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 h...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li><li>Kang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Organotypic+Cultured+Cardiac+Slices:+New+Platform+For+High+Throughput+Preclinical+Human+Trials.">Human Organotypic Cultured Cardiac Slices: New Platform For High Throughput Preclinical Human Trials.</a> Scientific Reports. 6, 28798 (2016).</li><li>Ou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Biomimetic+Culture+System+for+Pig+and+Human+Heart+Slices.">Physiological Biomimetic Culture System for Pig and Human Heart Slices.</a> Circulation Research. 125 (6), 628-642 (2019).</li><li>Jones, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NHLBI-sponsored+Consortium+for+preclinicAl+assESsment+of+cARdioprotective+therapies+(CAESAR):+a+new+paradigm+for+rigorous,+accurate,+and+reproducible+evaluation+of+putative+infarct-sparing+interventions+in+mice,+rabbits,+and+pigs.">The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.</a> Circulation Research. 116 (4), 572-586 (2015).</li><li>Crick, S. J., Sheppard, M. N., Ho, S. Y., Gebstein, L., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+pig+heart:+comparisons+with+normal+human+cardiac+structure.">Anatomy of the pig heart: comparisons with normal human cardiac structure.</a> Journal of Anatomy. 193 (Pt 1), 105-119 (1998).</li><li>Fischer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 117 (2019).</li><li>Franke, J., Abs, V., Zizzadoro, C., Abraham, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+the+effects+of+fetal+bovine+serum+versus+horse+serum+on+growth+and+differentiation+of+primary+equine+bronchial+fibroblasts.">Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts.</a> BMC Veterinary Research. 10, 119 (2014).</li><li>Vuorenpaa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+in+vitro+cardiovascular+constructs+composed+of+vascular-like+networks+and+cardiomyocytes.">Novel in vitro cardiovascular constructs composed of vascular-like networks and cardiomyocytes.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 50 (4), 275-286 (2014).</li><li>Qiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+slice+culture+platform+for+the+investigation+of+human+cardiac+tissue+physiology.">Multiparametric slice culture platform for the investigation of human cardiac tissue physiology.</a> Progress in Biophysics and Molecular Biology. 144, 139-150 (2018).</li><li>Watson, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 2168 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000ml, 0.22&#181;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>500ml, 0.22&#181;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 (CaCl2)</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><a href="https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/" target="_blank">https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/</a></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 (MgCl2)</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&#8482; 07.164.17</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199, Earle&#39;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><a href="https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/" target="_blank">https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/</a></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&#38;D Systems</td>
			<td>233-FB-025/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human VEGF</td>
			<td>R&#38;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 (NaHCO3)</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...

Watch this Scientific Journal Video about Slicing and Culturing Pig Hearts under Physiological Conditions at JoVE.com

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 ...

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.

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

Research

JoVE Journal

Medicine

13.9K 조회수.  University of Louisville. 이 프로토콜은 급성 심장 독성 검사뿐만 아니라 심부전 치료의 효능을 테스트하는 데 사용할 수있는 생리적 조건하에서 6 일 동안 심장 조직 섹션을 잘라내고 배분하기위한 것입니다. 이 배양 시스템은 임상 전 및 임상 테스트 결과 사이의 격차를 좁히는 급성 심장 독성 테스트를 위한 시투 모델에서 강력한 예측 인간이 될 가능성이 있습니다. 절차를 시연하는 것은 우리의 ?...