Organ Bath Systems
Contents
1. pump is built into the water bath to circulate the heated water and ensure even heat distribution throughout the entire system Manipulators micropositioners are supplied for accurate positioning of the transducers and applying pre tension to the tissue preparations prior to experimentation Tissue chamber Reservoir coil Gas diffuser Filling electrovalve Emptying electrovalyve Tissue holder Transducer Transducer micropositioner Heating rod Water circulator Ae TAN ONS gt Top view E D Physiological solution inflow i l Gas inflow hy 9 _ Physiological solution outflow Figure 2 Schematic diagram of a 2 chamber Panlab organ bath ADInstruments Panlab Organ Bath Systems The Panlab organ baths are supplied as complete ready to use research systems that include all of the necessary hardware and software to perform isolated tissue experiments Each research system includes e An 8 or 16 channel PowerLab data acquisition system e Multi chamber Panlab organ baths 4 8 or 16 chamber systems available e Bridge amplifiers Quad or Octal Bridge Amps e Isometric transducers e LabChart Pro including Dose Response All of these components may be purchased separately if required Single Chamber Organ Bath ADInstruments supply a single chamber organ bath by Panlab however it is only recommended for teaching applications and does not include a thermostat controller ADInstruments have a Thermoregulate2. short term and long term stimulations Frequently Asked Questions What physiological solution should I use The solution chosen will have an effect on the viability of the preparation Solutions such as donor blood may be used however there are limitations such as availability as well as the possibility of denaturing blood proteins Researchers would commonly use an artificial plasma like solution such as Ringer s or Krebs Henseleit which contain a buffer to maintain pH levels nutrients and supply oxygen to the tissue What should I use to aerate my physiological solution Tech Note Page 8 10 Bicarbonate buffers such as Krebs Henseleit require aeration with carbon dioxide therefore carbogen 95 O D CO is commonly used for aerating these artificial solutions The use of 95 O rather than atmospheric levels of 21 O is necessary to compensate for the lack of hemoglobin or other oxygen carriers in artificial perfusate solutions ensuring adequate delivery of oxygen to the tissue If donor blood is used then a lower O concentration may be used In traditional organ bath configurations using donor blood or a bicarbonate buffer the tissue is NOT perfused through its blood vessels Therefore if the tissue is not normally porous it relies on diffusion for the delivery of oxygen The researcher should ensure that the tissue sample is not too thick to avoid its core becoming hypoxic due to inadequate oxygen availability What size
3. tissue chamber should I use The size of the tissue chamber should be large enough to accommodate the tissue and any additional equipment such as stimulating electrodes The chamber must have an adequate amount of solution to cover the tissue used Minimizing the amount of physiological solution in the chamber will conserve the amount of drugs that are required to induce an appropriate response which may be an important factor How long does it take for the tissue to stabilize in the organ bath Depending on the tissue type storage and experimental procedures the period of equilibration can be from 20 to 60 minutes aft er the tissue has been placed in the organ bath It is best to consult the latest literature for exact equilibration periods for the tissue being investigated There are irregular artifacts in my recording The artifacts may be due to vibrations of the silk thread Make sure the tissue and silk threads are positioned outside of the bubble stream It is also recommended to have a bubbling rate in which the bubbles when they reach the liquid surface do not cause a boiling effect at the surface IMPORTANT Always ensure that the gas diffuser is turned off or has a constant stream of carbogen passing through it before filling the chamber with perfusate This is necessary to prevent solution entering the diffuser and avoids precipitation of salts which can block the diffuser Refer to the manual for further details about cleaning an
4. touch the tissue and have a current passed between them This method is effective provided that the tissue remains within the field of stimulation and is the most common technique used in organ bath preparations A field stimulating electrode is available from Radnoti It is constructed of platinum wire sealed in glass and designed to insert into Radnoti organ baths This electrode features a dual vertical spiral coil design with a 1 cm gap that provides a large uniform electrical field that surrounds the tissue It is suited for stimulating sections of blood vessels various other smooth or skeletal muscle preparations or any type of tissue that requires field stimulation and comes with two coil sizes that suitable for different chamber sizes which are e 158812 Field Stimulating Platinum Electrode 5 10ml e 158814 Field Stimulating Platinum Electrode 25 50ml Alternatively direct stimulation involves electrode contact with the tissue and is often used to stimulate the tissue via a nerve trunk that innervates it i e the isolated diaphragm dissected with the phrenic nerve attached to a stimulating electrode see Stimulating Electrodes Goad omy Figure 5 Field Stimulating Platinum Electrode Constant Current versus Constant Voltage Stimulation For most organ bath experiments researchers use constant current or constant voltage stimulation Constant current stimulation is often used as the effectiveness of a stimulus is directly related
5. Organ Bath Systems ADINSTRUMENTS Introduction The organ bath is a traditional experimental set up that is commonly used to investigate the physiology and pharmacology of in vitro tissue preparations Perfused tissues can be maintained for several hours in a temperature controlled organ bath After a suitable equilibration period the researcher can begin experiments Typical experiments involve the addition of drugs to the organ bath or direct field stimulation of the tissue The tissue reacts by contracting relaxing and an isometric or isotonic transducer is used to measure force or displacement respectively From the experimental results dose response curves are generated tissue response against drug dosage or stimulus potency ADInstruments supply a range of organ baths and transducers for pharmacological research LabChart software records displays and analyzes the data which may then be analyzed or exported to graphing programs such as GraphPad Prism Some of the tissues that may be studied with an organ bath system include Smooth Muscle Skeletal Muscle Cardiac Muscle Vascular e g arterial aortic rings Toad Abdominus Rectus Atrium Guinea pig ileum Chick Biventer Cervis Ventricle Vas Deferens secretory duct of the testicle Mammalian Diaphragm Uterine Colon Tissues are usually prepared in a petri dish containing physiological solution i e Kreb s solution The ends of the tissue are then attached to the mounti
6. d Water Pump Model MLE2001 that will work with up to four of the single chamber organ baths in series however there should be no more than 2 meters distance between the first and last organ baths If an external heating system is required then an immersion heater circulator is suitable i e the TU1 from Thermoline Scientific www thermoline com au When used with the Single Chamber Organ Bath the maximum flow rate of the circulator should not exceed 5 L min to avoid excessive pressure within the system The size of the support clamp length of the heating rod and intending heating volume should also be checked to ensure they are compatible with the dimensions of the water bath Stimulating Electrodes Electrodes are required if using a stimulator The platinum electrodes are recommended for most researchers using field stimulation see table and diagrams below All of the following stimulating electrodes are supplied with 2 mm plug connections ype amp Description Stainless Steel Single Ring 6 5 8 or 10 mm inner diameter 20 mm between ring and tissue holder pe Ee ee Double Ring 6 5 8 or 10 mm inner diameter 20 mm between rings 6mm above tissue holder MEANIMAMD cma 3 mm Length Pole 20 mm from tissue holder MLA0303 MLA0307 Double Ring 6 mm inner diameter 15 mm between rings 12 mm from tissue holder MLAJ404 MLAOS09 Specify ID 6 5 8 or 10 mm Key Benefits e Available in 2 4 8 chamber configuration e Sin
7. d care of the organ baths Glossary Common Terms in Pharmacology Affinity The strength of the reversible interaction between a drug and its receptor measured by its dissociation constant Kd Potency The concentration of a drug for a given response magnitude of the biological system The lower the concentration for a given response the higher the potency of that compound Efficacy Response magnitude of the biological system upon binding of the drug to its target receptors EC50 Half maximal effective concentration of a drug for producing a given effect Agonist A hormone neurotransmitter or drug that binds to a cell receptor to trigger an active response Antagonist A molecule that blocks a receptor and interferes with or prevents an agonist from eliciting a response Clearance The rate of elimination removal of a drug from the body Tech Note Page 9 10 Half Life The time taken for the concentration of a compound in blood circulation to decrease by 50 Tolerance Reduced effect of an agonist or antagonist following its prolonged administration resulting from the increased metabolism metabolic tolerance or decreased receptor sensitivity pharmaco or tissue tolerance Increased dosages are required to produce the desired effect Up regulation An increase in the number and or sensitivity of receptors to compensate for the decreased effect of an agonist MLS390 7 Dose Response Module for LabChart for Windows This Mod
8. es Quick Disconnect fittings e Large range of tissue organ baths volumes 5 10 25 50 100 200 300 ml Transducers Isometric Transducers Isometric transducers measure the change in force in grams g or milliNewtons mN produced by muscle relaxation contraction while the muscle length remains constant ADInstruments supply the following isometric transducers for use with ADInstruments range of tissue and organ bath models e MLT0201 Force Transducer 5 mg to 25 g e MLT0201 RAD Force Transducer 5 mg to 25 g for use with Radnoti Organ Baths e MLT0202 Sensitive Isometric Force Transducer 0 25 g ideal for isolated tissue studies and has been designed for operation with ADInstruments range of tissue and organ bath models e The MLT0210 Teaching Force Transducer is recommended for teaching applications It operates in the range from 10 mg to 25 g with a resolution of 5 mg and is recommended for use with the ML1110 Single Chamber Organ Bath and comes as o MLT0210 D Teaching Force Transducer MLT0210 D o MLT0210 A Teaching Force Transducer MLT0210 A e MLT0420 is a sensitive isometric force transducer suitable for measuring small forces up to 20 g The transducer is suitable for connection to ADInstruments Bridge Amps e MLTF500 ST Teaching Force Transducer A low cost amplified bridge type force transducer with measuring range 500 g The transducer provides a fixed 3 V excitation source for the internal sensor A range of digital
9. gle water bath provides controlled temperature regulation e Automated chamber filling and emptying using electrovalves e All in one design provides a compact portable unit Tech Note Page 3 10 Radnoti Organ Baths ADInstruments provide complete 4 8 and 16 chamber Radnoti tissue organ bath systems that include a PowerLab bridge amplifiers thermo bath circulator and transducers for the recording of data from isolated tissues Figure 3 PL3508B60 Radnoti 4 Chamber Tissue Organ Bath System Radnoti organ baths include a single water jacketed 2 L reservoir for the physiological solution The solution enters the tissue chamber via a preheating coil that is positioned within the water jacket of the High Tech Tissue Organ Bath The solution in the chamber can be emptied via a drain plug stopcock at the bottom of the tissue chamber for a single pass mode or may be recirculated in a constant flow mode In the constant flow mode the solution exits via an overflow outlet positioned at the top of the chamber Aeration of the physiological solution is provided via needle valves in each bath as well as a frit in the reservoir Each chamber has its own 5 mg to 25 g isometric transducer MLT0201 RAD and transducer positioner The modular construction of Radnoti systems permits the researcher to easily interchange tissue chambers and use different sized baths 5 10 25 50 100 200 300 ml Each organ bath is individually mounted via adjustable ring c
10. lamps and is connected to a sturdy steel base that has a resistive coating to avoid corrosion Radnoti Thermal Circulator Pump 170051B V This unit incorporates a high wattage heater for rapid warming of the water bath and a two stage force and suction pump that provides water circulation Circulation of the water is necessary to maintain a constant temperature in the reservoir and tissue chamber jackets When ordering the 170051B V please specify the mains power voltage V V 115 or 220 Note For countries with 115V power service the Thermo Circulator requies a 20 AMPS outlet For countries with 220V power service the Thermo Circulator requies a 15 AMPS outlet Tech Note Page 4 10 Specifications e Reservoir Volume 17 Liters 4 5 Gallons e Temperature range 12 C to 200 C e Temperature Stability 0 01 C e Maximum Pump Flow Rate 15 L min e Bright LED digital display e Stainless steel reservoir with drainage e Low water level safety shutoff I Figure 4 Thermo Bath Circulator Each ADInstruments Radnoti Tissue Organ Bath System Includes e 2L water jacketed reservoir e Transducer Positioners e Oxygenating Bubblers e Support Stand amp Hardware Kit e Force Transducers Isometric e Cable amp Connector Package e High Tech Tissue Organ Baths 5 300 ml User must specify size when ordering e Bridge Amplifiers e Thermo Bath Heater e PowerLab Data Acquisition System e LabChart Pro including Dose Res
11. low pass filters are built in to reduce signal noise and a software controlled DC offset adjustment is provided A detachable mounting bar is incorporated with the unit e MLTF050 ST Teaching Force Transducer A low cost amplified bridge type force transducer with measuring range 50g The transducer provides a fixed 3V excitation source for the internal sensor A range of digital low pass filters are built in to reduce signal noise and a software controlled DC offset adjustment is provided A detachable mounting bar is incorporated with the unit Tech Note Page 6 10 Isotonic Transducers Isotonic transducers measure the change in muscle length in millimeters mm or degrees traveled as it contracts relaxes against a constant force ADInstruments supply the following isotonic transducers for use with ADInstruments range of tissue and organ bath models e The MLT0015 High Grade Isotonic Transducer is suitable for the measurement of displacements with preadjusted loads in the range of 15 0 to 48 mm displacement e The MLT7006 Hall Effect Isotonic Transducer is a low inertia high performance isotonic transducer with a finely adjustable counterweight suitable for pre adjusted loads as small as 100 to 200 mg 0 to 60 mm displacement 15 Stimulation and Electrode Selection Stimulation of tissues may be achieved by a number of methods Field stimulation may be performed using ring or plate electrodes that are placed near but do not
12. ng hook and transducer using silk It is important to use non compliant material such as silk or fine wire to mount the tissue For tissue ring preparations i e arterial rings a custom tissue holder is required can be sourced by ADInstruments These may also be made using triangular shaped wire holders constructed of non compliant material and secured to the mounting hook on the Panlab tissue support rod Figure 1 Tech Note Page 1 10 Silk attached to transducer Tissue Support Rod gt Tissue preperation Triangular tissue supports Tissue Mounting Hook Figure 1 Schematic diagram of a custom made mounting system for tissue rings Panlab Organ Baths ADInstruments supply compact and robust yet versatile organ baths manufactured by Panlab Each organ bath can take 5 10 25 or 50 ml tissue chambers The tissue chamber can be easily changed by the user and any sized tissue chamber can be used in all the Panlab organ baths The tissue chambers are automatically filled and emptied using electrovalves with each tissue chamber also having it own gas diffuser which can be regulated A heating rod is built into each of the organ bath units and a thermostat controller is included to control the water temperature surrounding the tissue chambers Figure 2 Pre heating reservoir coils are also positioned within the water bath to heat the perfusate before entering the tissue chamber A highly efficient magnetic water
13. ponse These systems require manual refilling and emptying of tissue chamber Stimulating Electrodes Radnoti also provides several combination tissue support rods stimulating electrodes that are suitable for use in organ baths larger than 10 ml These allow both vertical and horizontal adjustment of the platinum electrodes in relation to the tissue sample to ensure maximal stimulation A general guide for the use of these electrodes is as follows e Tissue strips spirals Straight 160152 11 zig zag 160152 12 or coil 160152 13 electrodes e Small samples Coil 160152 13 or flat 160152 15 electrodes Tech Note Page 5 10 e Blood vessel rings L type electrode 160152 14 The tissue ring is slid on the lower support and attached to a tri shape tissue support 158817 and 158819 and placed under tension between the two horizontal platinum wire electrodes The above product codes are for combination universal support rods stimulating electrodes however the electrodes may be purchased separately to be interchanged on a universal support rod Key Benefits e Modular components enable tissue chambers to be easily removed and permit investigations with different sized chambers e Each chamber is individually water jacketed ensuring uniform circulation and heating e Reservoir is water jacketed to maintain solution temperature e Overflow port allows constant recirculation of perfusate to limit the amount of solution required e Incorporat
14. to the current That is current is a measure of the flow of energy into the tissue The more energy that goes into the tissue the greater the contraction An increase in current leads to recruitment of muscle fibers Tech Note Page 7 10 which contribute to an increase in contractile force Constant current stimulators are unaffected by changes in load due to factors such as electrode size contact resistance and polarization In constant voltage mode the stimulus voltage is gradually increased until the resultant current unknown is enough to induce a tissue response however the current itself is not known A disadvantage of using constant voltage is that polarization of the electrodes may occur in salt solutions i e a build up on one electrode that increases resistance The increased resistance between the electrodes means that a greater voltage is required to produce the same current Polarization of the electrodes may cause the researcher to believe that the tissue is dying when in fact the efficiency of the stimulus is reduced Stimulators ADInstruments provide a range of stimulators for organ bath studies They include the e ML155 Stimulator HC is a software controlled constant current stimulator with 100 V compliance designed for use in vitro with isolated nerve muscle or tissue samples The unit uses the analog output from the PowerLab to produce isolation and software adjustable constant current pulses of various durations freq
15. uencies and amplitudes up to 100 mA ML1001 Electronic Stimulator is designed to provide electrical stimulation in physiological and pharmacological research experiments It provides options for single pulse paired pulse pulse train and repeat pulse operation Biphasic stimuli are also supported The stimulator is equipped with a booster amplifier to allow field stimulation with a maximum output of 50V Input terminals also allow signal mixing and modulation with an external signal It can provide a constant current and or voltage stimulation with the ML1101 Stimulus Isolator ML1101 Stimulus Isolator insulates the output for the ML1001 Electronic Stimulator from ground This minimizes the area of stimulation to ensure greater precision while reducing stimulation artifacts and facilitating the observation of evoked action potentials This is not a stand alone unit and is configured for use with ML1001 Electronic Stimulator The STG4004 and STG4008 Stimulus Generators are multi channel 4 and 8 channels respectively general purpose stimulators which can be used with PowerLab for data acquisition as well as being controlled by a trigger signal from the PowerLab For each channel they can produce an output voltage range between 8 V to 8 V with a resolution of 1 mV or an output current range between 1 6 mA to 1 6 mA with a resolution of 100 nA They are designed to serve a wide variety of applications such in vitro and in vivo neurophysiology and
16. ule facilitates analysis of dose response type data from LabChart recordings The analysis can be done automatically or manually offline or online LabChart data recorded prior to the use of the Module can be readily converted for use by Dose Response The results of the analysis can be exported for use by other applications fu Dose Response Rat Blood Pressure adicht Dose Response Results View odlo Runs Ratle Rat2 Rat3 Rati Best fit values v 5 28 7 016 0 1333 0 0 6733 to 32 94 99 67 to 144 3 0 8981 to 0 0497 0 1264 to 0 8919 itt Response Degrees of Freedom R Squared Absolute Sum of Squares 99 18 Sy x i 5 75 analyzed 6 T T T T t T 2 0 L5 1 0 0 5 0 0 0 5 1 0 Log Dose ug Kg hd 7 Normalize graphs F Fix Hill slope at 1 0 Figure 6 The response points for the three channels and the fitted response curves are displayed in the Dose Response Results View Fitting parameters for a selected curve are shown in the list on the right Tech Note Page 10 10
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