Hollow Organ Bioreactor - Harvard Apparatus Regenerative
Contents
1. 2015 EE Name wem wem kee ea Ra EN Port 25 ea 1032581 BD crie Sealing Caps 5 ea 1032584 1031140 N OT 1031142 N OT 1033143 1031141 N OT 1032597 1031100 1031104 Reservoirs E 500 mL mn 1031105 WM tu mmm tu mm Revision 1 5 19 July 15 2015 Chapter 3 Care amp Maintenance Cross Infection Prevention Biohazardous Waste and Product Disposal 3 1 Cross Infection Prevention Universal Precautions All blood products or products potentially contaminated by blood or other body animal fluids should be treated as potentially infectious materials Personal protective equipment should be worn at all times when using the LARGE ANIMAL HOLLOW ORGAN Bioreactor to protect personnel from becoming contaminated as well as to help prevent cross infection and cross contamination Bench tops equipment and other potentially contaminated surfaces should be cleaned and disinfected according to the manufacturers and or the facility s procedures Any article used to clean potentially contaminated surfaces should be disposed of as Biohazardous Waste i CAUTION Failure to use the manufacturers cleaning and disinfecting procedure could result in damage to the surface or equipment Biohazardous Waste Dispose of biohazardous waste according to local Regulatory requirements 3 2 Cleaning Flush all fluid paths with deionized water and either follow with or wipe with 70 IPA The2. for aseptic cell processing It Is recommend that all manipulations of the unit once sterile are performed in a biological safety cabinet laminar flow hood Failure to provide a means to conduct aseptic cell processing may result in harmful contamination Revision 1 5 4 July 15 2015 Chapter 1 Introduction The LARGE ANIMAL HOLLOW ORGAN Bioreactor is a rotating double chamber bioreactor designed for cell seeding and culturing both surfaces of a tubular matrix and includes rotational movement of the scaffold around its longitudinal axis A polymeric chamber houses the biological materials throughout the culture period Cylindrical scaffold holders are constructed with working end of different diameters to house matrices of diverse dimensions and a central portion of smaller diameter to expose the luminal surface of the matrix for cell seeding and culturing A co axial conduit links the inner chamber to the external environment through the chamber wall This provides access to seed and feed the luminal surface of the construct Secondary elements moving with the scaffold holder induce continuous mixing of the culture medium to increase oxygenation and mass transport Revision 1 5 5 July 15 2015 1 1 Controller Connect the Communication Cable from the controller to the The power module 1032418 PARATUS bioreactor motor housing should be connected to the power REGENERATIVE TECHNOLOGY 1030280 port Bioreactor C
3. into consideration including minimizing e The number of times the sterile flow path is opened to minimize the risk of contamination e The volume of medium changed at a single time to not change the environment the cells are exposed due after they have conditioned the medium with the particular growth factors and cytokine cocktails they are accustomed to e The volume changed if the fresh medium temperature is different from the operating medium temperature One manner to deal with the considerations above is to have a medium feed bottle large enough supply the entire amount of medium required throughout the experiment This may slow the initial growth phase of the culture as it may take a bit more time for the cells to secrete the needed levels of cytokines to condition a larger volume of medium However in general this is the safest manner to minimize contamination A second manner is to pour a volume typically not more than 25 50 of the existing medium out and pour fresh medium in Considerations for aseptic technique when handling large mouth reservoirs should be seriously studied Another manner is to have an external bag s or large bottle s of medium and have the system automatically feed and remove small volumes of medium on a continuous basis Fittings can be used to allow multiple inlet and outlet lines to feed into a single bottle Clear Nut 1031141 Cap 1031140 T Fitting 1032597 Green Nut 1031142 Red Nut 103
4. 1143 Please note that it is recommended that the tubing inside the bottle be long enough to always be below the medium level Revision 1 5 6 July 15 2015 1 3 Equipment Components A Drive Motor Base Plate Plate that aligns the reservoir with the drive motor B Control Unit Independent controller of rotational speed The bioreactor may also be controlled using an ORCA Controller C Motor Drive Drive motor responsible for rotation of the Arbor and organ Hex Rod 1031573 IL Outlet 1032584 Organ Support Lock 1031510 O Ring Cut Stock 1031578 Dam 1031509 Support Outlet 1031587 Support Inlet 1031588 a Drive Shaft 1030196 EL Inlet IL Inlet EPDM O Ring 1032584 11032584 61031729 IL Port this is used when the DAM is to the inlet side of the reservoir and the desire is to run the IL reservoir dry Fitting 1032584 or Plug 1032416 EL Port this is used when the DAM is to the outlet side of the reservoir and the desire is to run the EL reservoir dry Fitting 1032584 or Plug 1032416 Revision 1 5 EL outlet Fitting 1032584 or Plug 1032416 4 vi BLU i 1031029 Seal Wash Ports July 15 2015 Chapter 2 Operating Instructions 2 1 Fluid Connections Flow Path Options
5. 30196 Insert the hex rod 1031573 and the driveshaft Revision 1 5 11 July 15 2015 Operating Instructions Prior to Starting Assure that all the requirements identified in this manual have been successfully met Personnel using the LARGE ANIMAL HOLLOW ORGAN Bioreactor should read through this proce dure in its entirety prior to using the device Personnel should train on all processes and equipment prior to operating the Bioreactor Failure to follow aseptic techniques and failure to train on all processes and procedures prior to using the bioreactor may result in critical delays contamination and other harmful events 2 4 Sterilization Bioreactor Equipment for Sterilization Sterile Pouch Equipment Bag 1 Culture vessel Other bags Organ Holder Parts Wipe down Base Plate DO NOT AUTOCLAVE Control Unit Power Supply Before autoclaving foil or autoclave wrap the following 1 Reservoirs and connectors 2 Tubing ends 3 Luer fittings of all bioreactor connections do not unscrew Then remove valves and drive shaft from the operating ports and place them into the storage ports Revision 1 5 12 July 15 2015 2 5 Surface Disinfection Equipment A NOTE Drive Motor amp Base Plate Two operators are recommended for unpacking Control unit disinfection and assembly CAUTION In the case where several bioreactors are being used simultaneously a unique identifier should be placed on each bio
6. It is optional to apply RTV Sealant 1030566 to the threads of every port and fitting A small amount the size of a match head should be applied to the thread to minimize the potential for leaks Do not use too much RTV a single drop will spread out and cover the entire area Do not get sealant on the top of the fitting or the lids When changing the fittings use a Q tip to clear debris while holding upside down to allow residue to fall out of chamber Seal Wash Ports should be connected to a source of distilled water An elevated inlet will provide enough pressure to allow flow through the ports This will continuously wash the drive shaft and keep medium from drying and leaving salt crystals behind the seal which can cause damage to the seals and leaks Be careful to insert the fittings gently and not force them or you could cross thread the fittings TE IL Inlet PSA IL Port this is used when the EL Port this is used when the DAM EL outlet Fitting Seal Wash Ports DAM is to the inlet side of the is to the outlet side of the reservoir 1032584 or Plug 1031029 reservoir and the desire is to run and the desire is to run the EL 1032416 the IL reservoir dry reservoir dry The level of the medium in the EL reservoir is selected by connecting a fitting to one of the three EL outlet ports on the reservoir or using the variable level outlet on the reservoir cover Place f
7. Large Animal Hollow Organ Bioreactor HARVARD APPARATUS REGENERATIVE TECHNOLOGY Regenerated Organs for Transplant 84 October Hill Drive S 11 Holliston MA 01746 1371 USA www HARTregen com 4 774 233 7300 cservice HARTregen com Contents Warning amp Caution Statements General Safety Requirements Chapter 1 Introduction 1 1 Controller 1 2 Medium 1 3 Equipment Components Chapter 2 Operating Instructions 2 1 Fluid Connections amp Flow Path Options 2 2 Medium Connections 2 3 Chamber Assembly 2 4 Sterilization Al e W N 2 5 Surface Disinfection 2 6 Post Sterilization Assembly 2 7 Affixing the Organ Scaffold 2 8 Equipment Components Chapter 3 Care amp Maintenance 3 1 Cross Infection Prevention Universal Precautions 13 14 16 Revision History 20 20 20 21 22 23 WS a ma a WE KE a WE E WS DE a KJE B Revision 1 5 2 July 15 2015 HARVARD APPARATUS REGENERATIVE TECHNOLOGY Harvard Apparatus Regenerative Technology HART partners with leading global scientists to provide specialized solutions The company is uniquely positioned to develop advanced instrumentation to accelerate regenerative medicine tissue engineering and cell therapy experimentation From the beginning HART works closely with leading global researchers to produce products with the highest levels of performance quality and support necessary for the new challenges of your life s
8. and Caution Statements The use of a WARNING statement in this User Manual alerts you to a potential safety hazard Failure to observe a warning may result in a serious injury to the user The use of a CAUTION statement in this User Manual alerts you to where special care is necessary for the safe and effective use of the product Failure to observe a caution may result in minor injury to the user or damage to the product or other property Intended Use The Hollow Organ Small Animal Bioreactor is a vessel used to support a hollow organ scaffold for the purpose of cell seeding it is intended for research use only NOT FOR HUMAN USE Use of this device in non research settings must be conducted under local Regulatory requirements consult your local Regulatory Authority The following conditions must be met prior to using the Bioreactor General Safety Requirements The Bioreactor should only be used by qualified personnel who have been trained by the manufacturer or other authorized representative Unauthorized use of this device is not recommended To prevent contamination aseptic procedures must be followed and personal protective equipment must be worn at all times when handling and using the Bioreactor Wherever blood products are used Universal Precautions must be followed DO NOT SUPPLY EXPLOSIVE GASES TO THE BIOREACTOR Facility Requirements Assure that the facility is able to provide a clean safe and suitable area
9. ber 1 2014 O Ring changes amp O Ring Tool 1 3 February 20 2015 Add sealing caps 1 4 March 10 2015 General Updates and picture with new black O Rings 1 5 April 21 2015 Add Spare Parts 1 6 July 15 2015 Correction on Tee fitting P N Revision 1 5 25 July 15 2015 HARVARD APPARATUS REGENERATIVE TECHNOLOGY 84 October Hill Drive S 11 Holliston MA 01746 1371 USA www HARTregen com 774 233 7300 cservice HARTregen com Revision 1 5 26 July 15 2015
10. cience research We look forward to working with you to develop new tools to assist you in solving these new challenges of regenerative medicine from the lab bench to the patient There are hundreds of publications in regenerative medicine and stem cell research utilizing HART products These products will serve the researcher and the physician to accelerate basic research and clinical translation Disclaimer Use of the LARGE ANIMAL HOLLOW ORGAN Bioreactor should be conducted by a trained operator Harvard Apparatus Regenerative Technology HART does not warrant unauthorized use of this product HART warrants the operation of the bioreactor for a period of one year under normal laboratory conditions HART reserves the right to change the instructions for use and any related products at any time without any prior notice and is not liable for any damages arising out of any change and or alteration of the contents or product This product is for RESEARCH USE ONLY and NOT FOR HUMAN USE Copyright 2014 Harvard Apparatus Regenerative Technology All rights reserved Harvard Apparatus Regenerative Technology owns the intellectual property rights to the LARGE ANIMAL HOLLOW ORGAN Bioreactor This material may not be reproduced displayed modified or distributed without the expressed prior written permission of the copyright holder U S international and foreign patent applications are pending Revision 1 5 3 July 15 2015 Warning
11. d to absorb the medium and cells faster than natural scaffolds so smaller droplets are recommended on natural scaffolds One loading procedure is to apply droplets across the horizontal axis of the scaffold Then rotate the arbor to the next number and repeat Cells will fall off into the EL reservoir They can be picked up with a pipette and the process repeated until the cells required by the protocol are at tached to the scaffold e How to seed the Intra Luminal cavity Protocols may dictate which type of loading procedure is used One method involves using a sterile stopcock and loading the cells directly into the IL via a syringe Once the volume of cells required is loaded the stop cock Is turned to trap the volume in the IL Rotating at 0 5 1 rpm may assist the adherence of the cells Some methods call for the selector valve to be set position 1 with no outlet of the IL fluid to assist in hold ing the medium in the IL Caution should be used as some seepage will occur through the scaffold walls Once the cells are attached the selector can be switched back to allow normal flow from the IL reservoir through the IL Some protocols connect two stopcocks one on the inlet and one on the outlet and allow a piece of by pass tubing to bypass the reser voir and minimize the fluid path volume in the initial stages of the culture after the cell seeding Revision 1 5 24 July 15 2015 Notes Date Changes 1 1 September 23 2014 1 2 Decem
12. ictate the dam location We recommend that you allow enough tubing length to not only complete the circuit to the IL outlet but with enough length to spare to allow the tubing to be moved an inch each week Moving the tubing an inch out of the head minimizes tubing wear on a single spot Revision 1 5 9 July 15 2015 IL Port this is used when the DAM is to the EL q inlet side of the reservoir and the desire is to outlet run the IL reservoir dry Sealing Ports pS Swabble Ports 1031081 are available in bulk These ports can be autoclaved once They allow a sterile barrier to be maintained that can be connected to Sterile Caps 1032581 are available These are shipped in individual sterile blisters Each cap has both a male and female dead end port Revision 1 5 EL Inlet IL Inlet July 15 2015 2 3 Chamber Assembly Insert the O ring 1031577 onto the Organ Support Outlet 1031587 Place the small O Ring 1030195 onto the end of the hex rod 1031573 Push O Ring in flush against the flex hole of the Support Inlet 1031588 Lock Organ Support 1031510 should be placed on the Dam 1031509 Organ Support with the dowel Pin The Lock should swivel parallel to the Dam 1032467 O Ring re E moval tool is used to re place O Rings on the drive shaft n Place three black EPDM O Rings 1031729 onto the driveshaft mm 10
13. itting into culture lid Add swabble port to the fitting Push cannula through the swabble port Adjust the height to provide the level of medium in the reservoir desired Cannula 1032413 Swabable Port 1031081 s Fitting 1031136 Culture Lid 1032414 Revision 1 5 8 July 15 2015 2 2 Medium Connections Peristaltic Pumps Depending on the configuration of the system and how you set up the medium feed and waste will dictate the number of pumps and channels you will require A typical configuration may be O EL Inlet tubing is selected according to the flow rate range required see chart The EL is normally fed from a bottle inside the reservoir O EL Outlet tubing is selected according to the flow rate range required see chart Typically the outlet flow rate is selected higher than the inlet to assure the unit does not over fill IL Inlet and outlet tubing is selected according to the flow rate range required see chart The IL is normally fed from a bottle inside the reservoir IL Outlet tubing is selected according to the flow rate range required see chart Typically the outlet flow rate is selected higher than the inlet to assure the unit does not over fill This type of configuration would require 2 pumps with two channels each gt Placing the drive unit of the peristaltic pump inside the incubator is usually recommended to minimize the tubing length and thermal effec
14. may be used to push the O Ring into place Place the organ on the other organ holder There are flat areas on each of the Organ Supports that allow the or gan to lie stationary Slide the driveshaft through the organ and into the initial organ holder Use of pins to attach the trachea may be enough to hold a synthetic scaffold in place Suturing may be needed to close the ends Revision 1 5 14 July 15 2015 If pins are chosen the notches indicate where the pins should be placed Use the forceps to place the pins one at a time in the four dedi cated locations An alternative is to suture the scaffold into place by wrapping the thread around the groove in the organ holder and pulling it tight Using a biopsy punch a sample of tissue can be taken post incubation for further analysis if desired This sample can now be used for testing Revision 1 5 15 July 15 2015 2 8 Equipment Components Part Name Included Part Chamber Ony 1081870 Drive Motor Assembly amp Controller ol 1082420 ll EECHER O Ring EPDM 375 x 25 x 065 Drive shaft fo each fo 1031729 Cima fm CET ef mee Foe TT dine i oe Fon o ra mem Revision 1 5 16 July 15 2015 Part Name Qty Part Fitting Luer Female Barb 1032298 m RR mm Fitting Luer Male Barb ies 25 1032295 CE ME NE Revision 1 5 17 July 15 2015 Part Name Qty Part Revision 1 5 18 July 15
15. n the standard unit from 16 24 mm For organs requiring 11 12 mm OD a custom organ holder can be made e Should I sterilize using plasma sterilization or does autoclaving work The materials of construction are a polycarbonate reservoir stainless steel baseplate driveshaft and valves silicone rubber O rings Kynar Luer fittings polycarbonate cover and PEEK arbor components All of these materials will withstand steam sterilization One CAUTION however Please disassemble all the valve and driveshaft parts from the block before steam sterilization The high temperature may cause the machined holes in the reservoir to become deformed by the parts pressed into them Reinstall the valves and driveshaft after the reservoir cools down The Kynar Luer fittings have been installed using silicone adhesive to hold them in place and prevent leaks It should not be necessary to remove these fittings from the reservoir because of the sterilization process e Can washing be done with reagent These materials of construction will withstand virtually all biological reagents and cold Sterilization agents such as Cidex Cidex OPA Mucasol etc e How does the seal wash inlet and outlet work This passage Is designed to take a non salt solution and flush it through a separate space between the two outside O rings on the driveshaft This will prevent any buildup of evaporated salt crystals from forming on the turning shaft during long period
16. ontroller 1 E POWER ALARM Alarm can be switched to the e E The controller can be used on various models of bioreactors Confirm that the switch is in the clockwise position Otherwise the shaft will be rotated out of the bioreactor ON position which allows the controller to verify that the motor is turning The TEST position will test the audible alarm The RPM switch allows selection of the revolutions per minute the shaft will be turned The switch is turned clockwise to increase the rotational speed from 0 5 to 5 rpm In order to select the rpm required it is recommended that a stop watch is used to measure the turns made by the drive shaft and the knob adjusted accordingly This should be done after the unit is in the incubator The motor tray is placed in an incubator with the connection wire normally fed through an access port or by compressing the door gasket If the door gasket is compressed to allow the wire to pass it is recommended that a piece of foam is glued to the wire to eliminate a space being formed above the wire and the inside door of the incubator Place the bioreactor on the motor plate with the drive shaft connected to the motor Note the clover leaf coupling should be in place 1 2 Medium Replenishing feed medium can occur in a number of ways When deciding on the manner of medium replenishment and removal that will be employed several factors should be taken
17. reactor component to avoid parts being exchanged Colored stickers have typically been used on each pouch Similar precautions should be taken with components post sterilization Failure to follow the defined sterilization procedure may damage the bioreactor and make it unsuitable for use 2 6 Post Sterilization Assembly Assembly Preparation The bioreactor parts are delivered in sterile pouches Unpacking and bioreactor assembly should be performed in a clean room environment provided by a laminar airflow cabinet CAUTION To prevent contamination aseptic procedures must be followed and personal protective equipment must be worn at all times when handling and using the Bioreactor Failure to strictly follow aseptic techniques may result in critical delays contamination and other harmful events Sterile Bioreactor Equipment Other Materials Bioreactor Arbor amp organ sterilized in sterile pouch Driveshaft Reservoir Cover Transport Sealing Cover Culture medium Inoculum Disinfected Bioreactor Equipment Control unit Power Supply Revision 1 5 13 July 15 2015 2 7 Affixing a Synthetic Scaffold Organ to the Organ Holder First lay out the relevant materials you will be needing in a laminar flow hood to protect sterility The bioreactor parts will be sterile after autoclaving It is Important to have the correct size organ holder If the or Place the O Ring onto the shaft A sterile pipette tip
18. s of use As an example an intravenous drip bag with water set to between 0 1mL and 0 5mL per minute to continuously flush the seal wash path e Can I use the bioreactor for natural and synthetic scaffolds and organs The bioreactor can be used for both types of organs Revision 1 5 22 July 15 2015 3 5 Frequently Asked Questions cont e Can I use Tygon tubing to save money Tygon tubing is not recommended but can be used The issue that arises all too frequently even with the autoclave grade Tygon tubing is that a piece of the tubing will crease and partially block during an autoclave cycle It is very difficult to undo the crease and can cause issues If Tygon tubing is used it is highly recommended that tubing be removed from the fittings during the autoclave process Otherwise the tubing should be taken off the fitting and the end clipped to present fresh tubing and then pushed back on the fitting Otherwise the fatigue experienced as the different plastics expand at differential rates may cause the tubing to come undone during an experiment e Autoclave cycle settings It is highly recommended that the temperature cycle not exceed 121 C as this can cause damage to various components of the bioreactor e What type of pump must I use for medium delivery There is not actual requirement on the type of pump selected In most cases people will use a peristaltic pump that allows the electronics to be outside the incubator with
19. se materials of construction will withstand virtually all biological reagents and cold steriliza tion agents Stainless steel parts may be sonicated 3 3 Specifications Size of Organ 0 7 9 mm ID up to 65 mm in length is standard Rotation Speed Positional Monitoring 100 240 VAC 50 60 Hz Revision 1 5 20 July 15 2015 3 4 Troubleshooting Arbor not turning Check to see if the reservoir is in place and the drive shaft is connected to the motor The retaining clamp is tightened during installation to maintain the connection Check to see if the medium blood being used has fouled the arbor This can occur in high fibrinogen solutions Excess Medium Reagent build up tn the reservoir Check for clogging or fouling of the reservoir outlet line or port especially during a decellularization procedure Medium level in the IL or EL flow path has gone dry Confirm that there is sufficient medium in the inlet reservoirs and that the inlet tubing inside the reservoirs is below the medium level Leaks DO NOT USE BLEACH WHEN CLEANING THE BIOREACTOR AS IT HAS BEEN SHOWN TO CAUSE FAILURE OF COMPONENTS DURING AUTOCLAVE OPERATIONS Also be sure to check O Rings frequently and replace as necessary Revision 1 5 21 July 15 2015 3 5 Frequently Asked Questions e What is the size of the standard organ holder The typical organ holder has an ID of the hole leading to the intraluminal space of 12 mm The OD can vary o
20. the drive motor inside If you are using multiple pumps in the incubator or multiple bioreactors with multiple pumps the ability of the incubator to maintain temperature may be an issue Many types of incubators only heat and the use of multiple pumps may cause too much heat inside the incubator If this is an issue select an incubator that will heat and cool or contact the customer service group as the power to the pump motor may be lowered and this may aid with minimizing the heat e How do I vary the length of the scaffold holder There is a sliding dam which divides the chamber into an IL and EL chamber The slide allows a maximum length of 140 mm of an organ The organ holder slides on a hexagonal drive rod The standard organ holder has a 12 mm ID opening There is a nipple where the organ is attached to is made with an OD diameter from 14 24 mm Custom organ holders down to 11 mm OD with a smaller ID can be designed Revision 1 5 23 July 15 2015 3 5 Frequently Asked Questions cont e What do I do if I want to use organs with an ID of less than 10 mm A small hollow organ bioreactor cnamber is available which can be used with the con troller and motor drive of the large hollow organ bioreactor e How to seed the Extra Luminal cavity Protocols will vary depending on many factors including the type of scaffold medium and cell types Typically the cells are introduced in droplets of 100 250 uL Synthetic scaffolds ten
21. ts of having liquid moving in and out of the incubator This also minimizes the number of tubes that have to go through an aces port or that have to be squeezed against a door gasket Connecting the EL Circuit 4 The EL inlet should be connected to a piece of Masterflex see chart below tubing through a Luer Barb Fitting to the EL reservoir It is recommended that the user allows enough tubing length to not only complete the circuit to the EL outlet but with enough length to spare to allow the tubing to be moved an inch once per week Moving the tubing an inch out of the head minimizes tubing wear on a single spot The EL outlet should be selected according the to the medium level desired in the reservoir The side of the chamber allows five choices There are two choices that will allow running of the chamber virtually dry 1 amp 2 below The number 2 position can have issues with tubing fitting under the drive shaft if the tubing is too large Both allow the exact functionality Positions 3 4 amp 5 allow the medium level to be fixed at three different levels The variable level EL outlet on the Lid 6 may also be used The level is selected by simply adjusting the level of the cannula tip 4 The IL Inlet should be connected to a piece of Masterflex see chart tubing through a Luer Barb Fitting to the IL reservoir The positions 7 or 8 are identical Position 7 may not be usable depending on the length of the scaffold which will d
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