TMAM is supported by NIH grant P30GM127607 and American Heart Association grant 16SDG29950012. RB is supported by P01HL78825 and UM1HL113530.

All animal procedures were in accordance with the institutional guidelines of the University of Louisville and approved by the Institutional Animal Care and Use Committee.
1. Preparation for Slicing (One Day Before Slicing)
<ol>
	<li>Preparation of the vibrating microtome
	<ol>
		<li>Place the ceramic blade into its holder by following these steps: after carefully unwrapping the blade, place the sharp edge first into the slot of the blade tool. Then, fit the blade into the holder by loosening the two screws on the arms of the holder and slide the blade under each washer and push it firmly back against the rear stops. Tighten the screws to secure the blade. 
		NOTE: The screws should not be overtightened.</li>
		<li>Calibrate the blade using the calibration protocol and tools according to the manufacturer&#39;s protocol. 
		NOTE: Briefly, in order to facilitate alignment of the blade with the axis of travel and to minimize the Z axis deflections, the vibrating microtome uses a demountable calibration device. When the calibration device is plugged into the instrument its presence will automatically be detected, and the vibrating microtome will take control of adjusting the amplitude and frequency settings of the blade to a magnitude which best allows the adjustment of the blade alignment error.
		<ol>
			<li>Press the Slice button to start the alignment process which will automatically move the blade so that the cutting edge is in optimal position relative to the calibration device for best alignment evaluation. Follow the on-screen instructions to make adjustments to the blade using the provided screwdriver to ensure that the Z-alignment is within 1 &#181;m.</li>
		</ol>
		</li>
		<li>Autoclave the inner bath and all the metal parts of the vibrating microtome.</li>
	</ol>
	</li>
	<li>Autoclave the large metal trays (for collecting the slices and soaking the electrical stimulation plate covers), any glass containers, regular rectangle blades, forceps, scissors, printer timing belt (cut them into 6 mm wide pieces), metal washers and 5 L of double distilled water (ddH2O).</li>
	<li>Sterilize one 1 L jar, one 500 mL jar and one plastic tray for the heart in situ and in vitro perfusion with the cardioplegic solution.</li>
	<li>Prepare 2 L of the slicing Tyrode&#39;s solution in a 2 L beaker.
	<ol>
		<li>For 1 L of the slicing Tyrode&#39;s solution, mix 3 g/L 2,3-butanedione monoxime (BDM), 140 mM NaCl (8.18 g), 6 mM KCl (0.447 g), 10 mM D-glucose (1.86 g), 10 mM HEPES (2.38 g), 1 mM MgCl2 (1 mL of 1 M solution), 1.8 mM CaCl2 (1.8 mL of 1 M solution), up to 1 L of ddH2O. Adjust the pH to 7.40 using NaOH. Then, filter the solution using a 1,000 mL, 0.22 &#181;m, vacuum filter/storage system and store at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Prepare 4 L of the cardioplegic solution.
	<ol>
		<li>For 1 L of the cardioplegic solution, mix 110 mM NaCl (6.43 g), 1.2 mM CaCl2 (1.2 mL of 1 M solution), 16 mM KCl (1.19 g), 16 mM MgCl2 (3.25 g), 10 mM NaHCO3 (0.84 g), 1 U/mL heparin, up to 1 L of ddH2O. Adjust the pH to 7.40 using NaOH. Then, filter the solution using a 1,000 mL, 0.22 &#181;m, vacuum filter/storage system and store at 4 &#176;C overnight. 
		NOTE: Add 1,000 U/L heparin in cardioplegia solution the day of slicing (see step 2.1).</li>
	</ol>
	</li>
	<li>Freshly prepare 500 mL of culture medium in the original bottle in a biosafety cabinet: mix medium 199, 1x insulin-transferrin-selenium (ITS) supplement, 10% fetal bovine serum (FBS), 5 ng/mL vascular endothelial growth factor (VEGF), 10 ng/mL FGF-basic, and 2x antibiotic-antimycotic. Filter sterilize through the 0.22 &#181;m, vacuum filter/storage system and store at 4 &#176;C overnight. 
	NOTE: Do not adjust pH, add VEGF and FGF freshly before use.</li>
	<li>Prepare 4% agar gel plates.
	<ol>
		<li>Add 200 mL of sterilized ddH2O in a 500 mL sterilized flask. Weigh 8 g of agarose, and gently pour into the flask. Boil for 2 min in a microwave, then cool for 5 min at room temperature.</li>
		<li>Pour 25 mL in each 100 mm Petri dish in a biosafety cabinet (prepare 6 dishes) and wait for 1 h to solidify. Then, seal the plates with paraffin film, wrap with clean wrap, and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 4 L of 70% ethanol by diluting the 200-proof absolute ethanol in autoclaved ddH2O.</li>
	<li>Prepare 2x antibiotic-antimycotic in sterilized ddH2O: mix 980 mL of sterilized ddH2O with 20 mL of antibiotic-antimycotic under the biosafety cabinet.</li>
</ol>
2. Pig Heart Perfusion
<ol>
	<li>Add 1 mL of 1,000 U/mL heparin to each 1 L of cardioplegic solution. Keep the solution on ice. 
	NOTE: At least 3 L are needed for the perfusion and preservation of the heart during the transfer to the tissue culture room.</li>
	<li>Take the following items to the pig surgery room: one large ice bucket full of ice, one sterilized metal tray (keep on ice bucket for heart perfusion), one sterilized 500 mL jar, one 1 L jar (to transfer the heart in cardioplegia solution back to the tissue culture room, on ice), and one sterilized plastic tray full of ice (to keep cardioplegic solution on ice).</li>
	<li>Prepare an in-situ heart perfusion system containing a 3-way stopcock, a 60 mL syringe, an 18 G butterfly needle catheter, and extension tubing to the cardioplegic solution reservoir.</li>
	<li>Anesthetize the Yorkshire male pig (2&#8722;3 month-old, 20&#8722;25 kg) first with an intramuscular injection of a ketamine (20 mg/kg)/xylazine (2 mg/kg) cocktail. Confirm proper anesthesia by the loss of jaw tension. Then, transfer the pig to the surgery room, intubate and mechanically ventilate the lungs as previously described14.</li>
	<li>Maintain anesthesia with (1.5&#8722;2%) isoflurane.</li>
	<li>Place the animal on the right lateral position and shave the fur from the chest area. Clean the skin with alcohol scrub, then sterilize the surgical site with 10% (w/v) povidone-iodine solution.</li>
	<li>Open the chest by left thoracotomy incision using a scalpel at the 4th intercostal space, and use a chest retractor between ribs to hold the chest open and expose the heart. 
	NOTE: Use all sterilized instruments for this procedure.</li>
	<li>Insert the butterfly needle catheter into the left atrium and fix the needle with a purse string closure. After intravenous injection of a bolus dose of heparin (100 U/kg), start to perfuse the heart in situ with 500 mL of ice-cold cardioplegic solution (to slow down the heart rate) via the left atrial catheter.</li>
	<li>Deeply anesthetize the pig with 5% isoflurane. Quickly excise the heart out of the chest with surgical scissors. Cannulate the aorta with an aortic cannula and perfuse the heart in vitro with another 1 L of ice-cold cardioplegic solution (100 mL/min) to flush out vasculature blood.</li>
	<li>Following heart perfusion, keep the heart in a 1 L jar filled with ice-cold cardioplegia solution and keep on ice during the transfer to the tissue culture room.</li>
</ol>
3. Pig Heart Tissue Slicing
<ol>
	<li>Set up the tissue bath on the vibratome, add ice to the tissue bath cooling jacket, then add Tyrode&#39;s solution into the tissue bath. Set up 1 L plastic jar to collect the disposal of melted ice from the tissue bath cooling jacket.</li>
	<li>Under the biosafety cabinet, transfer the pig heart to a tray containing 1 L of fresh cold cardioplegic solution.</li>
	<li>Dissect the heart to isolate the left ventricle using retractable sterile scalpels. Then, cut the left ventricle into blocks of 1&#8722;2 cm3 each using razor rectangle blades. Use one piece for slicing and keep the remaining pieces in a 50 mL tube in cold Tyrode&#39;s solution on ice for cutting later, if needed. 
	NOTE: Massage the tissue before slicing with hands, especially if this is the second block. Massaging helps to restore the correct muscle fiber configuration and prevents any stiffness within the heart block.</li>
	<li>Add 1&#8722;2 drops of tissue glue (Table of Materials) to the metal sample holder and stick a piece of the 4% agar block with a surface area of approximately 1 cm2.</li>
	<li>Add 1&#8722;2 drops of tissue glue on the agar.</li>
	<li>Stick the heart block to the agar with the cardiac epicardium side facing down on the tissue glue and make sure it is as flat as possible. Then, transfer the tissue holder with the heart block to its position in the slicing bath of the vibratome.</li>
	<li>Attach the oxygen tube to the slicing bath and the metal tray filled with Tyrode&#39;s solution for collecting the slices (oxygenated Tyrode&#39;s bath). Then, add 40 &#181;m cell strainers in the metal tray to collect the slices after cutting.</li>
	<li>Adjusting the slicing position
	<ol>
		<li>Using the vibratome operating software and the dashboard, adjust the height of the blade/sample. Select the height button and follow the on-screen instructions for adjusting the height. Ideally, have the blade close to the top of the tissue but below the papillary muscles and make sure there is liquid covering the tissue and the blade before slicing.</li>
		<li>Adjust where to begin slicing the tissue by selecting the advance button. Press the slice button and increase the speed using the knob to move the blade towards the edge of the tissue. Then, press slice again to stop, and press the advance button to inactivate the process.</li>
		<li>Adjust the cutting parameters: advance speed = 0.03 mm/s, vibration frequency = 80 Hz, and horizontal vibration amplitude = 2 mm. Then, start slicing by selecting the slice button.</li>
		<li>Let the vibratome slice until it reaches the end of the tissue but before it hits the back end of the specimen holder. At this point, press slice again to stop. Hit the Return button to go back to the start position.</li>
		<li>Select auto repeat (clicking it twice) which will allow the vibrating microtome to auto repeat the slicing process for up to 99 times.</li>
	</ol>
	</li>
	<li>Collecting slices
	<ol>
		<li>Wait until slices are full length and appear good (after getting past the papillary muscle layers), then start collecting the slices.</li>
		<li>Collect the slices using a plastic Pasteur pipette filled with cold Tyrode&#39;s solution to gently grab the tissue from the bath. If necessary, use forceps and spring scissors to dissociate the slice from the heart block if the tissue is still attached.</li>
		<li>Transfer the slice to one cell strainer in the oxygenated Tyrode&#39;s bath (see step 3.7) and use the liquid in the plastic Pasteur pipette to get the tissue to lie flat on one of the cell strainers, then add a metal washer on the top to hold the tissue down. 
		NOTE: The slices need to be incubated at least for 1 h in the Tyrode&#39;s solution before processing (this is for the BDM to relax the tissue).</li>
	</ol>
	</li>
</ol>
4. Culturing Heart Slices
<ol>
	<li>Preparing the slices for culture
	<ol>
		<li>Trim the slice from the edges to avoid any uneven edges. Then, glue it from both ends to sterilized polyurethane 6 mm wide printer timing belt with metal wires embedded using tissue glue.</li>
		<li>Transfer the supported heart slices to 6 well plates that contain 6 mL of culture medium in each well.</li>
		<li>Place the stimulation plate cover (Table of Materials) on the top of the 6 well plate and connect the cover to the cell culture electrical stimulator (Table of Materials). Adjust the stimulator to electrically stimulate the heart slices at 10 V, 1.2 Hz continuously all the time. 
		NOTE: After plugging in the stimulation, the heart slices will start to beat, and this movement will be visually obvious.</li>
		<li>Transfer the plates to an incubator and maintain at 37 &#176;C with humidified air and 5% CO2.</li>
	</ol>
	</li>
	<li>Change culture medium 3x per day and add 6 mL of culture medium per well during each media change. 
	NOTE: Culture medium is oxygenated for 5 min prior to each media change. Medium must be changed at least 1x per day; this is okay on weekends if necessary. Two days of only 1 media change per day is the maximum that is tested.</li>
	<li>Change the stimulation plate cover every day to prevent release of toxic graphite particles in the culture medium (usually during the mid-day media change).
	<ol>
		<li>Remove the stimulation plate cover from the culture dish and insert the white foam plug where the cover connects to the cable for the cell culture electrical stimulator, to prevent water damage to the electric circuit.</li>
		<li>Place the plate cover in a bath of autoclaved water with 2x antibiotic-antimycotic. Keep it in this bath (i.e., rinsing bath) overnight.</li>
		<li>The next day, move the plate cover into a 70% ethanol bath for 5&#8722;15 min to decontaminate. Shake off as much liquid from the device before switching baths.</li>
		<li>Then, transfer the plate cover to the third and final bath (i.e., clean bath), which consists of autoclaved water and 2x antibacterial-antimycotic, to rinse any remaining ethanol traces.</li>
		<li>After rinsing the plate cover in the clean bath, use a clean lint-free wipe (sprayed down with ethanol) to dry off any residual water on the plastic parts, then remove the white foam piece and carefully place the plate cover back on the culture plate. 
		NOTE: Do not touch the black graphite electrodes that go into the well, as the device will no longer be sterile.</li>
		<li>Using sterile forceps, make sure the tissue is in the center of the well so the plate cover does not touch the tissue. Ensure that the curved side of the plate cover matches up with the angled side of the plate and that the square corners line up. 
		NOTE: To minimize contamination of the stimulation plate covers, keep them in clean and empty 6 well plates after finishing the experiment.</li>
	</ol>
	</li>
	<li>Perform an MTT assay, calcium measurements and contractile function assessments on fresh pig heart slices (day 0), and after 6 days in culture according to Ou et al.13.</li>
</ol>

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TMAM holds equity in Tenaya Therapeutics. The other authors report no conflicts.

Here we describe the detailed video protocol for our recently published method for simplified medium throughput (processes up to 48 slices/device) method that enables culture of pig heart slices for a period sufficiently long to test acute cardiotoxicity13. The proposed conditions mimic the environment of the heart, including frequency of electrical stimulation, nutrient availability, and intermittent oxygenation. We attribute the prolonged viability of heart slices in our biomimetic stimulated cu...

Drug induced cardiotoxicity is a major cause of market withdrawal1. In the last decade of the 20th century, eight non-cardiovascular drugs were withdrawn from the market as they resulted in sudden death due to ventricular arrhythmias2. In addition, several anti-cancer therapies (while in many cases effective) can lead to several cardiotoxic effects including cardiomyopathy and arrhythmias. For example, both traditional (e.g., anthracyclines and radiation) and targeted (e.g., trastuzumab) breast cancer therapies can result in cardiovascular complications in a subset of patients3. A close collaboration between cardiologists and oncologists (via the emerging field of &#34;cardio-oncology&#34;) has helped make these complications manageable ensuring that patients can be treated effectively2. Less clear are the cardiovascular effects of newer agents, including Her2 and PI3K inhibitors, especially when therapies are used in combination. Therefore, there is a growing need for reliable preclinical screening strategies for cardiovascular toxicities associated with emerging anti-cancer therapies prior to human clinical trials. The lack of availability of culture systems for human heart tissue that is functionally and structurally viable for more than 24 h is a limiting factor for reliable cardiotoxicity testing. Therefore, there is an urgent need to develop a reliable system for culturing human heart tissue under physiologic conditions for testing drug toxicity.
The recent move towards the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in cardiotoxicity testing has provided a partial solution to address this issue; however, the immature nature of the hiPSC-CMs and the loss of tissue integrity compared to multicellular nature of the heart tissue are major limitations of this technology4. A recent study has partially overcome this limitation through fabrication of cardiac tissues from hiPSC-CMs on hydrogels and subjecting them to gradual increase in electrical stimulation over time5. However, their electromechanical properties did not achieve the maturity seen in the adult human myocardium. Moreover, the heart tissue is structurally more complicated, being composed of various cell types including endothelial cells, neurons and various types of stromal fibroblasts linked together with a very specific mixture of extracellular matrix proteins6. This heterogeneity of the non-cardiomyocyte cell population7,8,9 in the adult mammalian heart is a major obstacle in modeling heart tissue using individual cell types. These major limitations highlight the importance of developing methods to enable culture of intact cardiac tissue for optimal studies involving physiological and pathological conditions of the heart5.
Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete 3D multicellular system that is similar to the human heart tissue which could reliably reflect the physiological or pathological conditions of the human myocardium. However, its use has been severely limited by the short period of viability in culture, which does not extend beyond 24 h using the most robust protocols reported until 201810,11,12. This limitation was due to multiple factors including the use of air-liquid interface to culture the slices, and the use of a simple culture medium that does not support the high energetic demands of the cardiac tissue. We have recently developed a submerged culture system which is able to provide continuous electrical stimulation and optimized the culture media components to keep cardiac tissue slices viable for up to 6 days13. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing to close the gap between preclinical and clinical testing results. In the current article, we are detailing the protocol for slicing and culturing the heart slices using a pig heart as an example. The same process is applied to human, dog, sheep, or cat hearts. With this protocol, we are hoping to spread the technology to other laboratories in the scientific community.

<table><tbody><tr><td>1,000 mL, 0.22 &amp;micro;m, Vacuum Filter/Storage Systems</td><td>VWR</td><td>28199-812</td><td></td></tr><tr><td>2,3-Butanedione monoxime (BDM)</td><td>Fisher</td><td>AC150375000</td><td></td></tr><tr><td>500 mL, 0.22 &amp;micro;m, Vacuum Filter/Storage Systems</td><td>VWR</td><td>28199-788</td><td></td></tr><tr><td>6-well C-Dish Cover (electrical-stimulation-plate-cover)</td><td>Ion Optix</td><td>CLD6WFC</td><td></td></tr><tr><td>6-well plates</td><td>Fisher</td><td>08-772-1B</td><td></td></tr><tr><td>Agarose</td><td>Bioline USA</td><td>BIO-41025</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Thermo</td><td>15-240-062</td><td></td></tr><tr><td>C-Pace EM (cell-culture-electrical-stimulator)</td><td>Ion Optix</td><td>CEP100</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher</td><td>C79-500</td><td></td></tr><tr><td>Ceramic Blades for Vibrating Microtome</td><td>Campden Instruments</td><td>7550-1-C</td><td></td></tr><tr><td>Cooley Chest Retractor</td><td>Millennium Surgical</td><td>63-G5623</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher</td><td>D16-1</td><td></td></tr><tr><td>Disposable Scalpel #20</td><td>Biologyproducts.com</td><td>DS20X</td><td></td></tr><tr><td>Falcon Cell Strainers, Sterile, Corning</td><td>VWR</td><td>21008-952</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo</td><td>A3160502</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fisher</td><td>NC9475675</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>H3149-50KU</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-1</td><td></td></tr><tr><td>Histoacryl BLUE Tissue glue</td><td>Amazon</td><td></td><td>&lt;a href=&quot;https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/&quot; target=&quot;_blank&quot;&gt;https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/&lt;/a&gt;</td></tr><tr><td>Iris Spring scissors</td><td>Fisher</td><td>NC9019530</td><td></td></tr><tr><td>Iris Straight Scissors</td><td>Fisher</td><td>731210</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Piramal</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>ITS Liquid Media Supplement</td><td>Sigma-Aldrich</td><td>I3146-5ML</td><td></td></tr><tr><td>Ketamine HCl (500 mg/10 mL)</td><td>West-Ward</td><td>NDC 0143-9508</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher</td><td>M33-500</td><td></td></tr><tr><td>Mayo SuperCut Surgical Scissors</td><td>AROSurgical Instruments Corporation</td><td>AROSuperCut&amp;trade; 07.164.17</td><td></td></tr><tr><td>Medium 199, Earle&#039;s Salts</td><td>Thermo</td><td>11-150-059</td><td></td></tr><tr><td>Oxygen regulator</td><td>Praxair</td><td></td><td></td></tr><tr><td>Oxygen tanks -</td><td>Praxair</td><td></td><td></td></tr><tr><td>Plastic Pasteur pipettes</td><td>Fisher</td><td>13-711-48</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher</td><td>AC193780010</td><td></td></tr><tr><td>Printer Timing Belt</td><td>Amazon</td><td></td><td>&lt;a href=&quot;https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/&quot; target=&quot;_blank&quot;&gt;https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/&lt;/a&gt;</td></tr><tr><td>Razor rectangle blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>Recombinant Human FGF basic</td><td>R&amp;amp;D Systems</td><td>233-FB-025/CF</td><td></td></tr><tr><td>Recombinant Human VEGF</td><td>R&amp;amp;D Systems</td><td>293-VE-010/CF</td><td></td></tr><tr><td>Retractable scalpels</td><td>Fisher</td><td>22-079-716</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher</td><td>AC217125000</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>AC327300010</td><td></td></tr><tr><td>Vibrating Microtome</td><td>Campden Instruments</td><td>7000 SMZ-2</td><td></td></tr><tr><td>Xylazine HCl (100 mg/mL)</td><td>Heartland Veterinary Supply</td><td>NADA 139-236</td><td></td></tr></tbody></table>

slicing and culturing pig hearts under physiological conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

Using a commercially available cell culture electrical stimulator that can accommodate eight 6 well plates at once, we emulated the adult cardiac milieu by inducing electrical stimulation at the physiological frequency (1.2 Hz), and screened for the fundamental medium components to prolong the duration of functional pig heart slices in culture13. Since pig and human hearts are similar in size and anatomy15, we developed a biomimetic heart sl...

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Slicing and Culturing Pig Hearts under Physiological Conditions

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.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

slicing-and-culturing-pig-hearts-under-physiological-conditions

Research

JoVE Journal

Medicine

15.2K Views.  University of Louisville. Dit protocol is voor het snijden en kweken van hartweefsel secties gedurende zes dagen onder fysiologische omstandigheden die kunnen worden gebruikt voor acute cardiale toxiciteit testen en het testen van de werkzaamheid van hartfalen therapieën. Dit cultuursysteem heeft het potentieel om een krachtig voorspellend mens in situ-model te worden voor acute cardiale toxiciteitstests die de kloof tussen preklinische en klinische testresultaten dichten. Het aantonen van de procedures zal Qinghui Q...

Video: Slicing and Culturing Pig Hearts under Physiological Conditions

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Biology

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 &#181;m are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode&#39;s solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 &#176;C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Source: Ou, Q. et. al., <a href="https://www.jove.com/v/60913">Slicing and Culturing Pig Hearts under Physiological Conditions.</a> J. Vis. Exp. (2020)
This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

Source: Ou, Q. et. al., Slicing and Culturing Pig Hearts under Physiological Conditions. J. Vis. Exp. (2020)
This video demonstrates a vibratome-based ...

JoVE Encyclopedia of Experiments

Large Animal Models

Porcine

Porcine Heart Excision: A Protocol to Extract Porcine Heart for Cardiac Transplant

Source: Hatami, S., et al. <a href="https://www.jove.com/v/58430">Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism.</a> J. Vis. Exp. (2019).
In this video, we describe a step-by-step protocol to extract the heart from a porcine model for cardiac transplantation.

Source: Hatami, S., et al. Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism. J. Vis. Exp. (2019).
In ...

Ex Situ Heart Perfusion: A Technique to Preserve Isolated Porcine Heart Function

Source: Hatami, S. et. al., <a href="https://www.jove.com/v/58430">Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism.</a> J. Vis. Exp. (2019).
This video describes an ex situ technique for preserving an isolated porcine heart by perfusing it with an oxygenated nutrient-rich perfusate solution. The technique allows the comprehensive evaluation of cardiac performance under physiological conditions.

Source: Hatami, S. et. al., Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism. J. Vis. Exp. (2019).
This ...

Slicing and Culturing Pig Hearts under Physiological Conditions

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