oe Noting the new weight of the tank filled with water The difference between the first and the second measured weight is the amount of the kg water in the tubes connected with port 5 Because water has a density of 1 3 m 13 the weight in g in the tubes is equal to the volume in ml in the tubes Table 3 2 Series of measurements of the dead volume Old weight in g 50 1 50 2 50 1 50 3 New weight in g 48 1 48 1 48 0 48 3 Difference in g 2 0 2 1 2 1 2 0 Dead volume inmi 2 0 2 0 2 0 2 0 The concentration of the old liquid in the new liquid can b
22. for tissue engineering 1 aA 4 AVATA Fachhochschule Jena Page 55 EADS a University of Applied Sciences Jena SErium m Long Term Test Cavro Pump Ses Long Term Test Cavro Pump comi Connect Disconnect Number of cycles 20d x15min300 min Start 0 times executed Note Please ensure that the Port 1 is able to aspire liquid because without the syringe will be damaged Figure 3 37 MMI of the long term test of the Cavro Pump The sequence of this long term test program is the following e Open valve port 1 of the Cavro Pump drive 6000 steps down e Open valve port 1 of the Cavro Pump drive 6000 steps up This sequence can be performed as often as the user wants Further information can be found in section 3 1 2 1 3 2 8 Supply Procedure for an ECM This section describes how all parts of the Liquid Handling are working together to supply the ECMs Each ECM has three septa interfaces where it can be penetrated by the LHA e Spray Pump Septum e Waste Sample Septum e Bubble Trap Septum They can be seen in the Figure 3 2 and Figure 3 3 During the filling of the ECMs it can happen that some gas enters the FSC For this reason the Bubble Trap was implemented Now the gas in the Bubble Trap can be detected as soon as Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering aA H AYATA Fachhochschule Jena Page 56 EADS eet University of Applied S
23. is necessary Every 60 seconds one command will be sent This delay was integrated to let the Actuator Bridge fulfil its respective task The Figure 3 11 below shows the Man Machine Interface MMI in which execution settings of the test can be entered To perform the long term test two parameters have to be chosen the COM Port which is connected to the Actuator Bridge and the amount of times the sequence is to be executed The long term test begins when the Start Button is pressed Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 30 EADS a University of Applied Sciences Jena SErium Long Term Test Actuator Bridge com 4 x Connect Disconnect Number of cycles fioo x6 5min 650 The estimated time for the long term test 1 times executed Figure 3 11 MMI of the long term test of the Actuator Bridge The program creates a log file for each performed test The log file was implemented to proof the error free operation of the long term test So nobody has to be present during the test of the Actuator Bridge The next Figure 3 12 shows the log file and describes what the single messages are standing for P Actuator Bridge Loa txt Editor O x D Initialisation and t 24 COMMAND RECEIVED gt X58 x 2 Start of the MOTION STOPPED AND BUFFER CLEARED 24 debug mode SCOMMARND
24. number from 01 to 20 always 2 digits The last character specifies if an offset must be included R release offset O optical sensor offset Y spray pump offset Examples M12 MO8sy Internal Parameters R Move units relative to the current position A Move to the absolute position relative to zero point Examples MR 1500 MA2114 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVAVA Fachhochschule Jena Page 105 EAD ea University of Applied Sciences Jena SErium R Releasing Mechanism ECM Parameters 0 Lock 1 Unlock Example RO Y Spray pump Parameters number of times the spray pump will be activated 1 to 9 Example Y8 O Optical Sensor Parameters 0 Disengage 1 Engage Example 01 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVATA Fachhochschule Jena Page 106 EAD ea University of Applied Sciences Jena SErium Oscillatory Pump Parameters O Oscillate X Stop S Status Request See the details of the response in the section Status Responses Parameters G General Optional If only S is sent the General Status Response is returned B Oscillatory Pump Frequency C Oscillatory Pump Stagnation Time D Oscillatory Pump Offset Examples S S B
25. of Applied Sciences Jena SSErium The Peristaltic Pump is a commercial product with an eight rolls around one central axis The eight rolls will be driven by a planetary gear thereby no slip accurse between the rolls and the tube This has the following advantages e No axial stress of the tube e Long operating life The flow rates were tested by a student who formerly interned at Astrium only the serial interface of the Peristaltic Pump has to be examined Software was used to check the functionality to check the pump This software will be described in section 4 2 The Peristaltic Pump worked without any errors The correct pumping of the liquid cannot be completely confirmed until the final ECMs will be delivered to Astrium by the subsystem responsible subcontractor 3 2 Liquid Handling LH The Liquid Handling takes care of the storage and the handling of all the liquids used in the BMTC The Figure 3 22 gives a detailed overview of the Liquid Handling The important parts of the Liquid Handler are listed below e Cooler Unit o Stores the liquid at a constant temperature of 4 C e Manual Refilling Port o Makes it possible to refill the liquid storage bags during a BMTC operation e Cavro Pump o Transports the liquid control of volume and speed o Control of the 9 port valve e Liquid Handler Adapter LHA o Provides the interface to the ECMs o Can be handled by the Modular Support System see Chapter 3 3 e Storage Position for
26. of the TTL logic is not yet defined Servo Status Requested with the command SS This status response has 4 bytes Each one of them tells information about one of the servos A zero means that the servo is retracted that means in the position necessary for the free movements of the other components From left to right 1st ECM Unlocking Mechanism 2nd Optical sensor Mechanism 3rd Spray Pump 1 means also that we are currently spraying 4th Oscillatory Pump 1 means also that the pump is currently oscillating Position Status Requested with the command SP Returns the position of the Bridge in the Bridge s coordinate system See Position Description and Coordinate System Oscillatory Pump Values Status Requested with the command S followed by the needed variable as listed below they return the saved value Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVATA Fachhochschule Jena Page 104 EAD ae University of Applied Sciences Jena SSErium B C D Oscillatory Pump Frequency in s Period Oscillatory Pump Stagnation Time in s Oscillatory Pump Offset in Command Specification End user commands X Stop Bridge Also clears buffer in Command Queuing Mode H Go Home Reference If the bridge does not respond to this command read the document Setting up the TMCM M Move Parameters _ move to Position
27. optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 50 EADS a University of Applied Sciences Jena SErium To access this volume from the ECMs the LHA is connected with the Modular Support System and has mounted a Canula on its Luer Lock Adapter The Canula of the LHA penetrates the septum of the Waste Sample storage of the used medium and the sample is unloaded by aspiration of the Cavro Pump Afterwards the Modular Support System convey the LHA to the sample cooler I F where the test tube is stored There the Canula penetrates the septum of the test tubes and then the Cavro Pump will eject the sample into the test tube When the test tube is filled a transport system brings the test tube to the Sample Storage see Figure 3 31 Sample Storage Freezer 20 C Figure 3 31 Schematic sketch from the function of the Sample Storage These transport system is developed by KT The fixation of the Sample Storage is also in the responsibility of KT The Figure 3 32 shows the place of the I F where the Canula penetrates the septum of the test tube Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a H AVATA Fachhochschule Jena Page 51 EADS et University of Applied Sciences Jena SSErium test tube Figure 3 32 LHA in the position to fill the test tube 3 2 6 Waste Disposal Canula The Solid Was
28. procedures will be performed under the following cases If the reference run will be performed the first time then it is necessary to teach the first position manually But if the reference run was performed more than once the first position is already known by the Robot and so no user intervention is required Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 73 EAD a University of Applied Sciences Jena SErium Steps 1 Find Reference Position First time the reference run is done Switch all motors of the robot off Take the robot with the hand and place the gripper of the robot onto the I F sphere of the target Switch the motors of the robot on All reference runs after the first one 2 3 Oo a N om n 11 12 13 14 15 16 17 18 19 Move to the Robot Park Position Move to the position of the first ECM stored during first reference run Grip the sphere of the ECM Close the gripper with full power Switch all motors off apart from the gripper s one Close the gripper with full power Switch all motors on Store the new position Move 80 mm in Z Direction of the tool coordination system Move 30 mm in Y Direction of the tool coordination system Move 75 mm in Z Direction of the tool coordination system Grip the sphere with full power Switch all motors off apart from the g
29. the Liquid Handler Adapter LHA o Mechanical storage of the LHA o Provides an interface to a waste bag for cleaning the LHA Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AYATA Fachhochschule Jena Page 39 EADS et University of Applied Sciences Jena SErium e Pressure Sensor o Measures the pressure in the spiral tube between Cavro Pump and LHA o Used to detect defined operational states Cooler Unit Optional Syring Filter with a pores Medium B Channel 1 carers S emes Manual Refilling Port Pressure Sensor A Liquid Handler Adapter LHA Canula Rack Storage Position for the Liquid Handler Adapter di A Cavro Pump DIGITAL IN 1 DIGITAL IN 2 Power Supply Unit RS232 interface ji eu Z Figure 3 22 Sketch of the whole Liquid Handling system fluorescence Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AMA AVATA Fachhochschule Jena Page 40 EADS Le University of Applied Sciences Jena 3 2 1 Medium Storage Cooler Unit In the Medium Storage all liquids for the daily work of the BMTC are stored at a temperature of 4 C 9 The former design concept of the Medium Storage was improved and built during the making of this diploma thesis Manual Refilling Ports Refrigerator Figur
30. the Status Display Exit Finishing the program BMTC Control Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 81 EAD a University of Applied Sciences Jena SErium 4 2 Peristaltic Pump The tab Peristaltic Pump provides functions to setup the Peristaltic Pump and is shown in Figure 4 2 The upper box in the Peristaltic Pump tab enables the possibility to select which pump is to be turned into which direction The box left is called Read Parameters this box lists the parameters that are adjusted at the moment The parameter Calibrated Flow Rate cannot be adjusted and is calculated by the Peristaltic Pump based on the parameters of the Inner Tubing Diameter and the Rotation Speed On the right side in this tab is a box called Send Parameters This box enables to setup the Peristaltic Pumps The values that have to be adjusted are the Inner Tubing Diameter and the Rotation Speed in percent The rest of the fields in this box are filled in automatically by the program and is only for user information The selected parameters are submitted to the respective pump whenever the Send Parameter button is pressed Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AVA AVAVA Fachhochschule Jena University of Applied Sciences Jena Page 82 BMTC Control General Choose Pump
31. 0 University of Applied Sciences Jena 1 EADS __ SsErium 9 2 Annex B Cleaning Procedure Start Cleaning Aspiration 10 ml of Cleaning Solution from the tube which provides Seeding solution Expiration over the tube that is Aspiration 10 mi of Cleaning Solution from the tube which provides Start medium Expiration over the Ports 1 4 of the Cavro Pump 2 5 ml over each Port Aspiration 10 ml of Cleaning Solution from the tube which provides Priming Buffer Expiration over the Ports 5 8 of the Cavro Pump 2 5 mi over each Port and fill it with Sterilised Water Sterilised Water from the tube which provides Expiration over the tube that is Sterilised Water from the tube which provides Start medium Expiration over the Ports 1 4 of the Cavro Pump 2 5 mil over each Port Aspiration 10 ml of Sterilised Water from the tube which provides Priming Buffer Expiration over the Ports 5 8 of the Cavro Pump 2 5 ml over each Port ng e ba with Sterilis Water and connect a sterile Air Filter with Septum Aspiration 10 ml of Sterilised Air from the tube which provides Seeding solution Expiration over the tube that is Waste Bag Aspiration 10 ml of Sterilised Air from the tube which provides Start medium Expiration over the Ports 1 4 of the Cavro Pump 2 5 mi over each Port Aspiration 10 ml of Sterilised Air from the tube which p
32. 1 days had been simulated After the test passed successfully the Actuator Bridges was integrated by KT When the integration was completed all functions of the Actuator Bridge had to be tested again During these functional tests an error occurred which have never appeared before The problem was that the reference run had to be done twice before the Actuator Bridge had signalized the ready state The solution of this issue is presented in section 3 1 2 2 The writing of the long term test software for the Actuator Bridge as well as the solution of the above mentioned problem is part of this diploma thesis Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 29 EAD a University of Applied Sciences Jena SErium 3 1 2 1 Long term test of the Actuator Bridge To perform long term tests a software was written using the programming language Visual Basic VB The resulting program sends a sequence of commands to the Actuator Bridge in the following order 1 2 3 4 5 6 7 8 9 Reference run Drive to position 1 Drive to position 8 Test all servos Drive to position 0 Reference run Drive to position 3 Test all servos Drive to position 8 with an offset 10 Test all servos 11 Drive to position 0 This sequence can be performed as often as the user wants it So it is possible to test the Actuator Bridge as long as it
33. 3 26 Exploded view of the Canula Rack The equipping of the Canula Rack has to be done accordingly to the following steps 1 2 a Sh m oe Remove the screws of the cover plate Removal of the used aluminium foil and the unused Canulas Checking of the O rings after the O rings were used three times they have to be exchanged at the latest Loading of new Canulas into the holes of the Aluminium Block Placing a new aluminium foil onto the O rings Placing the cover plate over the aluminium foil Tightening the screws of the cover plate 3 2 3 Manual Exchange Interface Gate The Manual Exchange Interface is used to facilitate the exchange of the Canula Rack and the ECMs between the BMTC and the outer environment This became necessary because the BMTC should not be opened during an experiment The transport of the ECMs between the Manual Exchange Interface and the Peristaltic Pump is realised by the Modular Support System To record and examine the ECMs on the Manual Exchange Interface a web camera was placed within the BMTC This feature additionally allows checking the amount of gas Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 45 EADS University of Applied Sciences Jena SSErium that is collected in the Bubble Trap These parts are built by KT The Figure 3 27 shows the Manual Exchange Interface holding an ECM
34. 9 2 PANMOX Boae a E E a E E E E AA EE 110 9 3 ANIE A E E A E E E S AT E E 115 10 Own Work Declaration aseissrrrrrerriisrorurriirruiptnirittr arreir Sir EEEN EEFE EURER eE 116 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 aw ve AVATA Fachhochschule Jena Page 5 EAD University of Applied Sciences Jena poese aj SSErium Table of Abbreviations and Formula Symbols ECM Experiment Chamber Module I F Interface KT C CKaiser Threde Simplified Biotechnology Mammalian Tissue Culturing Facility S BMTC GD Ground Demonstrator VB Visual Basic MMI Man Machine Interface Astrium Astrium Space Transportation GmbH Optical MUX Optical Multiplexer FSC Fluid Storage Cassette IWSSsi lt lt i is s s dN Vitro Systems and Services ti i isSOSOSOSOS S S S LH Liquid Handling LHA Liquid Handling Adapter Robot Modular Support System Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 6 EAD a University of Applied Sciences Jena SErium Acknowledgements Special thanks go to Dr Ing Peter Kern who has given me the opportunity to work at this great and interesting project Furthermore am grateful for his trust and helpful influence by giving good advices and always having a great new idea in mind would also
35. Info o Shows general information about the communication with the components e Send o Shows the commands in ASCII that were sent to the components e Receive o Shows the commands in ASCII that were received from the components Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 79 EAD a University of Applied Sciences Jena SErium The program was written in Visual Basic The structure of BMTC Control is pictured in Figure 4 1 All subsystems that are connected to the serial I F are configured in the same way They use the following settings for the communication e Baud rate 9600 baud e Data Bit 8 e Stop Bit 1 e Parity Bit None The Control PC is the Master and the subsystems are the Slaves 4 1 General All components that can be controlled are listed in this tab Every component can be switched off when it is not required If a component is needed the Com Port for this device has to be chosen During the tests by Astrium the Com Ports of the subsystems had been changed so the Com Ports are arbitrary but in the end configuration by KT the Com Ports does not change anymore A list with the defined Com Ports shall be available from KT One laptop was setup in a way that he can operate with the RS232 switches from KT The Status Display shows if a component is ready not ready or not yet checked This check can be done fo
36. M storage Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a 4 AVATA Fachhochschule Jena Page 13 EAD a University of Applied Sciences Jena SErium 3 Test Analysis and Improvement of the BMTC In this chapter all important subsystems of the BMTC are explained as well as the long term test which was realised to guarantee an operationally robust function of all components at subsystem and system level 3 1 Cultivation Unit The Cultivation Unit is responsible for the cultivation and supply of the samples It consists of three parts with different tasks respectively which will be explained in the next paragraphs e Actuator Bridge e ECM e Peristaltic Pump In Figure 3 1 these parts are attached on the Mounting Plate Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 14 EADS University of Applied Sciences Jena SSErium Peristaltic Pump Actuator Bridge Mounting Plate Figure 3 1 Cultivation Unit Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 15 EAD a University of Applied Sciences Jena SErium 3 1 1 Experiment Chamber Module ECM The ECM is responsible for the cultivation liquid supply and the placement of the tissues The tissue is p
37. a Page 16 EADS a University of Applied Sciences Jena SErium These parts will be explained in the next chapters and in Table 3 1 The Figure 3 2 shows the ECM with the 2D reactor and the Figure 3 3 shows the 3D reactor These both ECMs are identical besides their reactors and their mounting plates for the reactor Although the 3D Reactor for cartilage and bone has the same shapes only in the inner there are some differences F for External Handler Bubble Trap Septum Optical Sensor I F Wast Sample Septum Spray Pump Septum Medium Spray ff Bioreactor 2D on dedicated mounting plate Fluid Storage Tube track Cassette Figure 3 2 ECM with 2D Reactor Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 17 EADS ee University of Applied Sciences Jena SErium External Handler I F Bubble Trap Septum Optical Sensor I F Waste Sample Septum Spray Pump Septum Fluid Storage aes ii Cassette Bioreactor 3D on dedicated mounting plate Tube Track Figure 3 3 ECM with 3D Reactor The Figure 3 4 shows the whole ECM in an exploded view Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena University of Applied Sciences Jena Page 18 EADS CEE Figure 3 4 Exploded view of the ECM Table 3 1 All parts of the ECM
38. ally connected with the reactors have to be connected to empty bags just as the tube that leads to the Waste bag This is provided to catch the waste that will be produced during the Cleaning Procedure A sketch of this configuration can be seen in Figure 3 48 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 76 EADS ce University of Applied Sciences Jena SErium 1 J Seeding Device RS232 Figure 3 48 Schematic sketch of the Seeding Unit for the Cleaning Procedure In Annex B Section 9 2 the Cleaning Procedure the Priming Procedure the Filling Procedure the Seeding Procedure and the Washing Procedure are displayed At the beginning of this diploma thesis the Seeding Unit was already available in its current form only some upgrades had to be realised e Integration of the Cavro Pump into a box to protect the electronics during the operational liquid handling e Fixation of the seeding unit to an infusion holder e Attachment of a Contamination Shield The same like by the Cavro Pump of the Medium Storage see Figure 3 36 e Mounting a hook to hold the Waste Bag e Reworking of the power supply and the communication connection new connection is described in Annex C section 9 3 Daniel Redlich S
39. as in sterile conditions The Figure 3 25 shows the Canula Rack in an explosion view Generally it is designed in such a manor that it can be reused and each Canula can be removed without contaminating other Canulas This is done to minimise the operational costs The cover plate is used to push the aluminium foil against the O rings Additionally the Cover Plate has a chamfer on every entrance of the holes This is implemented to minimise the clearance of the Modular Support System so the LHA finds the hole properly The holes of the Aluminium Block are slightly grooved to fixate the respective O rings The sterilisation of the Canula Rack will be realised with gamma radiation Silicon of the O rings can absorb 50 100 kGy 7 During one sterilisation an amount of 25 kGy 8 impacts on the Canula Rack and also on the O rings That means these rings have to be exchanged after three gamma ray radiations Before the Canula Rack is used again the O rings have to be examined if there are brittle If this event appears the closeness can not guarantee and the O rings have to be changed prematurely This additional check and a possible previous exchange ensure the total sterility of the Canulas Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 44 EADS cl University of Applied Sciences Jena SSErium Aluminium block Store adapter Figure
40. ase 3 joints 1 rotating gripper e Communication Serial I F Figure 3 43 Katan 6M180 robot The Katana robot is equipped with six harmonic drive joints each assisted by a digital encoder Its basic movements are based on the cylinder or sphere coordinate system Cartesian movements in x y z direction has to be assembles by using the basic coordination systems To make it possible that the Robot can grip the ECMs and the LHA the Gripper of the robot was modified The standard gripper was exchanged by a self build gripper The self made gripper has two holes in his sides and is shown in Figure 3 44 These holes are necessary to ensure a form closure and alignment between the gripper and the sphere which is attached on the ECMs and the LHA Thereby the three linear degrees of freedom are eliminated To protect the spheres against rabbles and to increase the holding friction a rubber protection pad was glued on the inner side of the gripper Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena Page 65 EADS e University of Applied Sciences Jena as Aluminium sheet Nut holder for the LHA Holes in the gripper Rubber protection pad Figure 3 44 First Version of the robot gripper Additionally a second alignment Nut holder for the LHA was implemented to eliminate the undesirable two tilting degrees of freedom
41. ation flow chart between stepper motor and Actuator Bridge controler sencia aT EEA E E NOAN TAO AOT cides 33 Figure 3 15 New communication flow chart between stepper motor and Actuator Bridge COMMPON OM aor na a a aa a a 33 Figure 3 16 3D CAD Model of the Optical MUX ssssssssssssssssrsseeressssrrssssresrsserensrereenssrrnessrenne 34 Figure 3 17 Section view of connection between the Fluorescence Sensors and the Light FIDES oren ieni enir ae daer ees ra aeaa e a E eiesdntnane rentaad 35 Figure 3 18 ISMATEC Peristaltic Pump in the original State ccscecssssececessseeeeesssneeeeeees 36 Figure 3 19 3D CAD model of the peristaltic pump including its additionS ceeseeeeee 36 Figure 3 20 Principle function of the Tube Track mounted on the peristaltic pump 37 Figure 3 21 3D section view of the Peristaltic Pump with locked ECM csscccsessteeeeeees 37 Figure 3 22 Sketch of the whole Liquid Handling system fluOresCeNnce cccsscceceeeeseeeeeees 39 Figure 3 23 Medium Store its inner parts are standing on the Plexiglas c cccccessseeeeeees 40 Figure 3 24 Section view of the inner of the Medium Storage ccccssssccccceeeeessessnseeeeeeeees 41 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 nd aa Fehinsactalg dena iia EADS on Figure 3 25 Canula Rack with slide and release gate ccccc
42. bes with the FSC a fitting was designed and integrated o The tube had to be connected with the FSC to ensure a proper flow of the cultivation medium 4 The form of the FSC was changed to avoid a bend of the tube o Inan earlier design the fitting was connected in the same angle like the fitting on the top of the right side in the figure below Thereby the tube bent and the angle of the fitting had to be changed The Figure 3 6 below shows half of the FSC model This design was used to build the final moulding form Figure 3 6 Half form of the FSC with changes Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 23 EAD aa University of Applied Sciences Jena SErium 3 1 1 3 Spray Pump The FSC can replace the used medium by fresh one Therefore the Spray Pump injects a small amount of new medium every few hours The reason why the whole cultivation medium is not exchanged in one step is that these cause a wild concentration fluctuation which will stress the culture and stimulate unwanted activation of genetic switches This is able to influence the results of the experiment Furthermore the design concept of the Spray Pump assures that no I F have to be broken to inject new medium and thereby a contamination will be avoided The volumetric equivalent of the added medium volume within the FSC will be displaced in the Waste Sample Volu
43. ched on at the same moment So the microcontroller of the Actuator Bridge has a zero in its buffer This is the reason why the reference run of the Actuator Bridge has to be executed three times To solve this problem the receive buffer is cleared every time before a command is sent to the stepper motor The right Figure 3 15 below shows the new communication flow chart for the communication between the Actuator Bridge and the stepper motor Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering ww 4 AVATA Fachhochschule Jena Page 33 EADS a University of Applied Sciences Jena SErium Clear the receive buffer Send command to stepper motor Send command to stepper motor Wait 5 ms Wait 5 ms Respons received Respons received Correct response Status not OK Status not OK Status OK Status OK Figure 3 14 Old communication flow chart between Figure 3 15 New communication flow chart stepper motor and Actuator Bridge controller between stepper motor and Actuator Bridge controller 3 1 2 3 Optical Multiplexer Optical MUX The Optical MUX is mounted on the Actuator Bridge see Figure 3 9 and connects the two Fluorescence Sensors of the ECM to the two Light Fibre I Fs of the optical fluorescence sensors The possibility to implement a third Sensor and Light Fibre is implemented but not in use at the moment Tho
44. ciences Jena SSErium the ECM is placed in the Manual Exchange Interface by a nearby located web cam The flow chart of the gas exhaustion process is shown in Figure 3 38 ervice every two or three days Pick up Liquid Handling Adapter from the Parking Position Take a new Canula move to the ECM penetrate the Bubble g Trap Septum w i sE E Exhaust the gas from the z Bubble Trap 25 5a G Remove the Canula from the ECM s Bubble Trap Septum Inject the exhaust gas and liquid into the waste bag that is connected with the Cooler Unit using the syringe pump Dispose used Canula Figure 3 38 Flow chart of the exhaustion of the gas Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering aA H AYATA Fachhochschule Jena Page 57 EADS a University of Applied Sciences Jena SSErium The Figure 3 39 shows the flow chart of a normal servicing cycle This has to be done every two or three days 10 to load the spray pump with fresh medium and to remove the same volume as added by the spray pump from the FSC Sample Storage volume ervice every two or three days Pick up Liquid Handling Spray Pump septum move Adapter from the Parking cannula through ECM waste Position ptum Take a new Canula move to the current ECM penetrate the Aspire the defined waste medium volume Spray Pump septum Empty the content of the Sp
45. ck provides 16 Canulas for the Liquid Handler Adapter LHA see chapter 3 2 4 supporting all 16 ECMs during one servicing event The Liquid Handler is moved by the Modular Transport System for taking a Canula out of the Canula Rack With this Canula the ECMs are supplied with cultivation liquid The Canulas have to be stored under sterile conditions because a soiled Canula can influence the results of the experiments The Canula has a rubber shield to protect it against contamination Every three days the ECMs are provided with fresh cultivation medium and therefore an unused Canula is necessary too Due to this fact the Canula Rack has to be changed every three days To avoid a complex replacement process the Canula Rack is mounted on the Manual Exchange Interface so it is possible to exchange the Canula Rack without opening the BMTC Only the small release gate of the Manual Exchange Interface has to be opened This is shown in Figure 3 25 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a F AVATA Fachhochschule Jena Page 43 EADS a University of Applied Sciences Jena SSErium Manual Exchange Interface Release Store gate adapter Figure 3 25 Canula Rack with slide and release gate At the beginning only the outer dimensions of the Canula Rack were defined and a functional model was available So the design of the Canula Rack had to be changed to store the Canul
46. d Sciences Jena SSErium BMIC Control General Peristaltic Pump Actuator Bridge Liquid Handling Voume of the Syring Initizalise 10 0 ml z Action of Syringe Source Destination Aspiration of syringe gt Port 1 Medium B Check Pressure ypa aeee Low High Pressure Pressure Go Home Start Position of the Syring in Aspired Volume in ml Incremnets Received Figure 4 5 BMTC Control tab Liquid Handling Table 4 4 lists all button and their functions in the tab Liquid Handling Table 4 4 Overview of the buttons in the tab Liquid Handling Button Function Initialise Finding the references of the Cavro Pump s Stepper Motor Check Pressure Manual checking of the pressure Go Home Drives to the reference position Start Starting the specified action with the selected parameters 4 5 Robot To gain access to the Modular Support System the tab Robot has to be selected As mentioned at the beginning of this chapter the software Katana 4D has to be executed parallel with the program BMTC Control to perform interactions with the robot Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 89 EADS za University of Applied Sciences Jena SErium The communication between the BMTC Control and Katana 4D works regarding the following principle based on the used
47. d volume 4 ml new dead volume 2 ml The following changes were realised on the Store Position e All Parts had to be redesigned completely new o The old construction was way too instable Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AYATA Fachhochschule Jena Page 49 EADS La University of Applied Sciences Jena SSErium e The Storage Position has been mounted with an incline of 20 o Inthe old accommodation the Modular Support System was not able to pull the LHA out of the Store Position because the geometrical workspace of the Modular Support System does not sufficient Without the modification a collision between the LHA and Storage Position is possible During the design of the parts for LHA it was important to ensure that the dead volume of theses parts is as low as possible Through this it was possible to minimise the dead volume from 4 ml to 2 ml The Figure 3 30 shows the new accommodation of the LHA and the Storage Position New Storage Position Figure 3 30 New Storage Position with LHA 3 2 5 Sample Storage Freezer Unit The Sample Storage looks like the Medium Storage The only noteworthy difference is that it has an inner temperature of 20 C 9 It is used to store and freeze test tubes filled with the liquid from the Waste Sample storage of used medium part of the ECM see chapter 3 1 1 2 Daniel Redlich System tests and
48. e 3 23 Medium Store its inner parts are standing on the Plexiglas The liquid bags which are used to store the liquids are mounted in the Medium Storage on the Bag Bar All these liquid bags are surrounded by a gastight bag to protect the liquid bags against oxygen which can diffuse into the liquids passing through the bag foils For this reason the gastight bag will be flooded by CO at first After that the CO will be exhausted to minimize the volume of the gastight bag The rest of the oxygen will be absorbed by the Oxygen Absorber RP K 6 which is attached on the Bag Bar The Oxygen Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering av AVATA Fachhochschule Jena Page 41 University of Applied Sciences Jena EADS a Absorber can absorb 63 ml of oxygen per package This amount turned out to be sufficient for the with CO flooded gastight bag The noted gastight bags are attached on the ground plate To connect these bags to the tubes of the LH the Modular Bag I F was built on this ground plate too Furthermore an I F for the gas exchange was implemented It consists of a gas inlet and a gas outlet The Figure 3 24 shows the principle structure inside the gastight bag Liquid bag Retaining Ring Modular Luer Lock Adapter Interface to connect the tube Notch to attach a locking ring Ground plate Figure 3 24 Section view
49. e Figure 9 6 images the pin layout of the plug and the Table 9 1 shows the pin configuration Figure 9 6 Schematic sketch of the a 9 pin D Sub male plug Table 9 1 Pin allocation of the 9pin D Sub plug for the Cavro Pump Pinnumber Abbreviation Name 1 Not connected 2 Rx Receive data 3 TX Transmit data 4 Not connected 5 GND Ground computer 6 GND Ground Power Supply Unit 7 24V 24 V from the Power Supply Unit 8 Not connected 9 Not connected Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 10 Own Work Declaration Declaration hereby declare that this diploma thesis has been written only by the undersigned and without any assistance from third parties Furthermore confirm that no sources have been used in the preparation of this thesis other than those indicated in the thesis itself Erklarung Ich erkl re dass ich die vorliegende Diplomarbeit selbstst ndig und nur unter Verwendung der angegebenen Quellen und Hilfsmittel angefertigt habe Ort Datum Unterschrift
50. e calculated with the following formula c C DeadVolume V beadvotume C new i an Spraypump _ V beadvotume View Cspraypump The new concentration of the old liquid in the Spray Pump DeadVolume The old concentration of the old liquid that was in the Spray Pump Cnew The concentration of the old liquid in the new liquid 0 Vpeadvonme The amount of the old liquid this is injected in the Spray Pump Dead Volume of the tubes to the LHA 2ml Vrew The amount of new liquid that is in injected in the Spray Pump 5ml Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 60 EAD a University of Applied Sciences Jena SSErium To decrease the concentration of the old liquid the exhaustion and filling steps have to be repeated The Table 3 3 shows the concentration of the old liquid in the Spray Pump after one to five filling cycles Table 3 3 Concentration of old liquid after one to five filling cycles Filling cycles 1 2 3 4 5 Concentration of old liquid 28 6 8 2 2 3 0 7 0 2 The decision on the finally best suited filling protocol is up to scientist It will be primarily driven by the cost of compounds solved in the new medium 3 2 9 Cleaning Procedure for the Liquid Handler The Cleaning Procedure has to be done before an experiment starts This is necessary to guarantee a sterile and c
51. e tab Actuator Bridge which is displayed in Figure 4 3 As you can see there are four boxes each with a special task The box on the top allows the selection of the Actuator Bridge that shall be used has to be selected With the box below the chosen Actuator Bridge can be driven to a position and an offset for a Servo can be adjusted Additionally the reference run can be started here and in case some problems appear one of two different debug modes can be selected The second last box is called Servos and is providing options to control the movements of the servos individually The Teach in box is used to teach the Actuator Bridge its current positions the bottom box is designated Additionally a Button was implemented to open the window see Figure 4 4 for setting the servo positions by teaching Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering aA 4 AVATA Fachhochschule Jena Page 84 EADS p University of Applied Sciences Jena SErium BMIC Control General Peristaltic Pump Actuator Bridge Liquid Handling Choose Pump Cultivation Unit 1 Cultivation Unit 2 Go to Position Position Offset Debugmodus 1 m X No Offset v Debugmodus 2 Start Stop Reference Debug Modus Off Servos ECM Sensor Spray Pump Spray Pump pe Teach In Steps to Move Save Current Position C1 10 100 1000 X Forward Backward Save Servos Received
52. ease Servo unlocks the Locking Mechanism 7 Robot drives a bow to tilt the ECM 8 Move backwards and upwards to pull the ECM out of the Peristaltic Pump 9 Move to the position where the ECM shall be placed 10 Catch the Pivot bar with the ECM 11 Push the ECM down on the Peristaltic Pump the first latching level locks the ECM 12 Release the ECM from the gripper 13 Use the closed gripper to push down the ECM position behind the Spray pump 14 Move robot back to the Robot Park Position Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 71 EADS University of Applied Sciences Jena SSErium Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AYATA Fachhochschule Jena Page 72 EADS et University of Applied Sciences Jena SSErium 10 Figure 3 46 Illustration of the procedure to interact with the ECM 3 3 4 Automated teaching of the Robot positions The exact dimensions of the ECMs are not 100 known at the moment Therefore the Peristaltic Pump was designed insofar as to reduce the position shift of the ECMs To minimise the manual teaching effort a procedure was developed to find the sphere of the ECMs automatically and save the new positions There are two different variations of this procedure and there are only different in the steps two to four The different
53. ept and functional verification and test of the whole ECM and IVSS for its production We decided to produce the FSCs by injection moulding because a big amount of them is supposed to be needed The moulding form is very expensive and produced only once So the development of this part has to be done very well To produce such a difficult form it was necessary to cut the FSC in half lengthwise Both sides of the FSC will be covered by a bio foil which enables the gas exchange with the surrounding atmosphere while the cultivation liquid stays inside the FSC The following changes had to be done in collaboration with IVSS and can be seen in Figure 3 6 1 The interface for attaching the Spray Pump was implemented o The connection to the Spray Pump was defined depending on its dimensions o The sealing had to be tested 2 The interface for installing the fluorescence sensors was extended by an Insert o It is complicated to produce a thread by moulding that is accurately fitting Now the use of an Insert provides more opportunities to implement these threads o The thread size had to be changed from M 5 to M 4 because the Insert did not fit o The Insert allows implementing a possibly required third sensor Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Ma AVATA Fachhochschule Jena Page 22 EADS pee University of Applied Sciences Jena SBsSErium 3 To connect the reactor I F tu
54. gle parties are shown in the next overview e Kaiser Threde o Development of the incubator and of the environmental control system providing the proper test environment o Integration of all devices even if they are developed by other companies o Development of the BMTC control software e EADS Astrium Responsible for all units which are in direct contact with the biological sample or are providing operational services o Development of the operational and technical concepts o Development of the Experiment Chamber Module ECM o Development of the hardware to transport ECMs in the BMTC o Development of the liquid supply for the ECMs o Provision of contamination free on line medium monitoring Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 9 EAD paner University of Applied Sciences Jena SErium e In Vitro Systems and Services IVSS o Production of the ECMs Reactors e Prof David Jones o Development of a microscope to analyse the samples o Development of an actuator to simulate stress for the tissue 2 2 System Concept The Figure 2 1 shows the general system concept overview There will be 16 Experiment Chamber Modules ECMs These are divided into two groups each with 8 ECMs the control group and the experiment group Every ECM consists of two parts the reactor and its supply part To cultivate tissue in laboratory
55. gy facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 87 EAD a University of Applied Sciences Jena SErium e Determination of Residual Volume O O The Cavro Pump aspirates a small amount from port 5 connected with LHA see Figure 3 22 and after a short break checks the actual pressure in this tube Whenever the liquid in the Residual Volume is exhausted the pressure in this volume begins to fall If the pressure falls under a specified value See section3 2 7 the Cavro Pump stops the aspiration and the aspirated value will be displayed Required parameters Wait time Speed Volume Below the destination caption the port of the Cavro Pump which is connected with syringe can be selected The following ports are available Port 1 Port 2 Port 3 Port 4 Port 5 Port 6 Port 7 Port 8 Port 9 Medium B Washing Solution Medium A Sterilisation Solution LHA Fluorescence Solution A Fluorescence Solution B Not Connected Waste Wait and Speed can be adjusted either by using the slider or writing the amount into the textbox located behind Additionally to the volume slider the actual amount in ml is displayed behind The volume that is currently provided in the pump can be metered by Instantaneous Volume Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 88 EADS aan University of Applie
56. h the Liquid Handler ccsscccessececeessneeeeesseeeeesseeeeeeeaes 68 Interaction with the Experiment Chamber Modules ECM c0s seeeeees 70 Automated teaching of the Robot positions eecsecceeesseeeeeesteeeeeeeenaeeeeeees 72 Seeding Unit sas sicaveie Go ivstens ti lasteavientes cothdos Me ssensnddensss Wa ETES 74 Development of a Verification Software eeecceeeenteceeesseeecessteeeeeeeeaeeeeees 78 GSE al a A E E E E EE 79 Peristaltic PUMP ccccccccccccccsssssssseeceesceeseeseeeceeeessseseeaeseceseesseeaeeeeeeeesesessaaeas 81 Act at r Bridgeriin a a aE KEENAN Ea 83 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 RA Fachhochschule Jena Page 4 EAD y pee University of Applied Sciences Jena SErium 4 4 tiguid Handling sisca on meine he rent Riera Ree corre nt ery et nen oT ences ene meet 86 4 5 ROD OES ocra AAE vase desethscctau vers bahaeishediaaviticiusebcceee 88 5 Conclusa Sesnstoeksenendevencchatesachecoh sechisdydahetOel duceadoesdabhesdenienstieectaaeds 92 6 Bibliography issenensis a Gdvsasodieee wie viesdadteandiee E 93 7 ESE OP PIB UVES jccsanseidecacsdtecsenesanaglocelanasehesansonsedlneguadiniadiiceennatoeaeaalacuusauiebiaagese 96 8 Listol Tables cccccccciasececerisceccsncudwedcacnstsedctemsactebatiee detincieectincuSwecctraawtcteeutseatieentiendas 99 9 ANNEX aoa Sustdeucnosts E E 100 9 1 ANNEX A sern aE TEREE E E E ETE 100
57. ility for tissue engineering A av AVATA Fachhochschule Jena Page 11 EADS sae University of Applied Sciences Jena Figure 2 2 BMTC overview initial concept of the project Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AVA AVATA Fachhochschule Jena University of Applied Sciences Jena Page 12 1 EADS pose aj SSErium The Table 2 1 below only gives an overview of all the subsystems of the BMTC The listed parts will be described more detailed in the further sections of this diploma thesis Table 2 1 Overview over the BMTC concept Subsystem Part Function ID Number 1 Ground plate Provides accommodation space Which device is chosen depends on the tissue that will be used ZETOS Device for uni axial mechanical 2 ZETOS or Microscope stimulation of the tissue samples Microscope Optical observation and analysis of bioreactors Handling device to provide external services 3 Handling robot to the ECM 4 Sample Storage Freezer Unit Used for storage of medium samples 5 Medium Storage Used for storage of the operational medium 6 Canula Rack Provides Canulas for the Liquid Handling Cultivation Unit Peristaltic Provides elementary experiment services and 7 Pump Actuator Bridge on line medium monitoring 8 Liquid Handler Provides the Liquid Handling Experiment Chamber Module Provides medium circulation and tissue 9 EC
58. ion Of the gaS ssssssssesssessssssssrsrrsssrrnsreeeenssrrnsssreens 56 Figure 3 39 Flow chart of the normal supply cccccecesssececseseeecesseneeceessaaeceeseueeeeeeseeeeeesaaes 57 Figure 3 40 Sketch of the test setup for the measurement of the dead volume 664 58 Figure 3 41 Schematic sketch of the Cleaning Unit ssssssssssssssssssssssssssssrrssrrrrsseersrssrrnsssrens 61 Figure 3 42 Flow chart of the Cleaning Procedure csscccssssccecssssneececssnaececssseeecseseeeeeeesnes 63 Figure 3 43 Katan 6M180 robot sircssisirssieii ironii onks t irii Ea a EEKE EAR ENRE EEE Ei 64 Figure 3 44 First Version of the robot gripper ssssssssesrrsssesseersrssssesrrereessssosreerrensssssnerreenesne 65 Figure 3 45 Illustration of the procedure interaction with the Liquid Handler 69 Figure 3 46 Illustration of the procedure to interact with the ECM c cccccccccsssssssseeeeeeees 72 Figure 3 47 Schematic sketch of the Seeding Unit ssssssssssessessssssssrsssrerrssrrrnserressssrrnsssrenns 75 Figure 3 48 Schematic sketch of the Seeding Unit for the Cleaning Procedure c00 76 Figure 3 49 Seeding Unit in the final layout ssssssssssssssssesresseerssssrrrsssrrerssrrrnssreerssssrennssrrens 77 Figure 4 1 BMTC Control tab General lt i sccicsciisesccicszeadsctesccatacdessaccasczsddascesenancoeeationesveanaaaveacens 80 Figure 4 2 BMTC Control tab Peristaltic PUMp
59. iseenese naa EE a aa a Raaen 13 Experiment Chamber Module ECM cvcscsscavesennesiesasvensnasecdanvereoneveadtedacsavneanines 15 Actuator Bridge wrscccuccsssdccasunitaoniedeanagaivadabesadidacbaadudedeslicdsaasl hedmnaeievesensalicsadioens 26 Peristalti PUMP cciaieadantececudenszectatinntncenciiessensienedvbsdsecbindleidecietenkdeutuuneebuudbievsndsan 35 Liquid Handling CLA sisevasicinnaarca auseienitncstadlesbnsisecegenisedasvanadaxedundgceisencaustedtuagndnnenuiax 38 Medium Storage Cooler Unit scccccssecsncssadesentesenscexartaneseraaansonseqannenonncenaaae 40 Canula Rack enerne a e a E RE E R 42 Manual Exchange Interface Gate ccssseeccosssensecteennrcccassseecsssreneccesenaraceeaes 44 Liquid Handler Adapter LHA ssssssssesssssssesssressoerssenssenssrensornseensesressressrnsee 45 Sample Storage Freezer UIE si sijstiaspuiaiguina atari iiaienaiautnnanpuabunnnen 49 Waste Disposal Canula ccc cau sices dale ese ves sed evant eae the dd nao a Nate aaed en 51 Cavro XEP 6000 PUMP csser oriai ierasta idaan vessinbexedanagd eediaaedediaieedeceniageds 52 Supply Procedure for an ECM ccccsscccccccecessesssececeeceesesseaeeeceeeesssessseseeeeees 55 Cleaning Procedure for the Liquid Handler sccccsssseceecssneececsssteeeesssenees 60 Modular Support System RODOt c ccccssccssssecssececsseecsseeecsseeecseeeeseeeesaees 64 Control Program of the Modular Support System Kantana 4D 006 67 Interaction wit
60. it the following parts had to be added e Locking Mechanism e Guidance Plate for the ECMs e Pivot Bar as block for the ECMs The modified Peristaltic Pump including the just mentioned additions is schematically displayed in Figure 3 19 Locking Mechanism Guiding Plate for the ECMs Pivot bar supports the locking of the ECMs Figure 3 19 3D CAD model of the peristaltic pump including its additions Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 37 EADS a University of Applied Sciences Jena SSErium To ensure a proper function of the peristaltic the standard ISMATEC Tube Track was implemented in the ECM see Figure 3 2 The principle functionality of this Peristaltic Pump can be seen in the Figure 3 20 At the point of contact the cylinders displace the liquid in the tube and when the pump is activated the cylinders began to turn and push liquid through the tube Figure 3 20 Principle function of the Tube Track mounted on the peristaltic pump The Figure 3 21 below shows the Peristaltic Pump in a sectional view with a locked ECM Locking Mechanism Figure 3 21 3D section view of the Peristaltic Pump with locked ECM Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 38 EAD a University
61. ke the LHA out of the Storage Position 5 Move to the Canula Rack 70 mm above the Canula 6 Take a Canula out of the Canula Rack 7 Move with the LHA to the ECM that shall be supplied 8 Penetrate the Spray Pump with the Canula 9 Penetrate the Sample Storage with the Canula 10 Move to the Solid Waste 11 Remove the Canula from the LHA 12 Move to the Storage Position 13 Put the LHA back into the Storage Position 14 Move to the Robot Park Position of the Robot Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena Page 69 EADS University of Applied Sciences Jena SSErium Figure 3 45 Illustration of the procedure interaction with the Liquid Handler Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 70 EAD a University of Applied Sciences Jena SErium 3 3 3 Interaction with the Experiment Chamber Modules ECMs This section explains how the handling of the ECMs works To handle an ECM it is necessary to use the Actuator Bridge Steps 1 Searching Reference Position of the robot and the Actuator Bridge 2 Move to the Robot Park Position 3 Drive the Actuator Bridge to the position of the ECM with offset for the Release Servo 4 Move Robot over the sphere of the ECM that should be griped 5 Grip the sphere of the ECM 6 Rel
62. laced inside the reactor of the ECM and surrounded by a culture medium To get a fortunate concentration of the culture medium a Peristaltic Pump see chapter 3 1 3 transports the medium through the reactor Because the ECM is a closed system the medium has to be permanently reground This happens through a gas permeable foil that allows passing gas through it but no liquids In the inner of the BMTC predominates an atmosphere of nitrogen and of low oxygen concentration down to 5 A certain CO2 level has to be maintained to control passively the pH level in the medium These three gases are important for the growing of the tissue But the reground is not endless possible therefore is the Spray Pump foreseen which can inject small amounts of new medium in the FSC In the reactors the tissue will be cultivated the rest of the ECM has only supporting tasks The ECM consists always of the same parts only the reactors will be changed for the different types of tissue An ECM consists of 1 Common services e ECM housing e Spray Pump e Fluid Storage Cassette FSC o With medium storage and gas exchanger e Tubes e Tube track 2 Experiment specific equipment e Reactor chambers only one is usable at a time o Reactor A 2D for 2 layer tissue o Reactor B 3D for Bone o Reactor C 3D for Cartilage Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 Ma 4 AVATA Fachhochschule Jen
63. lean environment for the liquid transport This procedure was developed during the work on this diploma thesis Before the cleaning procedure can start it is required to replaces the old bags in the Medium Storage by a cleaning device This device has to be connected to the six I F of the Refilling Port see Figure 3 23 and the six I F in the hull of the Cooler Unit see Figure 3 24 The surface of the septa has to be decontaminated One possibility is to use a disinfectant cloth which is impregnated with a Sterilisation Solution Furthermore sterile Canulas have to be installed on the Luer Lock adapters The Cleaning Unit has also a 3 way cock to switch the connection between the Septum and the Canulas of the Cleaning Unit Besides a sterile stirring filter is attached on the bottle to avoid a negative pressure in the bottle due to the removal of the cleaning solution The Cleaning Unit can be seen in the Figure 3 41 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena Page 61 EADS LF University of Applied Sciences Jena SErium Cleaning Solution Septum to connect the Agent Storage with the Cleanig Unit Cannula to connect the Refilling Port with Cleaning Unit Sterile stirring filter with 0 2 ym size 3 Way Cock Figure 3 41 Schematic sketch of the Cleaning Unit Daniel Redlich System tests and optimisation of an automated
64. like to thank my tutor Prof Dr Jorg Grabow for the supervision of my diploma thesis Many further thanks go to my colleagues at Astrium who came up with useful proposals when was dealing with complicate issues and always provided a comfortable work environment at all Last but not least would like to thank the supreme persons in my life my girlfriend family and friends who have always been my real energy for their love and support Especially want to thank my brother David and Gabor Siket for proofreading my diploma thesis Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 7 EAD a University of Applied Sciences Jena SErium 1 Objective of the Diploma Thesis The objectives of the diploma thesis are the testing and optimisation of the experiment facility on system level to achieve an operational reliable setup prior start of biological experiments This includes the planning definition and execution of the tests and the implementation and verification of corrective actions The diploma thesis shall cover the following aspects e Inspection and familiarisation with the existing status of the Tissue Cultivation Unit o Identification and analysis of the basic functions and of their requirements o Identification of the technical and operational interfaces on hardware and software side e Definition of the test plan e De
65. mated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 24 EADS LF University of Applied Sciences Jena SSErium Figure 3 7 Front side of a ECM in the current design The Figure 3 8 shows the backside of a complete ECM Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering KA AVATA Fachhochschule Jena Page 25 EAD University of Applied Sciences Jena SErium Figure 3 8 Back side of a ECM without mounted back plate Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 26 EAD eh University of Applied Sciences Jena SErium 3 1 2 Actuator Bridge The Actuator Bridge is responsible for the supply and control of the ECMs see section 0 It has a linear drive unit powered by a stepper motor With this the Actuator Bridge can move to each ECM and fulfils there tasks by mechanical multiplexing Through this assembling it is not necessary to build actuators for every ECM The tasks of the Actuator Bridge are e Pressing the Spray Pump down e Unlocking of the ECMs e Connecting the Light Fibres to the sensors Each of these tasks is performed by a dedicated servo For this reason an offset can be adjusted to ensure the servo is at the correct position To communicate with the Actuator Bridge an RS 232 I F was assembled The protocol
66. me This part is shown in Figure 3 5 A visualisation of the Spray Pump can be found in Figure 3 3 3 1 1 4 Fluorescence Senor To measure the pH and the O value of the cultivation liquid Fluorescence Sensors are used These sensors consist of a Light Fibre with a diameter of 2 mm and a fluorescence ink spot at the bottom see Figure 3 17 Their exact functionality is described in the related manuals 2 3 3 1 1 5 Reactor Chamber The cultivation itself of the tissue is performed within the Reactor Chamber There are three kinds of reactors at the moment Reactor A Reactor B and Reactor C Reactor A is a 2D reactor see Figure 3 2 and the Reactors B and C are 3D reactors Figure 3 3 The working reactor is connected to the rest of the common ECM by tubes As it can be seen in the Figure 3 7 below the lower tube of ECM side leads to the higher reactor port Astrium is only responsible for the outer dimensions of the reactors and their operational compatibility with the overall ECM and BMTC concept In fact Prof David Johns had separately designed the inner dimensions of the reactors He has to ensure that the tissue inside the reactors gets enough cultivation liquid He also has to ensure that his designed reactors supply the tissue with enough cultivation liquid Further information of the respective reactors and their belonging cartilages can be found in the chapter 3 1 1 Daniel Redlich System tests and optimisation of an auto
67. n of an automated biotechnology facility for tissue engineering aA AVAVA Fachhochschule Jena University of Applied Sciences Jena d EADS SsErium Page 113 Seeding Procedure Start Seeding Aspiration of an amount of the Seeding Solution Aspiration of an amount of the Seeding Solution Expiration the Expiration the Seeding Solution Seeding Solution in a Reactor in a Reactor Seeding A Seeding B All Reactors filled Wait unit the cells Wait unit the cells in the seeding in the seeding solution are _ solution are sinking to the floor sinking to the floor End Seeding Figure 9 4 Flow chart of the Seeding Procedure Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Ma AVAVA Fachhochschule Jena University of Applied Sciences Jena Page 114 EADS _ PERS SSErium Washing Procedure Start Washing Aspiration an amount of Start Medium Espiration of the Start Medium in a Reactor are filled End Washing Figure 9 5 Flow chart of the Washing Procedure Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVAVA Fachhochschule Jena Page 115 EAD ee University of Applied Sciences Jena SErium 9 3 Annex C To connect the Cavro Pump of the Medium Storage and the Seeding Unit with the Computer and the Power Supply Unit a 9 pin D Sub male plug has to be used Th
68. nces Jena SSErium ioii Fie Options View Help Disconnect Port com4 z R Baud 9600 z Data fe 7 Stop 1 gt Parity None z I CTS Flow control rx 10 Reset T 9 Reset cotf 0 9 Reset Newline at None gt Vv Aik Clear received jf M ascii V Hex Dec T Bin Save output z H Clear at 0 o te Nenni ayen o M Autoscroll Show errors eo l fll 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Normal responds of the reference run The zero that is occurred when the stepper motor was just switched on Normal length of a respond Input control x m Input options Clear transmitted M ascii MV Hex Dec F Bin Send on enter Jone None Send file DIR RTS Type ex x F ASend l Tranomitied date Command to start the reference run 27 28 29 30 31 32 33 34 35 36 37 38 History 3 10 Connect to COM4 b 9600 d 8 s 1 p None a Figure 3 13 Communication between stepper motor and computer after the stepper motor was switched on The Figure 3 14 shows the old flow chart of the communication between Actuator Bridge and stepper motor In the Astrium configuration mentioned O problem does not matter because the controller of the Actuator Bridge is the last device that is powered But in the end configuration of the BMTC all power lines are connected to one supply unit This means all controllers are swit
69. neral Peristaltic Pump Actuator Bridge Liquid Handling Cassette Handling Old Position New Position Cultivation Unit 1 Cultivation Unit 2 Cultivation Unit 1 Cultivation Unit 2 06 X 02 X Start Transport Luiquid Handling C Cultivation Unit 2 Take LHA Take Needle Penetrate Spray Pump Penetrate Wasteport Discharge Cannula Return LHA Choose a Needle Choose a Spraypump Choose a Wasteport Position Teaching Pump 1 ECM Handling Pump 2 ECM Handling Storage Position CannulaRack Received Figure 4 6 BMTC Control tab Robot The Table 4 5 explains the buttons of the tab robot Table 4 5 Overview of the buttons in tab Robot Button Function Starts the Transport of an ECM from a selected position to another Start Transport selected position The robot removes the LHA from the Storage Position and moves to the Take LHA Robot Park Position Removing the selected Canula from the Canula Rack and moves to the Take Needle Robot Park Position Penetrates Spray The robot penetrates the Spray Pump Septum of the selected ECM in the Pump box below Depenetrate The robot extracts the Canula from the septum of the Spray Pump and Spray Pump moves back to the Robot Park Position Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 aw ve AVATA Fachhochschule Jena Page 91 EAD U
70. ng 1 a A AVATA Fachhochschule Jena Page 99 EAD a University of Applied Sciences Jena SErium 8 List of Tables Table 2 1 Table 3 1 Table 3 2 Table 3 3 Table 4 1 Table 4 2 Table 4 3 Table 4 4 Table 4 5 Table 9 1 Overview over the BMTC CONCEt cccsssccecesssseececssnececssseeeeceesenaeceeseaeeeseseneeees 12 All partsof the ES UM esi cesstcestspccce nice natvencneeteepeduasolesteonizacene tiveanedsinpeeddaciadaceeledeabeeunet 18 Series of measurements of the dead volume cececesecesescceseseeeesseeesssneeesseeeesseees 59 Concentration of old liquid after one to five filling cycles eeeeseeeessereeeeeeenees 60 Overview of the buttons in the tab General 0 e eee eeeeeesseeeeseeeesneeeesaeeeeneeeeaaees 80 Overview of the buttons in the tab Peristaltic PUMP csssccssseeceeessteeeeeeenaes 82 Overview of the buttons in the tab Actuator Bridge cccssscccsssseeecesseeeeeeesnaes 84 Overview of the buttons in the tab Liquid Handling ccssccccesssceeesseeeeeesenaes 88 Overview of the buttons in tab RODOT cece essseeesseeeeseeeeseeeesseeresaeeeeneeeeenaees 90 Pin allocation of the 9pin D Sub plug for the Cavro PUMP cssceeeesteeeeessteeeees 115 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVATA Fachhochschule Jena Page 100 EAD ae University of Applied Sciences Jena SEri
71. niversity of Applied Sciences Jena 2triom Penetrates The robot penetrates the Waste Port Septum that was selected in the Waste Port box below Depenetrate The robot extracts the Canula from the septum of the Waste Port and Waste Port moves back to the Robot Park Position Discharge Canula The Canula is discharged into the Solid Waste and the Robot moves to the Robot Park Position Return LHA The LHA will be returned to the Storage Position Pump 1 ECM Handling The automated teaching procedure for the ECM positions in the Cultivation Unit 1 is started Furthermore information to fulfil the teaching procedure will be displayed from the program The automated teaching procedure for the ECM positions in the Pump 2 ECM Handling Cultivation Unit 2 is started Furthermore information to fulfil the teaching procedure will be displayed from the program Storage Position The automated teaching procedure for the Storage Position is started Furthermore information to fulfil the teaching procedure will be displayed from the program The automated teaching procedure for all 16 Canulas in the Canula Rack Canula Rack is started Furthermore information to fulfil the teaching procedure will be displayed from the program Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 92 EAD a University of Applied Science
72. ns to reach a position They are explained in the next overview e Point o Points are defined by manual teaching and the robot arm can move towards e Grid o A group of points that are on one plane and have the same distance to its neighbouring points e Paths o The robot can be moved along a 3 dimensional path by hand while the software stores the respective points So the stored path can be automatically replayed repeated afterwards Not used by BMTC e Object recognition o By using neuronal networks the robot can find objects and drive towards them Not used by BMTC There are also some special Methods which are executed as soon as their specified event appears for example after a collision or during the robots initialization To see a full overview of the functionality of the Katana 4D software please have a look at the Katana 4D manual 12 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 68 EAD a University of Applied Sciences Jena SSErium 3 3 2 Interaction with the Liquid Handler In this section the actions of the robot are described when taking a LHA and supplying an ECM The following three procedures chapter 3 3 2 to chapter 3 3 4 were developed and tested during the work on this diploma thesis Steps 1 Find Reference Position 2 Move to the Robot Park Position 3 Move to the Storage Position 4 Ta
73. of script files 1 BMTC Control writes the command in the file toKatana txt in the C root and waits for the file fromKatana txt 2 If the toKatana txt file appears Katana 4D opens this file and compares the command with the names of known methods 3 If Katana 4D finds a match the responding method will be executed 4 If the method has been completed successfully the Katana 4D software writes the command done into the file fromKatana txt 5 BMITC searches after the file fromKatana txt until it recognises this Then the file fromKatana txt opened If the command done is read the file toKatana txt and fromKatana txt are deleted The Figure 4 6 gives an impression how the Robot tab looks like On the upper side of the tab Robot the box Cassette Handling is located The task of this box is to show whether or not the transport of ECMs between the Cultivation Units works The procedure of this task is named in section 3 3 3 The box below where the single steps of the procedure in section 3 3 2 can be activated is called Liquid Handling The bottom box is called Position Teaching Here the automated teaching of the position for the robot can be performed This is executed like introduced in section 3 3 4 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AMA A AVATA Fachhochschule Jena Page 90 EADS a University of Applied Sciences Jena SSErium BMTC Control Ge
74. of the communication bug During the functional tests by KT the Actuator Bridge did not show the same behaviour as it did during the functional tests by Astrium As it can be seen in Figure 3 10 the Actuator Bridge needs a 5 V and a 12 V power supply unit During the tests by Astrium two different power supply units had been used Because of the test setup the 5 V power supply unit had to be switched on before the 12 V power supply unit In the end configuration of the BMTC build from KT only one power supply unit with a 5 V and a 12V output is used The communication between the Actuator Bridge controller and the Stepper Motor works after the master slave principle whereas the Actuator Bridge controller is the master and the stepper motor is the slave Normally the stepper controller driver of the Stepper Motor sends an answer not until he received a command of the Actuator Bridge microcontroller but when the 12 V power supply unit is switched on the controller of the stepper motor sends a 0 to the controller board of the Actuator Bridge To demonstrate this strange behaviour the stepper motor was directly connected with a computer and a terminal program was used to record the communication The terminal program is shown in the Figure 3 13 below Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena Page 32 EADS ee Ay University of Applied Scie
75. of the inner of the Medium Storage Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 42 EAD a University of Applied Sciences Jena SErium On the bottom of the ground plate fittings and tubes are mounted which shall connect the Cavro Pump and the liquid bags Due to the fact that the Liquid bags have to be exchanged for any experiment an interface was established To connect the liquid bag with the tubes a septum has to be screwed on the upper side of the liquid bag It also has to be plugged into a hull with a Canula that is mounted on the ground plate The Canula is fastened on the Ground Plate by a Luer Lock Adapter This kind of adapter is a standardised I F in medical technologies for further information please see DIN EN 1707 1996 To ensure that the septum on the bag can not slide out of the hull a locking ring has to be insert in the slots on the upper side of the hull The whole construction is accommodated in the refrigerator to provide a constant temperature of 4 C for the liquids Normally the cover of the refrigerator was designed to be opened by a swinging board Due to the fact that the construction for the liquid bags was mounted to the cover of the refrigerator see Figure 3 23 the closing had to be changed Now it is possible to take the cover and the bags upwards out of the refrigerator 3 2 2 Canula Rack The Canula Ra
76. p of the Medium Storage This part of the BMTC Control was included from another software called Liquid Handler Cavro Pump It was created by Youssef Aidi during its internship at Astrium Because the code was copied into the BMTC Control program to minimise the programming effort the layout of the MMI was adopted and slightly adapted The resulting tab Liquid Handler is pictured in the Figure 4 5 How the ports are connected can be seen in Figure 3 22 For the configuration of the pump the key parameter is the Syringe Volume at full stroke All operational volumes are derived from this value by linear interpolation with 6000 steps The pump has a rotary valve with 9 external ports Which can be used as Source or Destination Below the caption Source the action to be performed is selectable The different actions and a short explanation of these are listed below e Aspiration of Medium o The Cavro Pump moves down and aspires the medium from the selected Source port o Required parameters Speed Volume e Expiration of Medium o The Cavro Pump moves upward and expires the medium to the selected port o Required parameters Speed Volume e Priming o The Cavro Pump aspires and expires medium from one bag to fill the tubes with liquid and eliminates the bubbles from the bag that is connected to this Destination port o Required parameters Wait time Speed Volume Repeats Daniel Redlich System tests and optimisation of an automated biotechnolo
77. pressure of Port 5 This part is necessary to know how many liquid was aspirated out of the Spray Pump For this reason the Exhaust Foil Tube was implemented because this enables the possibility to exhaust the Spray Pump completely The rest volume of the Spray Pump is needed to calculate the volume in the Sample Waste Port of the FSC Because all excess volume will be flow in this part and when the volume in this part is know the right amount of used liquid can be exhaust In Figure 3 35 a detailed description of the sensor board can be seen Two comparators are used on this board Comparator one is used for the detection of the low pressure status and immediately sends a signal when the pressure in the tube falls under a setup pressure This happens every time when the liquid will be exhaust but if the pressure is after a delay still under the setup pressure the Spray Pump shall be empty The second comparator is used for high pressure and it will send a signal when the system is blocked These two logical values are connected to the two logical inputs of the Cavro Pump Pin 7 and 8 With this design it is possible to read the pressure status by the communication with the Cavro Pump Two potentiometer to adjust the threshold value Comparator 2 high pressure sends a signal when the system is blocked Pressure Sensor Comparator 1 low pressure sends a signal when the Spray Pump is empty Figure 3
78. ps no parity 1 stop bit no flow control Male connector in the Bridge For connecting a Type B Bridge a physical connection between pins 1 and 9 of the interface cable connector is needed See more under Bridge Types Protocol Description Start and Stop Bytes The line starts with the symbol and ends with a carriage return OxOD Checksum The last two bytes before the carriage return contain the sum from the significant characters without the start and stop bytes in hexadecimal ASCIl Characters Modes In normal mode default only one command is received at a time Any other incoming commands will be rejected if the Bridge is not yet ready In Command Queuing mode the Bridge will receive all commands and save them in a Buffer for sequential execution Restrictions Only one command can be sent per line Sending commands with less than 250 ms between them can result in unexpected behaviour Bridge Response ap The Bridge responds with the starting characters gt Command executed Command ignored or Unknown command After this character an echo from the command comes in the same line After a successfully received status request command the response will also include the requested status as explained in the Status codes section Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a AVAVA Fachhochschule Jena Page 102 EAD ae
79. ptum of Canula of Connection to Spiral tube the I F the I F the Cavro Pump Interface box Luer Lock Adapter to connect Canula Short threaded DS cap nut O bold Figure 3 28 LHA with spiral tube and I F At the front of the LHA a Luer Lock adapter is mounted because the LHA has to pick up Canulas from the Canula Rack To get a Canula the Robot has to puncture the Aluminium foil which protects the Canula against contamination During the pick up process it has to be made sure that no parts of the aluminium foil are enters the I F cone of the Canula This would result in a leak and loss of sterility This aspect is controlled by the distance of the aluminium foil above the stored Canulas in the stored position The spiral tube is used because the LHA will be carried by the Modular Support System to supply the ECMs in the BMTC To avoid a caught up of the tube between the ECMs a spiral Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 47 EAD a University of Applied Sciences Jena SErium tube is used for this connection Using a normal inflexible tube instead could entail the mentioned caught up so the tube may finally burst The interface for the Modular Support System is located on the upper side of the LHA This is needed because the LHA has to be fixated directly on the Modular Support System to allow for proper positioning
80. r Debug modus off The Debug modus will be switched off First time pressed The ECM servo pushes the Locking Mechanism of the ECM ECM Second time pressed The ECM servo moves in its initial position and the Locking Mechanism relocks the ECM First time pressed The Sensor servo pushes the Optical MUX on its Counter Part on the ECM Sensor Second time pressed The Sensor servo moves back to its initial position an disconnects the Optical MUX The Spray Pump servo pushes the Spray Pump and drives in its initial Spray Pump position as often as it selected in the field next to this button Let the Stepper Motor turn the above selected steps to drive the servos Forward forward and teach the Actuator Bridge an ECM Position Let the Stepper Motor turn the above selected steps to drive the servos Backward backward and teach the Actuator Bridge an ECM Position Saving the current position of the Actuator Bridge as the above chosen Save position number Servos Opening the window to teach the positions of the servos Set Min Setting the initial position of the left mentioned servo Set Max Setting the end position of the left mentioned servo Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 86 EAD a University of Applied Sciences Jena SErium 4 4 Liquid Handling The tab Liquid Handler enables to control the Cavro Pum
81. r all components except for the Katana Robot The Robot works with its own software so this software can be started if the button Start Katana will be pressed The Figure 4 1 gives an impression how the General tab looks like Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AMA AVATA Fachhochschule Jena University of Applied Sciences Jena Page 80 BMIC Control Katana Roboter Start Katana Cultivation Unit 1 Peristaltic Pump if Actuator Bridge Liquid Handling After you clicked Start Katana please wait until this window come back Status Ismatec Pump Actuator Bridge Carvo Pump ON C OFF Not Checked ON OFF Not Checked Status ON OFF Not Checked Cultivation Unit 2 Status Ismatec Pump Actuator Bridge ON F OFF Not Checked ON e OFF Not Checked Status Check Connect Received Figure 4 1 BMTC Control tab General The Table 4 1 gives an overview of all usable buttons in this tab Table 4 1 Overview of the buttons in the tab General Button Function Start Katana Starting the software Katana 4D and the program BMTC Control in Katana 4D Connect Opening the com ports for the communication Disconnect Closing the com ports Starting an request if the components are available displaying the occurring Status Check results in
82. ray Pump until the pressure drops below a defined value Remove the Canula from the ECM s waste septum Cannula withhin the Waste Port of the ECM Inject the liquid sample into the corresponding liquid sample vial Note the removed liquid volume pump the waste liquid to the liquid waste station Aspire 7 ml of the medium from Dispose used Canula Medium A or B Drive Liquid Handler Adapter back to the Storage Position and rinsing the LHA Cannula within the Spray Pump Fill up the Spray Pump with fresh medium Perform the whole procedure Repeat the exhaustion and fill for every ECM of the Spray Pump whenever until the concentration of the old medium to the new medium is under a value that is defined Figure 3 39 Flow chart of the normal supply The tube that connects the Cavro Pump to the LHA has a volume of 2 ml This is called the dead volume of the LH systems because if old media is exhausted and new media is injected the old volume that is in the tube will be injected in the Spray Pump again The exact measurement of the dead volume and the calculation of the old medium concentration had to be done during the work on this diploma thesis Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering wa 4 AVATA Fachhochschule Jena Page 58 EADS ee University of Applied Sciences Jena SSErium The measurement of the dead volume was executed after the
83. remove the not unattached cells o Pumping a solution with nutrients and oxygen into the reactors to start the experiment The same type of Cavro Pump is used in the Seeding Unit as well as in the Sample Storage For further information please see section 3 2 7 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 75 EADS a University of Applied Sciences Jena SErium The Figure 3 47 shows the schematic sketch of the Seeding Unit Valve 1 Valve 2 Valve 3 E lt Q C a ae wee K l GND ULN 20658 24V DOTTL1 93 DOTTL2 OY DOTTL3 Seeding Device RS232 interface Figure 3 47 Schematic sketch of the Seeding Unit For the Cleaning Procedure shall be performed the configuration of the Seeding Unit has to be changed The bag with the Cleaning Solution has to be connected to the tube of the Seeding Solution Priming Buffer and Start Medium Additionally a sterile syringe filter 0 2 um pore size filter material must be hydrophilic has to be connected to this bag because it has to be filled or refilled with Sterile Water or Sterilisation Solution and when the cleaning solution is filled in a glass bottle the syringe filter takes care that the pressure in the bottle drops to deep All tubes that are norm
84. ripper s one Grip the sphere with full power Switch all motors on Store the new position repeat the steps 10 to 17 for all ECMs Move to the Robot Park Position Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 74 EAD a University of Applied Sciences Jena SErium 3 4 Seeding Unit At the beginning of an experiment with the Reactor A it is necessary to seed the reactors with living cells The seeding is performed by sedimentation of the suspended cells on the cover glass of the reactor To achieve this the Cultivation Unit is turned by 90 The seeding process is supported by a dedicated Seeding Unit It provides the following programmable and computer controlled services e Cleaning the Seeding Unit o Dispelling the rests of the old media o Sterilising the tubes and the Cavro Pump e Priming of all tubes o Filling all the tubes of the Seeding Unit with solutions o Removing the bubbles from the store bags of the solutions e Filling all reactors with the Priming Solution o Filling of the Reactors with a cell compatible solution to avoid the dead of the cells because of an unhealthy environment e Filling all reactors with the Seeding solution o Pumping a solution consisting of cells oxygen nutrients and water into the reactors this can be repeated e Filling all reactors with the Start Solution o Purging of the reactor to
85. rovides Priming Buffer Expiration over the Ports 5 8 of the Cavro Pump 2 5 ml over each Port Connect new Infusion Sets to the Ports 1 8 of the Cavro Pump End Cleanig Figure 9 1 Flow chart of the Cleaning Procedure System tests and optimisation of an automated biotechnology facility for tissue engineering Aa AVATA Fachhochschule Jena Page 111 University of Applied Sciences Jena 4 EADS _ SsErium Priming Procedure Start Priming Aspiration of the Seeding Solution Expiration to the bag with the Seeding Solution Aspiration of the Priming Buffer Expiration to the Waste bag Aspiration of the Priminig Buffer Expiration to the bag with the Priming Buffer Aspiration of the Start Medium Expiration to the Waste bag Figure 9 2 Flow chart of the Priming Procedure Aspiration of the Start Medium Expiration to the bag with the Start Medium End Priming Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering aA AVATA Fachhochschule Jena Page 112 EADS Ca University of Applied Sciences Jena SErium Filling Procedure Start Filling the Reactors Aspiration of an amount of Priming Buffer Expiration to the Reactors All Reactors filled ith Priming Buffer End Filling the Reactors Figure 9 3 Flow chart of the Filling Procedure Daniel Redlich System tests and optimisatio
86. s Jena SErium 5 Conclusion At the end of my diploma thesis the BMTC project achieved the point where all problems are solved and only some solutions have to be finally implemented All components have been successfully tested and their full robust functionality could be proven The Astrium subsystems will now be integrated into the S BMTC on System level After functional verification on system level the S BMTC GD will be used for intensive biological testing The objective of my diploma thesis to gain a high operational robustness was achieved Additionally developed a number of S W modules to control the fast functional testing of the Astrium subsystems and to support the trouble shooting during system tests It was a great opportunity to write my diploma thesis at Astrium GmbH because it gave me a great insight how experiments for space application are conceptually designed and manufactured how operational robustness can be achieved and how its project management is built on It was a great experience to apply and improve my theoretical skills that gained during the studies of mechatronics at the FH Jena Thereby the most remarkable circumstance is the cover of all fields of mechatronics Mechanical Engineering Electrical Engineering and applied Computer Sciences Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVAVA Fachhochschule Jena Page 93
87. se Light Fibres again are connected with a 4 channel Fibre Optic pH Meter and a 4 channel Fibre Optic Oxygen Meter With these devices it is possible to measure contact less the oxygen concentration and pH value in the medium circulation loop of each ECM Both measurement devices are connected with a PC through an RS 232 I F Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 34 EADS See University of Applied Sciences Jena SSErium The Optical MUX itself was not test ready at the beginning of this diploma thesis A preliminary design was available but it was not functional ready and did not fit into the Actuator Bridge So the Optical MUX was completely revised and the functional capability was demonstrated during the making of this diploma thesis The Figure 3 16 shows the Optical MUX in the final configuration The Servo is used to push the Connector Port on its counterpart on the ECM side to establish the connection When the Servo goes back to its start position the spring pushes back the Light Fibre Receiver and the Connector Port and disconnects them from the ECM The Guidance for the Light Fibre Receiver is used to attach the Optical MUX on the Actuator Bridge see Figure 3 9 Two mandatory Light Fibre One optional Light Fibre Push back spring Guidance for the Light Fibre Receiver Counterpart of the Connector Port
88. see Figure 3 44 The fixation was upgraded by adding one screw to every side Thus this was the position of the alignment fixed and the LHA could not change its position anymore The exchange of the LHA body material was necessary because the liquids that flow through it have minerals included and these cause corrosion on the aluminium The new material of the LHA is stainless steel and can be seen in Figure 3 28 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 67 EAD a University of Applied Sciences Jena SErium 3 3 1 Control Program of the Modular Support System Kantana 4D The robot is controlled by the Katana 4D software This software has two different working modes the Administrator Mode and the User Made In the User Mode only programs that were built in the Administration Mode can be launched For that reason only the Administration Mode is describe within this diploma thesis In the Administration Mode Projects can be created Every Project consists of different Programs Sequences and Methods can be defined in the Programs When a Program is started then all the Sequences were executed one after another In the Sequences the single steps the Robot should fulfil had to be defined Often used steps are usually developed as Methods to easily execute them more than once within a Sequence The robot provides four different optio
89. sssscccececesssseseceeeeesessessaeseeeeeens 43 Figure 3 26 Exploded view of the Canula Rack cccsssccccssssceceesseeececssaeceessseeecseseneeeeesenes 44 Figure 3 27 Manual Exchange Interface With ECM ccccccsssseceeeeesssssesececeeeeessesssaseeeeeeesees 45 Figure 3 28 LHA with spiral tube and I F saccveccpsicsdusaracthdransedteawicsceassdcccavetaeveinecuannedivcusuous 46 Figure 3 29 Old Storage Position with LHA and Modular Support SysteM cccccceesteeeeees 48 Figure 3 30 New Storage Position with LHA ccccccccssssececsesseeeceesseeeceessaeeeessueeeceeseeeeeeeanes 49 Figure 3 31 Schematic sketch from the function of the Sample Storage cccceeeeesteeeeeees 50 Figure 3 32 LHA in the position to fill the test tube eee cceeeceesseeececssnaeceesseeecessteeeeeesenes 51 Figure 3 33 Disposes the Canula into the Solid Waste ccscccccsssseececssnececssseeeeeesseeeeeeeeeas 52 Figure 3 34 Cavro XLP 6000 PUMP ee eeeeesecceececececccececececeaeaeaeececececececececececececececeeeeeeeeeeetens 52 Figure 3 35 Sensor board to measure the pressure ON port 5 2 eecececessesssscceeeesessessneeeeeeeens 53 Figure 3 36 Cavro Pump with adapter and tubular film to make the syringe contamination E E E O enlace aan E E E E enserenedae ame 54 Figure 3 37 MMI of the long term test of the Cavro PUMP sssssssssssssssersessssesrrerersnssesrerreene 55 Figure 3 38 Flow chart of the exhaust
90. t von Kunststoffen 1 Auflage Carl hanser Verlag M nchen 2007 p 840 EADS Space Transportation GmbH Simplified BMTC Ground Demonstrator TN 2 Design Concept and Identification of Critical Technologies Issue 2 p 73 EADS Space Transportation GmbH Simplified BMTC Ground Demonstrator TN 2 Design Concept and Identification of Critical Technologies Issue 2 p 16 Neuronics AG Katana 6 M Specifications data sheet Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 95 EAD a University of Applied Sciences Jena SErium 12 Neuronics AG Katana 4D Manual Version 4 1 25 10 06 13 Dipl Ing FH Ulrich Fischer Dipl Gwl Roland Gomeringer Tabellenbuch Metall 43 Auflage Verlag Europa Lehrmittel 2005 p 117 Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 96 EAD a University of Applied Sciences Jena SErium 7 List of Figures Figure 2 1 System concept of the BMI CL sie sccacesssccasvesaicaseessabccnussascannn caacaives sascebeveacnanenaaaas 10 Figure 2 2 BMTC overview initial concept of the project cecsessssececeeeeesesseeeeeeeeeeeees 11 Figure 3 1 Cultivation Unit seia cciiccts iespieseatevaseseenieseagteaetsinuius cieceeane nda pavateaeeseaesoateuasdeudaneeeseueaes 14 Figure 3 2 ECM
91. te is a device to strip off the Canula from the LHA The robot positions the Canula under the strip off plate and moves the LHA upward So the used Canula is strippe off This can be seen in Figure 3 33 The hole in the Ground Plate leads to a container to collect safely the used Canulas Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena University of Applied Sciences Jena Page 52 EADS SSErium Stripp off plate KIXI KIAJ Figure 3 33 Disposes the Canula into the Solid Waste 3 2 7 Cavro XLP 6000 Pump The Cavro XLP 6000 Pump performs any liquid transport within LH Important Data 5 e Type Cavro XLP 6000 Pump e Principle Syringe Pump e Data l F RS232 e Power supply 24 V e Rotary valve with 9 ports with UNF 28 thread e Power Output 5V Pin 13 e Logical input 2 lines TTL level e Logical output 3 lines TTL level e Used syringe 10 ml e Accuracy lt 1 0 at full stroke 6000 Figure 3 34 Cavro XLP 6000 Pump steps on full stroke Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a 4 AVATA Fachhochschule Jena Page 53 EADS a University of Applied Sciences Jena SSErium To use the Cavro Pump in this special environment an additional sensor board was installed on the top of the pump Thereby it is possible to measure the
92. the FSCs 3 1 1 2 Fluid Storage Cassette FSC The FSC reground and stores the liquid For sterility reasons it is considered as a disposable and will only be used for one cultivation cycle To use the FSC in the peristaltic pump see chapter 3 1 3 the housing of the ECM is responsible and provides all interfaces to connect the ECM with the FSC The concept of a cassette as modular unit allows achieving reproducible results on extern cultivation platforms That enables independent testing and validation The Figure 3 5 shows the Fluid Storage Cassette FSC Its functional parts are 1 Waste Sample volume for used medium o The used cultivation Medium will be stored here 2 Bubble Trap o The Bubbles that are in the FSC will be caught here and can be blown off 3 Mixer o Is responsible for a proper stirring of the old Cultivation Liquid with the freshly added medium from the spray pump 4 Gas exchanger o Provides the required surface for an adequate gas exchange Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 21 EADS ee University of Applied Sciences Jena SSErium Waste Sample volume for used medium Bubble removal access Bubble Trap Gas exchanger Figure 3 5 Early 3D CAD model of the FSC The Figure 3 5 shows the FSC at the beginning of this diploma thesis Astrium is responsible for the design conc
93. timisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 108 EAD za University of Applied Sciences Jena SErium SLY Set upper limit for the Spray Servo in OCR value between 115 for 1 ms and 230 for 2 ms SPR Set the current position for the Release Servo in OCR value between 115 and 230 SPO Set the current position for the Optical Servo in OCR value between 115 and 230 SPY Set the current position for the Spray Servo in OCR value between 115 and 230 Examples gt P05 gt R500 gt Y 0350 Wait In Command Queuing Mode is useful when we want the Bridge to pause after a command and wait for an event before continuing with the command buffer processing In Normal mode this command would make the Bridge ignore all commands before the command Go is sent Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering AMA f AVATA Fachhochschule Jena Page 109 EAD mee University of Applied Sciences Jena SSErium G Go Resume L Light Gate Returns the current status of the LightGate either open or closed This commands are executed immediately They are not ignored in Normal Mode and they skip the queue in Command Queuing Mode Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 11
94. um 9 Annex 9 1 AnnexA S BMTC GD Actuator Bridge User Manual 05 06 2009 Bridge Types Due to an exception in the symmetry of the S BMTC GD layout the Bridge Type A and Type B are defined The difference is the place where the Bridge will place its arm during the servicing process The Bridge Type A will move its arm over the motor of the pump while Bridge Type B will move it to the opposite side This is done to avoid collisions during the servicing The servicing position is denoted with the number 9 See Position Description and Coordinate System Position Description and Coordinate System All positions are denoted with numbers These positions are saved and can be modified with the teaching commands The position number is always denoted with two digits 00 Home position 01 to 08 ECM Cassettes 1 to 8 09 Servicing position 10 Optical Sensor Reference position 11 to 20 Custom positions The zero point is where the arm rests against the wall at motor s side The positive direction goes away from the motor The coordinate units for the Bridge s commands are defined as follows Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVAVA Fachhochschule Jena Page 101 EAD za University of Applied Sciences Jena aserium 1 unit 10 motor steps 18 of rotation Motor QMot QSH 4218 0 15 mm The Connection RS 232 9600 b
95. velopment of required test tools and test criteria e Execution of tests on the system level and documentation of the occurring results e Iterative improvements e Documentation of the final results Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a 4 AVATA Fachhochschule Jena Page 8 EAD a University of Applied Sciences Jena SErium 2 The Simplified Biotechnology Mammalian Tissue Culturing Facility Ground Demonstrator S BMTC GD 2 1 General Information To study the biochemical and physiological principles of tissue cultivation the S BMTC GD is to be developed The focus of interest is to provide a huge amount of different tissue kinds to be cultivated In the current development stage it is possible to use three different kinds of tissue bone cartilage and two layer tissues In the following paragraphs S BMTC GD will be dubbed BMTC GD stands for ground demonstrator That means the facility is used on earth for developing and experimental sampling of different technologies These technologies shall be used in space later The S at the beginning means simplified because the preparations of the experiment have to be done by an operator human and are not performed from the BMTC The BMTC will be developed in cooperation with other companies whereas the prime contractor of this project is Kaiser Threde KT The respective responsibilities of the sin
96. was defined in an earlier diploma thesis see Annex A In the Figure 3 9 the Actuator Bridge is displayed Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering a AVATA Fachhochschule Jena Page 27 EADS eet University of Applied Sciences Jena SSErium Servo to actuate the Spray Pump Servo to connect the Light Fibre Servo for the unlocking of the Stepper Motor Linear Drive Unit Figure 3 9 CAD Model of the Actuator Bridge The Figure 3 10 shows how the communication control I Fs between the Actuator Bridge and the host computer besides it displays how the components of the Actuator Bridge is organised Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering L a 4 AVATA Fachhochschule Jena Page 28 EADS a University of Applied Sciences Jena SSErium Servo to connect the Servofor Servo for Optical unlocking of pushing the Interface the ECM Spray Pump Stepper Motor with controller board for the Linear Drive Unit Power Supply Unit Perse Power Supply Unit eu z u e iC e O O HyS Ai Figure 3 10 Sketch of the Actuators communication The standard operating time of the BMTC is 21 days without interruption To guarantee a stable function a long term test of 3 days was performed During these 3 days the movement amount of the 2
97. with 2D REACtON iacantcadnecicyutsuubecndeasanetedeesaudedeuuiiwaneasadyesdeuiuuideetupeedsrudaatouratves 16 Figure 3 3 ECM with 3D Reactor cccccccccccccssssssssecesescesseseeseseeeeeesseeseaseaesecseseeeseasaeeeeeeeeeees 17 Figure 3 4 Exploded view of the ECM ccccccsssssscececeeeseessseceeeeeeeseesseaeeeeeeeesseeseaeaeeeeesseseees 18 Figure 3 5 Early 3D CAD model of the FSC ccsscccccssssececseseeeeceesseeecesssaeeeeseaeeeceeseneeceesenes 21 Figure 3 6 Half form of the FSC with Changes cccccssssecccsssseeecessseeceesssaeeeesssaeeeceeseneeseenaes 22 Figure 3 7 Front side of a ECM in the current GeSIgN cccssccccesssseeceeseneeceeseseeecesseeeeeeesaes 24 Figure 3 8 Back side of a ECM without mounted back plate ccccecssssececseseeeeeessseeeeeeees 25 Figure 3 9 CAD Model of the Actuator Bridge ccccsssccecssssececeesseeceessnaeeecseaeeeceeseneeeeesanes 27 Figure 3 10 Sketch of the Actuators COMMUNICATION cccceeeeesseteeceeseeeceessneeeeeseneeeeesnes 28 Figure 3 11 MMI of the long term test of the Actuator Bridge cccsssccecesssneeeeeeneeeeeees 30 Figure 3 12 Cut out of the log file of the long term test program for the Actuator Bridge 30 Figure 3 13 Communication between stepper motor and computer after the stepper motor was switched ON 2 eeecccceeecccccececcceccsscececeeseececeeescesecaeeeeecueeeeecuueaeeesaueners 32 Figure 3 14 Old communic
98. ystem tests and optimisation of an automated biotechnology facility for tissue engineering 4 KA Fachhochschule Jena Page 77 EAD pee University of Applied Sciences Jena SBsSErium The Figure 3 49 displays the seeding Unit in the final configuration Bags for operation liquids Waste collection bag Infusion holder Figure 3 49 Seeding Unit in the final layout Daniel Redlich System tests and optimisation of an automated biotechnology facility for tissue engineering 1 a A AVATA Fachhochschule Jena Page 78 EAD a University of Applied Sciences Jena SErium 4 Development of a Verification Software To test the BMTC on sub system and system level a software package was written that is able to control the BMTC built by Astrium This software is called BMTC Control and allows to launch single actions of all BMTC components as well as sequences of multiple actions The following tabs with their respective functions are available within the BMTC Control software e General o The settings for all components are selected in this tab e Peristaltic Pump o With this tab it is possible to command the Peristaltic Pump e Actuator Bridge o Here are options to control the Actuator Bridge e Robot Handling o This tab provides the handling of the Robot carrying Cassettes or the LHA e Liquid Handling o Commands to actuate the Liquid Handling The BMTC Control software also has three windows to display the current state of execution e
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