Slicing and Culturing Pig Hearts under Physiological Conditions
March 20th, 2020
This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.
This protocol is for slicing and culturing heart tissue sections for six days under physiological conditions which can be used for acute cardiac toxicity testing as well as testing efficacy of heart failure therapies. This culture system has the potential to become a powerful predictive human in situ model for acute cardiac toxicity testing that closes the gap between pre-clinical and clinical testing results. Demonstrating the procedures will be Qinghui Qu, a lab manager from our laboratory.
To obtain pig heart tissue slices, first add ice to the tissue bath cooling jacket of a vibratome and add Tyrode solution to the bath. Use a one liter plastic jar to collect the melted ice runoff from the tissue bath cooling jacket and transfer the pig heart to a tray containing one liter of fresh cold cardioplegic solution in a biosafety cabinet. Use retractible sterile scalpels to dissect the heart to isolate the left ventricle and use rectangular razor blades to cut the left ventricle into one to two cube centimeter blocks.
Set aside one tissue block for slicing and place the remaining tissue block pieces into a 50 milliliter tube of cold Tyrode solution on ice. Gently massage the reserved tissue block and add one to two drops of tissue glue to the metal sample holder of the vibratome. Stick a piece of 4%agar block with a surface area of approximately one square centimeter to the glue and add one to two drops of tissue glue to the agar.
Attach the heart block to the agar cardiac epicardium side down with the tissue as flat against the holder as possible and place the tissue holder with the heart block into the slicing bath of the vibratome. It is crucial to stick the heart block to the agar with the cardiac epicardium side facing down on the tissue glue and to make sure the tissue is as flat as possible. Then attach the oxygen tube to the slicing bath and the metal tray filled with Tyrode solution and place 40 micrometer cell strainers onto the metal tray to collect the heart tissue sections as they're sliced.
Use the vibratome operating software to adjust the height of the blade and sample so that the blade is close to the top of the tissue but below the papillary muscles and liquid is covering the tissue and the blade. Select advance to adjust where to begin slicing the tissue. Press slice to increase the speed and use the knob to move the blade toward the edge of the tissue.
Press slice again to stop and advance to inactivate the process. Then adjust the cutting parameters as indicated and press slice to begin slicing the tissue. When the blade reaches the end of the tissue but before it hits the end of the specimen holder, press slice again to stop and press return to go back to the start position.
When the slices reach full length and appear to be of good quality, use a plastic Pasteur pipette filled with cold Tyrode solution to gently collect the tissue from the bath using forceps and spring scissors to dislocate the slice from the heart as necessary. Place each collected slice in a single cell strainer in the oxygenated Tyrode bath and use the solution in the pipette to press the tissue on the cell strainer surface. Then place a metal washer onto the top of the tissue to hold it down.
After at least one hour in oxygenated Tyrode solution, trim the outside of each slice to remove any uneven edges and glue the end of each slice onto a six millimeter wide piece of sterilized polyurethane printer timing belt with embedded metal wires. Place the supported heart slices into six-well plates containing six milliliters of culture medium per well and use sterile forceps to make sure that the tissue is in the center of the well. Take care that the plate cover will not touch the tissue and place the plate onto the culture plate so that the curved side of the plate cover matches up with the angled side of the plate and that the square corners line up.
Place the slices into a 37 degree Celsius and 5%carbon dioxide incubator. Replace the culture supernatants with six milliliters of fresh oxygenated culture medium three times per day. Connect the cover to the cell culture electrical stimulator and adjust the stimulator to continuously deliver 10 volts of electricity and a 1.2 hertz frequency to the heart slices.
After plugging in the stimulation, the heart slices will begin to beat. Everyday during the midday media change, replace the stimulation plate cover from the culture dish to prevent the release of toxic graphite particles into the culture medium. Insert the white foam plug where the used cover connects to the cable for the cell culture electrical stimulator to prevent water damage to the electric circuit and place the plate cover into a bath of autoclaved water supplemented with 2X antibiotic/antimycotic.
The next day, decontaminate the plate cover in a 70%ethanol bath for five to 15 minutes before transferring the cover into a fresh autoclaved water bath supplemented with 2X antibiotic/antimycotic for three minutes. After rinsing the plate cover, use an ethanol-sprayed clean lint-free wipe to remove any residual water from the plastic parts and remove the white foam plug. The cover is then ready to be placed back into the culture plate.
Using this new biomimetic culture setup as demonstrated, pig heart slices viability was maintained for six days as assessed by MTT assay. In the first six days, similar to the responses observed for fresh heart slices, there are no spontaneous calcium transients within the pig heart slice cardiomyocytes. The cardiomyocytes did respond to external electrical and beta-adrenergic stimulation.
In addition, the contractile force and responses to isoproterenol were maintained in heart slice culture for up to six days similar to that observed in fresh heart slices. Several structural and functional assessments can be performed on the heart slices including MTT viability assays, immunostaining, electron microscopy, calcium transient measurements, contractile function assessments, metabolic assessments, and analysis. We are currently using this culture system to test the acute cardiac toxicity and efficacy of drugs and gene therapies of interest in pig and human heart slices.
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Overview
This protocol describes the slicing and culturing of heart tissue sections for six days under physiological conditions. This system can be utilized for testing the efficacy of heart failure therapies and assessing acute cardiac toxicity.
Key Study Components
Area of Science
- Cardiac research
- Tissue culture
- Pharmacology
Background
- Heart failure is a significant health issue requiring effective therapies.
- Acute cardiac toxicity testing is essential for drug development.
- Current models may not accurately predict human responses.
- This protocol aims to bridge the gap between pre-clinical and clinical testing.
Purpose of Study
- To provide a reliable method for heart tissue culture.
- To evaluate the efficacy of novel heart failure therapeutics.
- To establish a predictive model for acute cardiac toxicity testing.
Methods Used
- Use of a vibratome for slicing heart tissue.
- Preparation of cardioplegic solution for tissue preservation.
- Isolation of the left ventricle for culturing.
- Culture maintenance under physiological conditions for six days.
Main Results
- Successful culture of heart tissue sections for six days.
- Potential for testing heart failure therapies demonstrated.
- Acute cardiotoxicity testing model established.
- Protocol provides a reproducible method for future studies.
Conclusions
- The developed culture system is a valuable tool for cardiac research.
- It enhances the predictive accuracy of drug testing.
- This method could lead to improved therapeutic strategies for heart failure.
Frequently Asked Questions
Executive Industry Relevance
Current in vitro and in vivo models poorly predict human cardiac liabilities, leading to costly late-stage failures in drug development. This protocol provides a biomimetic heart slice culture system that preserves viability and function for six days under physiological conditions, enabling more accurate assessment of acute cardiotoxicity and therapeutic efficacy. By using multicellular adult heart tissue from pig (and translatable to human) sources, the model addresses a critical gap in preclinical safety testing and supports better go/no-go decisions in cardiovascular drug discovery.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses in multicellular adult heart tissue that reflects human myocardium physiology.
- Operational Value: Supports functional target validation by measuring contractile force and responses to beta-adrenergic stimulation over six days in culture.
Screening & Assay Development
- Scientific Value: Provides a standardized, reproducible platform for preparing validated heart slices for downstream compound evaluation.
- Operational Value: Supports assay readiness through consistent tissue slicing, mounting, and culture under electrical stimulation and oxygenation.
Translational & Preclinical Research
- Scientific Value: Uses adult cardiomyocytes with physiological properties distinct from immature hiPSC-CMs, improving translational relevance.
- Operational Value: Enables continuity from discovery through preclinical validation by maintaining structural and functional biomarkers for up to six days.
Pipeline & Workflow Integration
The method fits within the discovery continuum from early target validation to preclinical assessment, supporting hypothesis testing and lead identification through functional readouts.
- Discovery Biology: Supports pathway clarification and biological de-risking by enabling testing in intact multicellular tissue under near-physiological conditions.
- Screening: Delivers assay readiness and reproducibility via standardized slicing, mounting, and culture procedures with electrical pacing.
- Analytics: Provides quantitative outputs including MTT viability, contractile force, calcium transient measurements, and metabolic assessments for comparative condition analysis.
- Translational Research: Connects to preclinical continuity through preservation of adult cardiomyocyte function and structural integrity over six days.
- Enterprise Reuse: Establishes a reusable platform applicable to pig, human, dog, sheep, or cat heart slices for cardiotoxicity and efficacy testing across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by modeling human-like multicellular heart tissue responses to drugs and gene therapies.
- Operational Value: Ensures reproducibility through standardized vibratome slicing, tissue mounting, and culture under electrical stimulation (1.2 Hz, 10 V) and oxygenation.
- Strategic Value: Reduces late-stage biological risk by improving early detection of cardiotoxicity, supporting capital-efficient advancement decisions.
- Portfolio Impact: Enables risk-adjusted prioritization of compounds based on functional heart slice responses, improving go/no-go criteria in cardiovascular portfolios.
Implementation Considerations
- Requires expertise in tissue handling, vibratome operation, and sterile technique for heart dissection and slicing.
- Depends on specialized equipment including vibratome, cell culture electrical stimulator, oxygenation setup, and incubators with 5% CO2 at 37°C.
- Necessitates cross-team standardization of tissue preparation, mounting on polyurethane belts, and media change protocols to ensure slice viability.
- Involves adaptation considerations for different species (pig, human, dog, sheep, cat) and tissue block sizing (1–2 cm³) to maintain slicing quality.
- Includes practical limitations such as the need for daily media changes, stimulation plate maintenance, and avoidance of graphite particle contamination during electrical stimulation.
