Organ Bath Experiments
Dose response studies are typically conducted to assess concentration-response relationships in isolated tissue preparations. Traditionally, tissue-organ baths are used for in vitro dose response experiments to investigate the physiology and pharmacology of tissue preparations from various species (e.g. chick, toad, rabbit, rat, guinea-pig, etc.). Tissue-organ baths are used to maintain the integrity of the tissue for several hours, in a temperature-controlled environment, while physiological measurements are performed. 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).
Some of the more common tissues that are studied with an organ bath system include:
- Aorta rings
- Heart tissue (papillary muscle, left ventricles)
- Arteries (mesenteric arteries)
- Gastric antral muscle
- Tracheal rings
- Phrenic diaphragm
- Pulmonary arterial smooth muscle
- Lung parenchyma
Other isolated tissue preparations include urinary bladder, penile muscle strips and prostate.
Isolated tissue-organ experiments can generally be run in groups of 2, 4, 8 or more sample preparations, thereby enabling a high throughput in most laboratories. These in vitro preparations are also more readily instrumented and can be easily subjected to controlled changes in perfusate, oxygen availability, drug administration etc than is otherwise possible in the intact animal.
A water-jacketed organ bath provides a stable and easily adjustable way of temperature control. Substrates and other nutrients that are required to sustain tissue function are provided via a physiological solution, which allows the study of evoked tissue responses to:
- Pharmacological drug/agents (Dose Response studies)
- Electrical stimulation
- Both pharmacological and electrical stimulation
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 mounting hook and transducer using silk or fine wire (non-compliant) to mount the tissue. The tissue placement may vary depending on the tissue type such as:
- Ring/Vessel Preparation
- Strip Preparation
Organ Bath Apparatus
ADInstruments offer the following range of organ baths manufactured by Panlab and Radnoti.
Compact “all-in-one” Organ Bath systems by Panlab which allow;
- Immersion of all tissue chambers and perfusate warming coils in a single temperature controlled bath
- Individual or simultaneous filling and emptying of tissue chambers
- Compact design to save on bench space
- Bath capacity options of 5 to 50 mL
Modular Organ Bath systems by Radnoti which feature;
- Water-jacketed reservoirs and tubing to ensure perfusate temperature stability
- Easy addition and substitution of components
- Modular design allows easy customization of glassware placement and type
- Bath capacity options of 5 to 300 mL
The recorded responses are the contraction/relaxation of muscle in the tissue in the form of:
- Force (isometric)
- Displacement (isotonic)
ADInstruments Single, Quad or Octal Bridge Amplifier models are software controlled preamplifiers, providing a range of filter and amplification settings for isometric or isotonic transducers. A range of Isometric and Isotonic Transducers of varying grades and specifications for recording tension are also available.
The electrical stimulus can either be constant current or constant voltage and can be used in:
- Field Stimulation - The response evoked by electromagnetic field generating membrane depolarization and Ca2+ influx resulting in contraction and/or relaxation
- Point Stimulation - The response evoked by local charge stimulus spreading from one excitable cell to another through the sarcolemma or adjacent cell membranes
For experiments where stimulation is required, the PowerLab output can be used to provide the stimulation or a trigger signal that controls the frequency and pulse duration of an external stimulator. ADInstruments also supplies a variety of External Stimulators for use with isolated tissue studies that supply voltage and/or current stimulation.
A range of Stimulating Electrodes for various types of tissues and experimental studies are also available for use in organ baths larger than 10 ml. They include:
- Single or double ring stainless steel or platinum electrodes for tissue strips
- Straight or zig-zag electrodes for tissue strips and spiral samples
- Coil and flat electrodes for use with very small tissue
- L-shaped electrodes for vascular (i.e. blood vessel) ring preparations
PowerLab can also control perfusion or syringe pumps or auto-injectors via eight TTL compatible digital outputs.
In some isolated tissue-organ bath studies, a myograph is used. A myograph is a device used for determining and recording the intensity of muscular contraction (force) or velocity generated at the different stages of a contracting muscle. Typical myography techniques include:
- Wire Myography- an in vitro technique that examines the functional responses and vascular reactivity of isolated small vessels and other tubular tissues with internal diameters of 60 - 400 µm.
- Muscle Strip Myography- for measuring the myo-mechanical properties of strips of muscle including skeletal and cardiac muscle and larger pieces of smooth muscle.
Refer to the Wire Myography application page for more information.
Related Areas of Interest
- Radnoti Tissue/Organ Bath Principles Technique Note: Information about various techniques and principles related to isolated tissue/organ research applications. Download
- Glassware Maintenance and Post-Experimental Cleaning Technique Note: Information about maintaining and cleaning glassware and other components manufactured by Radnoti Technology Inc. Download
- MLT0015 Isotonic Transducer Calibration and Operation Technique Note: Information about adjusting preload, measuring units of force and measuring units of displacement. Download
The LabChart Advantage:
(may require additional Modules and Extensions)
- Units Conversion for easy calibration into appropriate units such as grams or milli Newtons
- Comments can be added in real time, viewed or moved at a later stage
- The Dose Response Module allows for the calculation of dose response type data from LabChart recordings, automatically or manually, offline or online.
- The Peak Analysis Module provides automatic detection and analysis of multiple, but not overlapping, signal waveforms from recordings, online or offline.
- The DMT Normalization Module provides an easy method for the calculation of optimal pretension conditions for microvascular or any small tubular tissue research.
- Fast data extraction, analysis and export to other applications using Timed Add to Data Pad and Multiple Add to Data Pad (Note: export as .csv or .txt into GraphPad Prism®)
- The LabChart GLP Client and GLP Server software offers a comprehensive audit trail, secure signing and a centralized user authorization system required for GLP and 21 CFR Part 11 (FDA) compliant environments.
LabChart software (for Windows and Macintosh) together with a PowerLab data acquisition system offers up to 32 channels of real-time data acquisition, data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Additional acquisition and analysis functionality is provided with the use of specialized LabChart Extensions and LabChart Modules. Modules are available as part of LabChart Pro while Extensions are free for download from the website for existing LabChart users.
For most experiments the degree of muscle contraction can be logged into the LabChart's Data Pad. The results in the Data Pad can be exported as a .csv or .txt file to GraphPad Prism® (Windows and Macintosh) for further analysis and to prepare dose response graphs (including Schild plots).
Dose Response Module
The Dose Response Module (Windows) provides easy analysis of dose response type data (response to stimulation by chemical, electrical or physical agonists) recorded in LabChart from various studies including:
- Muscle contraction
- Enzyme activity
- Hormone secretion
- Heart rate
- Blood pressure
- Membrane potential
This module provides:
- Real-time or offline analysis
- Automated or manual modes of analysis
- Fast analysis of raw data to dose response parameters
- Fast comment detection and conversion to dose response markers
- Easy options for calculating different response parameters
- Instant single or multiple dose response curves (Hill-curves) generation
- Instant calculation of EC50 and Hill slopes
- Export options to other software applications for further analysis
Peak Analysis Module
The Peak Analysis Module (Windows) provides automatic detection and analysis of multiple, but not overlapping, signal peaks in acquired waveforms.
- Real-time or offline analysis
- Fast parameter extraction and analysis from single or multiple peaks
- Automatic peak detection of an entire channel or a selection within a channel
- Detection of positive or negative peaks
- Provides several analysis presets suitable for general and specific types of waveforms
- Evoked Response, Population Spike, Synaptic, Action Potential and Cardiac Action Potential
- Instant tabulation of extracted parameters that can be exported to other applications
- Height, width, area of the peak and time to peak
DMT Normalization Module
The DMT Normalization Module (Windows and Macintosh) is designed for use with PowerLab data acquisition units, LabChart software and DMT Wire Myographs, and calculates the optimal pretension conditions for microvascular or any small tubular tissue research.
- Real-time and offline data recording and analysis including;
- Stimulation amplitude
- Easy method of determining optimal microvascular pretension when using DMT myographs
GLP and 21 CFR Part 11
For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.
PowerLab Data Acquisition Systems
The PowerLab is a high-performance data acquisition unit capable of recording at speeds of up to 400,000 samples per second continuously to disk (aggregate). PowerLab units are compatible with instruments, signal conditioners and transducers supplied by ADInstruments, as well as many other third-party companies. In addition to standard single-ended BNC inputs, 4 differential Pod ports are also available for direct connection of Pod signal conditioners and appropriate transducers. Research PowerLab units include:
- PL3504 PowerLab 4/35 - 4 Channels
- PL3508 PowerLab 8/35 – 8 Channels
- PL3516 PowerLab 16/35 – 16 Channels
Compact "All-in-One" Organ Bath Systems
- PL3508B5/C-V Panlab 4 Chamber Organ Bath System or
- PL3508B6/C-V Panlab 8 Chamber Organ Bath System or
- PL3516B7/C-V Panlab 16 Chamber Organ Bath System
- Systems include:
- Tissue chambers available in 5, 10, 25 or 50 mL
- PowerLab Data Acquisition Units (with LabChart software)
- MLS260/7 LabChart Pro software
- Bridge Amp(s)
- Isometric force transducers
- Thermostat Controller
Modular Organ Bath Systems
- PL3508B60/C-V Radnoti 4 Chamber Tissue-Organ Bath System or
- PL3508B61/C-V Radnoti 8 Chamber Tissue-Organ Bath System or
- PL3516B62/C-V Radnoti 16 Chamber Tissue-Organ Bath System
- Systems include:
- PL3508B21 Single Wire Myograph System - Single Wire Myograph (maximum chamber volume = 10 ml) suitable for isometric experimentation.
PL3508B22 Dual Wire Myograph System - Dual Wire Myograph and is ideal for investigating two isolated tissues simultaneously in isometric conditions (the 10 ml chamber can be divided to give 2 x 5 ml).
PL3508B23 Auto Dual Wire Myograph System - Auto Dual Wire Myograph which is similar to the dual chamber wire myograph; however, this system has motorized micropositioners (the 10 ml chamber can be divided to give 2 x 5 ml).
PL3508B24/TS Multi Chamber Myograph w/Touch Screen System – Multi Chamber Myograph with four chambers (maximum chamber volume = 8 ml) that allow the user to conduct up to 4 separate experiments. It is ideal for work requiring high throughput such as drug screening. This system also includes Tissue Pins as well as the jaws allowing larger tissues such as aorta to be easily mounted.
PL3508B25 Confocal Wire Myograph System - Single Confocal Wire Myograph that is specifically designed for use with confocal microscopic techniques. Enables highly resolved images of fluorescent dyes in living tissues to be obtained with a laser-scanning confocal microscope (LSCM). The chamber has a maximum volume of 10 ml. Note: Microscope is not supplied by ADInstruments.
Organ Baths (not available as pre-configured research systems)
- ML0126/C-V Panlab Two Chamber Organ Bath – includes 2 tissue chambers, pre-heating reservoir coils, gas diffusers, tissue holders, micropositioners, a water pump, and thermostat controller. Note: force transducers are not supplied.
Muscle Strip Myograph (not available as pre-configured research systems)
- DMT820MS Muscle Strip Myograph w/Touch Screen - Multi Chamber Myograph with four chambers for recording tension in isolated skeletal or other muscle preparations, mounted as a strip (15 mm in length) between clamp supports.
- FE221 Bridge Amp: For connection of a single transducer
- FE224 Quad Bridge Amp: Connects up to 4 transducers
- FE228 Octal Bridge Amp: Connects up to 8 transducers
Transducers and Accessories
Isometric Force Transducers
For measuring small changes in force:
- MLT0202 Sensitive Isometric Force Transducer (0-25g)
- MLT0420 Force Transducer (up to 20g)
- MLT0402 Force Transducer (up to 2g)
Research-grade, suitable for use where larger forces are expected:
- MLT0201 Force Transducer (5 mg-25g)
- MLT0201/RAD Force Transducer (5 mg-25g)
- 159901A-V Force Transducer with amplifier
Wide-range, semi-isometric strain-gauge transducers:
- MLT1030/D Wide Range Force Transducer (10mg - 1kg)
- MLT1030/A Wide Range Force Transducer (10mg - 1kg)
Isotonic Force Transducers
For measuring displacement against a constant force:
- MLT0015 High Grade Isotonic Transducer
- MLT7006 Hall Effect Isotonic Transducer (Low inertia, high-performance)
- 160152-11 Universal Holder with Straight Electrode
- 160152-12 Universal Holder with Zig-Zag Electrode
- 160152-13 Universal Holder with Coil Electrode
- 160152-14 Universal Holder with L-Shaped Electrode
- 160152-15 Universal Holder with Flat Electrode
- MLA0301/ID Single Platinum Ring Stimulating Electrode
- MLA0302/ID Double Ring Platinum Stimulating Electrode
- MLA0303 Platinum Stimulating Electrode
- MLA0304 Double Ring Stimulating Electrode With Rings
- MLA0305/ID Stainless Steel, Single Ring Stimulating Electrode
- MLA0306/ID Stainless Steel, Double Ring Stimulating Electrode
- MLA0307 Stainless Steel Stimulating Electrode
- MLA0308 Stainless Steel Double Ring Stimulating Electrode
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