Name: Preparation of Human Myocardial Tissue for Long-Term Cultivation (Video) | JoVE Journal
Uploaded: 2022-06-02T14:10:00.000+00:00
Duration: 10 min 58 s
Description: Learn to cultivate human ventricular myocardial tissue ex vivo using a biomimetic chamber with pre- and afterload application. This method enables continuous assessment of contraction parameters for drug screening in cardiovascular research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Research was funded by DZHK grants 81Z0600207 (JH, PS, and DM) and 81X2600253 (AD and TS).
The authors would like to thank Claudia Fahney, Mei-Ping Wu, and Matthias Semisch for their support in preparing the set-ups, as well as for the regular maintenance of the tissue cultivation.

Tissue collection for the experiments described here was approved by the Institutional Review Boards of the University of Munich and the Ruhr-University Bochum. Studies were conducted according to Declaration of Helsinki guidelines. Patients gave their written informed consent prior to tissue collection.
1. Tissue acquisition
<ol>
	<li>Obtain human tissue from patients undergoing heart transplantation or cardiac surgery.</li>
	<li>Before procuring the tissue, prepare 2 L of cardioplegic solution (further referred to as slicing buffer (Table 1)).</li>
	<li>After obtaining a 4 cm x 4 cm sized transmural left ventricular (LV) biopsy, place the tissue immediately (within 5 min after excision) in a closable plastic single-use sterile beaker containing approximately 70 mL of cold (4 &#176;C) slicing buffer. Keep at 4 &#176;C. 
	&#8203;NOTE: The slicing buffer used in this protocol allows for cold storage (4 &#176;C) of the tissue for up to 36 h. This permits cold transport of the tissue from clinics which are not close to the laboratory. However, transport times &#8804; 24 h have proven to be optimal.</li>
</ol>
2. Preparing agarose and the vibratome
<ol>
	<li>Make sure sufficient cultivation chambers are prepared and sterilized (Figure 1A).
	<ol>
		<li>Submerge the chambers and graphite electrodes in 1 L of a 10% isopropanol solution and agitate overnight. The following day, transfer the chambers to a 100% isopropanol solution for 3 min and autoclave the graphite electrodes at 120 &#176;C for 10 min.</li>
		<li>Let the chambers and graphite electrodes air-dry under a laminar flow hood.</li>
		<li>Attach a circuit board to each of the chambers according to the available positions on the rocker. Place two graphite electrodes to the circuit board as per manufacturer&#39;s instructions.</li>
		<li>Place a 35 mm Petri dish lid on top of the chamber to prevent contamination.</li>
	</ol>
	</li>
	<li>Set up the Myodish cultivation system (see Table of Materials) in an incubator at 37 &#176;C and 5% CO2 by connecting the system to a computer (Figure 1B).</li>
	<li>Prepare the 4% low-melt agarose in slicing buffer (without glucose; Table 2). The agarose can be stored at 4 &#176;C for 6 months.
	<ol>
		<li>On the day of the experiment, melt a stock volume of agarose solution using a water bath set at 80 &#176;C. Depending on the quantity, melting takes approximately 30 min.</li>
		<li>When the agarose is liquid, draw up 8 mL of liquid agarose into a 10 mL syringe, with an additional 2 mL of air. Close the syringe with a sterile cap and place it upside down in a 37 &#176;C water bath for 20 min, to prevent the agarose from solidifying, and to equilibrate its temperature to prevent hyperthermia damage of the sample.</li>
	</ol>
	</li>
	<li>If present, turn on water cooling device of the vibratome at least 30 min prior to tissue slicing to allow for sufficient cooling capacity. Set the temperature of the water circulation to 4 &#176;C. 
	NOTE: The cutting tray and cooling plate used here both contained a built-in water circulation. This is highly recommended for cooling and to lower the contamination risks. It is however also possible to use ice as a cooling technique. Also, the water-cooling device does not need to be connected to the vibratome&#39;s cutting tray and/or cooling plate at this point in the protocol.</li>
	<li>Clean the vibratome&#39;s slicing tray and sample plate by flushing all surfaces with 100% isopropanol for at least 3 min. 
	NOTE: Depending on the vibratome system used, the vibratome set-up might differ. Refer to the manual of the vibratome present in the laboratory for information about the set-up and blade calibration methods.</li>
	<li>Fill the cutting tray up to 90%-95% with slicing buffer. In the currently used set-up, this corresponds to approximately 400 mL. Connect the tubing of the water-cooling device to the valves on the vibratome&#39;s cutting tray and cooling plate.</li>
	<li>Disinfect all tools needed for the preparation by submerging them in a 100% isopropanol solution for 5 s. Remove the tools from the isopropanol solution and air-dry under the laminar flow hood.</li>
</ol>
3. Trimming and embedding the samples
<ol>
	<li>Transfer the tissue sample to a 100 mm Petri dish filled with cold slicing buffer. Keep the dish on a cooling plate at 4 &#176;C (connected to cooler or placed on ice).</li>
	<li>Remove endocardial trabeculae by holding the endocardium with tweezers and using scissors to cut away approximately 3 mm of endocardial tissue. In the same way, remove excessive adipose tissue underneath the epicardium if present.</li>
	<li>Fixate the cut tissue sample, endocardial side up, to a 2 cm x 2 cm rubber patch using four 0.9 mm x 70 mm 20 G needles that are fixed in a square position (0.9 cm x 0.9 cm; Figure 1C, D). Make sure that the diagonal edge of each needle tip is pointing inward. This enhances fixation and prevents damage to the myocardium. 
	CAUTION: The orientation of the above-mentioned square should be orthogonal to the expected predominant myofiber direction.</li>
	<li>Cut away all the excess tissue outside of the four needle square using a scalpel. If the size of the original sample allows, use two myocardial tissues samples during this preparation from the same raw sample.</li>
	<li>Using tweezers, place the trimmed sample on a sterile piece of tissue to remove any excess slicing buffer left on the sample.To prevent drying out of the sample, do not keep the sample on the tissue for longer than 10 s.</li>
	<li>Place the sample(s) in a 35 mm Petri dish, such that the blade cuts perpendicular to the cardiomyocyte alignment and the epicardium is facing downwards. If the preparation includes two samples, make sure the samples are centered and do not touch each other.</li>
	<li>Take the agarose syringe from the water bath and submerge the sample(s) in agarose. (Figure 2A). Let the agarose solidify for 5 min on a cooling plate. The sample(s) must stay in contact with the Petri dish, which will ensure that the cutting plane will be parallel to the predominant myocardial fiber direction. 
	&#8203;CAUTION: Do not fully empty the syringe, in order to prevent air bubbles. Pay attention to the amount of agarose that is left in the syringe during submerging of the samples. In the case of air bubbles in the dish with the samples, carefully pull air bubbles back into the syringe.</li>
</ol>
4. Placing the samples on cutting tray
<ol>
	<li>Remove the solidified agarose containing the sample(s) from the 35 mm Petri dish using a spatula or similar tool, by wedging it in-between the sample and the side of the Petri dish. Cut away some of the agarose using a scalpel while keeping the samples covered. 
	CAUTION: Do not remove too much of the agarose. There should be at least 5 mm of agarose left on the long and short sides in the X/Z plane. 
	NOTE: Steps 4.2-4.4 need to be performed in rapid succession (i.e., maximum of 5 s). Have all required tools within reach prior to starting. No repositioning of the sample is possible after placement. Try to limit the exposure of the glue to air and moisture. Contact with air or fluids will solidify the glue, making it unusable.</li>
	<li>With a pipette, place and distribute 60 &#181;L of glue in and around the center of the cutting platform.</li>
	<li>Place the epicardial side of the sample contained in agarose on top of the glued area using tweezers. Do not reposition. The endocardial side of the sample must be visible in the agarose. Let the glue solidify for 1 min. Gently press the agarose containing the sample from the top with a blunt tool (e.g., tweezers) while preventing cutting into or damaging the agarose.</li>
	<li>Place the sample platform in its designated position in the cutting tray of the vibratome, filled with slicing buffer.</li>
</ol>
5. Starting the vibratome
<ol>
	<li>Set vibration amplitude to 1 mm and the initial cutting speed to 0.07 mm/s. Set the thickness of the slice to 300 &#181;m to cut slices.</li>
	<li>As long as the blade only cuts the agarose, increase the cutting speed to its maximum, which in this case is 1.50 mm/s. As soon as the vibratome starts cutting the tissue, decrease the speed to 0.07 mm/s immediately. 
	&#8203;NOTE: If the tissue is not sliced smoothly, for example if there are large fibrotic areas in the sample, it may help to increase the cutting amplitude up to 1.5 mm and to reduce the cutting speed to 0.04 mm/s.</li>
</ol>
6. Medium and incubator preparation during slicing procedure
<ol>
	<li>Fill each cultivation chamber with 2.4 mL of complete cultivation medium (Table 3).</li>
	<li>Place the cultivation chambers filled with medium on the cultivation system in the incubator set at 37&#160;&#176;C, 5% CO2, 21% O2, and a humidity of 80%. Equilibrate the medium for at least 20 min.</li>
	<li>Connect the cultivation system with a computer and start the corresponding software program.</li>
	<li>Set the rocker speed to 60 rpm and preset the stimulation parameters (stimulation pulses and frequency). For human cardiac slices, set the standard stimulation to biphasic impulses with 50 mA current, each consisting of 3 ms positive current, a 1 ms pause and a 3 ms pulse of inverted current, at a pacing rate of 30 beats per min (BPM). 
	NOTE: In well preserved tissues, the typical stimulation threshold is around 15 mA. To ensure reliable stimulation and to account for any possible increase of the stimulation threshold, it is recommended to set current to a value that exceeds the stimulation threshold by two- to three-fold.</li>
	<li>Check the electrode indicators of the software to verify that the electrodes of the cultivation chambers are working correctly. 
	&#8203;NOTE: Action is needed whenever the channel indicator in the cultivation software turns red. In this case, the bipolar pulse charges are not balanced.</li>
</ol>
7. Preparing the slices
NOTE: Initial subendocardial slices are commonly not suitable for tissue cultivation and need to be discarded because of uneven morphology. After the first five to 10 slices, slice texture and morphology improve. The ideal slice is at least 1 cm x 1 cm, has no or only limited fibrotic patches, is not fragmented, and has homogeneous fiber alignment (Figure 2B, D). Interstitial fibrosis, located between the myocyte fibers, is often present in failing human myocardium. Surprisingly, this is not a negative predictor of cultivation success.
<ol>
	<li>Pour an adequate amount of cold slicing buffer in a 5 cm Petri dish lid to ensure slices will not dry out. Place the slices in the Petri dish lid containing the cold slicing buffer.</li>
	<li>Separate the agarose from the tissue by using tweezers. Prevent touching the tissue. Handle the tissue carefully as any damage to the tissue will reduce the success rate of the cultivation.</li>
	<li>Determine the direction of the myocardial fibers by close inspection against a light source. This is of importance when attaching the plastic triangles to the tissue in step 7.6. 
	NOTE: Steps 7.4 and 7.5 need to be performed in quick succession, within 5 s.</li>
	<li>Attach two plastic triangles to a sample using glue in order to anchor the tissue inside the cultivation chambers.
	<ol>
		<li>Place 1 &#181;L of glue on a sterile Petri dish lid. Use a hooked tweezer to pick up one of the autoclaved plastic triangles. Quickly dip the front edge of the triangle into the glue and paste the triangle onto the sample, perpendicular to the cardiomyocyte alignment. Repeat for the other triangle.</li>
	</ol>
	</li>
	<li>Trim off tissue exceeding the triangle width with a scalpel (Figure 2C). Place the slice with the two mounted triangles back into the cutting tray&#39;s slicing buffer. 
	NOTE: Repeat steps 7.2 to 7.5 until enough slices are prepared to fill the cultivation chambers. We recommend preparing a few additional slices to allow for the replacement of slices with poor contraction.</li>
</ol>
8. Mounting the slices
NOTE: The afterload is determined by the stiffness of the spring wire in the cultivation chambers. Three different types are available, based on the thickness of the spring wire.
<ol>
	<li>Take a medium-filled cultivation chamber from the incubator. Select one of the prepared slices and insert it into the chamber by connecting one triangle to each pin.</li>
	<li>Adjust the distance between the mounting pins according to the sample size. Make sure the sample is submerged in medium. Place the chamber back into its designated socket of the cultivation system in the incubator. 
	CAUTION: Do not overstretch the tissue and do not over-bend the spring wire!</li>
	<li>Set the preload tension after placement of the dish onto the rocker.
	<ol>
		<li>Decrease the preload by turning the adjusting screw counterclockwise. Do this until the baseline of the corresponding graph on the computer screen does not change anymore.</li>
		<li>Carefully increase the preload (i.e., increase the tension) by turning the adjusting screw clockwise. For the chambers with the highest stiffness, continue until the corresponding baseline in the graph has increased by 1000-1200 units, which corresponds to 1 mN of preload. 
		&#8203;NOTE: The exact adjustment will depend on the individual spring constant of a cultivation chamber, which can be determined by calibration prior to an experiment according to the manufacturer&#39;s instructions. The recording software permits consideration of the individual chamber calibration so that forces will uniformly be displayed in &#181;N. It is recommended to apply electrical stimulation from the start of the cultivation. As such, it is well possible that the slice starts contracting during the preload adjustment. In this case, focus on the diastolic baseline to assess the preload.</li>
	</ol>
	</li>
</ol>
9. Changing the medium
<ol>
	<li>Prepare cultivation medium according to the recipe in Table 3. Shortly before use of the medium, add 50 &#181;L of &#946;-mercaptoethanol (50 mM) to a 50 mL tube. Store at 4 &#176;C.</li>
	<li>Once every 2 days, partly exchange the cultivation medium. Refresh the medium every 2 days, if long term cultivation of the myocardial slices is desired.</li>
	<li>Pre-warm the fresh medium in a water bath or hot-air incubator at 37 &#176;C for 30-45 min. Remove a cultivation chamber from the incubator and place it under a laminar flow hood. 
	CAUTION: It is essential to keep the chamber, as well as the medium, warm at 37 &#176;C (&#62; 35 &#176;C) to prevent hyper contractures due to low temperature. Less distinct damage may be present as an increase of diastolic tension within a few hours after medium exchange. This may seem to recover, but repeated stress might result in accumulating deterioration.</li>
	<li>Remove medium from the cultivation chamber, leaving approximately 0.8 mL in the chamber. Add 1.6 mL of fresh medium to the same chamber. The total volume of medium should be around 2.4 mL per chamber.</li>
	<li>Place the cover of the chamber back and place the cultivation chamber back into its respective position.</li>
</ol>

<ol><li>George, S. A., Brennan, J. A., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+cardiac+electrophysiology+assessment+by+dual+voltage+and+calcium+optical+mapping+of+human+organotypic+cardiac+slices%5BTitle%5D)+AND+%22Journal+of+Visualized+Expereiments%3A+JoVE%22%5BJournal%5D)">Preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices.</a> Journal of Visualized Expereiments: JoVE. (160), e60781(2020).</li>
<li>Lu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+stretch+enhances+growth+and+maturation+of+3D+stem-cell-derived+myocardium%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.</a> Theranostics. 11 (13), 6138-6153 (2021).</li>
<li>Pontes Soares, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology%2C+adhesion+junctions%2C+presence+of+myofibrils+and+protein+expression%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS one. 7 (5), 38147(2012).</li>
<li>Klumm, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+cultivation+of+human+atrial+myocardium%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">Long-term cultivation of human atrial myocardium.</a> Frontiers in Physiology. 13, 839139(2022).</li>
<li>Krane, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+defects+in+cardiac+lineage+commitment+and+maturation+cause+hypoplastic+left+heart+syndrome%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome.</a> Circulation. 144 (17), 1409-1428 (2021).</li>
<li>Miller, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+slice+culture+system+reliably+demonstrates+clinical+drug-related+cardiotoxicity%5BTitle%5D)+AND+%22Toxicology+and+Applied+Pharmacology%22%5BJournal%5D)">Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.</a> Toxicology and Applied Pharmacology. , 406(2020).</li>
<li>Fischer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 1-12 (2019).</li>
<li>Kang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+organotypic+cultured+cardiac+slices%3A+new+platform+for+high+throughput+preclinical+human+trials%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials.</a> Scientific Reports. 6 (1), 1-13 (2016).</li>
<li>Ou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slicing+and+culturing+pig+hearts+under+physiological+conditions%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Slicing and culturing pig hearts under physiological conditions.</a> Journal of Visualized Experiments: JoVE. (157), e60913(2020).</li>
<li>Ou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+biomimetic+culture+system+for+pig+and+human+heart+slices%5BTitle%5D)+AND+%22Circulation+research%22%5BJournal%5D)">Physiological biomimetic culture system for pig and human heart slices.</a> Circulation research. 125 (6), 628-642 (2019).</li>
<li>Watson, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 1-15 (2019).</li>
<li>Abu-Khousa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+degree+of+t-system+remodeling+predicts+negative+force-frequency+relationship+and+prolonged+relaxation+time+in+failing+human+myocardium%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">The degree of t-system remodeling predicts negative force-frequency relationship and prolonged relaxation time in failing human myocardium.</a> Frontiers in Physiology. 11, 182(2020).</li>
<li>Bojkova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SARS-CoV-2+infects+and+induces+cytotoxic+effects+in+human+cardiomyocytes%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.</a> Cardiovascular Research. 116 (14), 2207-2215 (2020).</li>
<li>Esfandyari, D. A. -O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-365+regulates+human+cardiac+action+potential+duration%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">MicroRNA-365 regulates human cardiac action potential duration.</a> Nature Communications. 13 (1), 1-15 (2022).</li>
<li>Moretti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Somatic+gene+editing+ameliorates+skeletal+and+cardiac+muscle+failure+in+pig+and+human+models+of+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.</a> Nature Medicine. 26 (2), 207-214 (2020).</li>
<li>Pitoulis, F. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remodelling+of+adult+cardiac+tissue+subjected+to+physiological+and+pathological+mechanical+load+in+vitro%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.</a> Cardiovascular Research. 118 (3), 814-827 (2021).</li>
<li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nifedipine+blocks+Ca2%2B+store+refilling+through+a+pathway+not+involving+L-type+Ca2%2B+channels+in+rabbit+arteriolar+smooth+muscle%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> The Journal of Physiology. 532 (3), 609-623 (2001).</li>
<li>de Weille, J. R., Schweitz, H., Maes, P., Tartar, A., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calciseptine%2C+a+peptide+isolated+from+black+mamba+venom%2C+is+a+specific+blocker+of+the+L-type+calcium+channel%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.</a> Proceedings of the National Academy of Sciences. 88 (6), 2437-2440 (1991).</li>
<li>Schleifer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+molecular+modelling+study+of+the+calcium+channel+blockers+nifedipine+and+black+mamba+toxin+FS2%5BTitle%5D)+AND+%22Journal+of+Computer-Aided+Molecular+Design%22%5BJournal%5D)">Comparative molecular modelling study of the calcium channel blockers nifedipine and black mamba toxin FS2.</a> Journal of Computer-Aided Molecular Design. 11 (5), 491-501 (1997).</li>
<li>Thomas, G., Chung, M., Cohen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dihydropyridine+%28Bay-K8644%29+that+enhances+calcium+currents+in+guinea+pig+and+calf+myocardial+cells.+A+new+type+of+positive+inotropic+agent%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">A dihydropyridine (Bay-K8644) that enhances calcium currents in guinea pig and calf myocardial cells. A new type of positive inotropic agent.</a> Circulation Research. 56 (1), 87-96 (1985).</li>
<li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myocardial+slices+come+to+age%3A+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li>
<li>Watson, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li>
</ol>

JH, PS, DM, and KL have nothing to disclose. AD and TS are shareholders of InVitroSys GmbH, which provides the Myodish cultivation system.

In the past, cardiovascular research has made great advances in the cultivation of cardiomyocytes. However, the 3D cultivation of cardiomyocytes with intact geometry is not yet well-established. Compared to previous protocols applied for ex vivo cultivation of myocardial tissue, the protocol that we described here resembles the in vivo environment of the tissue more closely. Moreover, the application of pre- and afterload allows for a more biomimetic environment. We are able to fully analyze and underst...

In the past decade, in vitro cultivation of myocardial cells has made great advances, ranging from 2D and three-dimensional (3D) techniques to the use of organoids and induced pluripotent stem cells differentiated into cardiac myocytes1,2,3. Ex vivo and primary cell cultivations have shown to be of great value, especially for genetic studies and drug development4,5,6. Using human tissues improves the translational value of the results. Long-term 3D cultivation of myocardial tissues with intact geometry, however, is not well-established. The intact geometry is a key feature to mimic in vivo conditions, as proper cardiac function, communication between different cells, as well as cell-matrix interactions are prerequisites. Myocardial tissue cultivation went through various phases of development. The success rate and stability of ex vivo myocardial tissue cultivation were initially quite low, but recent approaches have yielded promising results7,8,9,10,11.
Among those, Fischer et al. were the first to demonstrate that viability and contractile performance of human myocardial tissue can be maintained in ex vivo cell cultivation for many weeks7. Their technique was based on thin tissue slices cut from explanted human myocardium, which were mounted in newly developed cultivation chambers that provided defined biomechanical conditions and continuous electrical stimulation. This cultivation method closely resembles the in vivo function of myocardial tissue, and has been reproduced by several independent research groups2,12,13,14,15. Importantly, the chambers used by Fischer et al. also enabled continuous registration of developed forces for up to 4 months, and thus opened unprecedented opportunities for physiological and pharmacological research on intact human myocardium7.
Similar techniques were independently developed by other groups and applied to human, rat, porcine, and rabbit myocardium7,10,11. Pitoulis et al. subsequently developed a more physiological method, which reproduces the normal force-length relation during a contraction cycle, but is less suitable for high-throughput analysis16. As such, the general approach of biomimetic cultivation can be regarded as a further step into the reduction, refinement, and replacement (3R) of animal experiments.
However, exploitation of this potential requires standardized procedures, high content analyses, and a high throughput level. We present a technique that combines automated slicing of living human myocardium with in vitro maintenance in a biomimetic cultivation system that has become commercially available (see Table of Materials). With the proposed approach, the number of individual slices that can be generated from a single transmural myocardial specimen is only limited by the processing time. A specimen of sufficient size and quality (3 cm x 3 cm) often yields 20-40 tissue slices being conveniently cut with an automated vibratome. These slices can be placed in cultivation chambers belonging to the system. The chambers allow for electrical stimulation, the parameters of which can be modulated (i.e., pulse duration, polarity, rate, and current), as well as the adjustment of pre- and afterload, using spring wires inside the chambers. The contraction of each slice is registered from the movement of a small magnet attached to a spring wire and displayed as an interpretable graph. Data can be recorded at all times and analyzed using freely available software. Aside from the constant baseline pacing, scheduled protocols can be performed to functionally assess their refractory period, stimulation threshold, post-pause-potentiation, and force-frequency relation.
This long-term biomimetic cultivation of multiple myocardial slices from an individual heart paves the way for future ex vivo research in both human and animal tissue, and facilitates the screening for therapeutic and cardiotoxic drug effects in cardiovascular medicine. It has already been applied to various experimental approaches2,12,13,15. Here, we give a detailed step-by-step description of the preparation of human tissue and provide solutions for frequently encountered cultivation problems.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose Low melting point</td><td>Roth</td><td>6351.2</td><td></td></tr><tr><td>Bay-K8644</td><td>Cayman Chemical</td><td>19988</td><td></td></tr><tr><td>BDM (2,3-Butanedione monoxime)</td><td>Sigma</td><td>B0753-1kg</td><td></td></tr><tr><td>CaCl2*H2O</td><td>Merck</td><td>2382.1</td><td></td></tr><tr><td>Calciseptine</td><td>Alomone Labs</td><td>SPC-500</td><td></td></tr><tr><td>Glucose*H2O</td><td>AppliChem</td><td>A3730.0500</td><td></td></tr><tr><td>H2O</td><td>BBraun</td><td>3703452</td><td></td></tr><tr><td>HEPES</td><td>AppliChem</td><td>A1069.0500</td><td></td></tr><tr><td>Histoacryl</td><td>BBraun</td><td>1050052</td><td></td></tr><tr><td>Isopropanol 100%</td><td>SAV LP GmbH</td><td>UN1219</td><td></td></tr><tr><td>ITS-X-supplement</td><td>Gibco</td><td>5150056</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1.04933.0500</td><td></td></tr><tr><td>Medium 199</td><td>Gibco</td><td>31150-022</td><td></td></tr><tr><td>MgCl2*6H2O</td><td>AppliChem</td><td>A1036.0500</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886-1KG</td><td></td></tr><tr><td>NaH2PO4*H2O</td><td>Merck</td><td>1.06346.0500</td><td></td></tr><tr><td>Nifedipine</td><td>Sigma</td><td>N7634-1G</td><td></td></tr><tr><td>Penicillin / streptomycin x100</td><td>Sigma</td><td>P0781-100ML</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>AppliChem</td><td>A1108.0100</td><td></td></tr><tr><td>&lt;strong&gt;Laboratory equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flow cabinet</td><td>Thermo Scientific</td><td>KS15</td><td></td></tr><tr><td>Frigomix waterpump and cooling + BBraun Thermomix BM</td><td>BBraun</td><td></td><td>In-house made combination of cooling and heating solution.</td></tr><tr><td>Incubator</td><td>Binder</td><td>CB240</td><td></td></tr><tr><td>MyoDish culture system</td><td>InVitroSys GmbH</td><td>MyoDish 1</td><td>Myodish cultute system</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200s</td><td></td></tr><tr><td>Water bath 37 degrees</td><td>Haake</td><td>SWB25</td><td></td></tr><tr><td>Water bath 80 degrees</td><td>Daglef Patz KG</td><td>7070</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mL plastic single-use beaker</td><td>Sarstedt</td><td>75.562.105</td><td></td></tr><tr><td>Filtration unit, Steritop Quick Release</td><td>Millipore</td><td>S2GPT05RE</td><td></td></tr><tr><td>Needles 0.9 x 70 mm 20G</td><td>BBraun</td><td>4665791</td><td></td></tr><tr><td>Plastic triangles</td><td></td><td></td><td>In-house made</td></tr><tr><td>Razor Derby premium</td><td>Derby Tokai</td><td>B072HJCFK6</td><td></td></tr><tr><td>Razor Gillette Silver Blue</td><td>Gillette</td><td>7393560010170</td><td></td></tr><tr><td>Scalpel disposable</td><td>Feather</td><td>02.001.30.020</td><td></td></tr><tr><td>Syringe 10 mL Luer tip BD Discardit</td><td>BBraun</td><td>309110</td><td></td></tr><tr><td>Tissue Culture Dish 10 cm</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>Tissue Culture Dish 3.5 cm</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Tubes 50 mL</td><td>Falcon</td><td>352070</td><td></td></tr></tbody></table>

preparation of human myocardial tissue for long-term cultivation

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

The contraction of the myocardial slices was displayed on the computer screen after insertion of the cultivation chamber into its corresponding connector (Figure 3). Contraction of the human myocardial slices started immediately upon stimulation. The slices hypercontracted for 5-10 min. This was visible as an increase of diastolic forces, caused by a tonic contracture of damaged tissue fractions. This process was reverted to varying degrees within 1-1.5 h. After stabilizing, human LV tissue ...

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

preparation-of-human-myocardial-tissue-for-long-term-cultivation

Research

JoVE Journal

Medicine

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

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.

Slicing and Culturing Pig Hearts under Physiological Conditions

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&lt;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Learn to cultivate human ventricular myocardial tissue ex vivo using a biomimetic chamber with pre- and afterload application. This method enables continuous assessment of contraction parameters for drug screening in cardiovascular research.

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Explore More Videos