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>

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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 understand the continuous recording of the contraction data and contraction parameters of the tissue.
The number of ex vivo cardiovascular tissue cultivation techniques using similar set-ups are limited11,21,22. A similar technique for myocardial slice cultivation that has been published uses a 6-well plate for the cultivation of myocardial slices10. However, an important limitation of this particular set-up is that the slices are not exposed to pre- and afterload, nor is it able to yield a detailed view of the contraction over time without the need for handling the tissue. The method we described here reduces the risk of tissue damage or infection. Furthermore, it gives a comprehensive view of the possible changes in contractile force whenever a substance of interest is added to the cultivation. In addition to the analysis of the normal contraction force of each slice, the current set up allows to regularly run pre-defined protocols. This allows the data collection of different relevant parameters of the cultivated tissue.
Critical steps within the protocol 
Tissue damage must be avoided, and this can already occur during the explant surgery. If the tissue is not immediately transferred to the cold storage solution after excision, this may result in damaged tissue samples. The described protocol contains several steps that are critical to obtain reliable and reproducible results. The agarose tissue embedding compound must be prepared using the slicing buffer without glucose, to prevent possible caramelization of the glucose during the melting process before the start of the protocol. It is important to avoid damage due to hyperthermia caused by using agarose directly from the 80 &#176;C water bath, instead of incubating the agarose in a syringe at 37 &#176;C. Temperature should be 4 &#176;C during storage and cutting to reduce metabolism and minimize ischemia. Also, tissue should be handled with care, by grabbing the agarose rather than the tissue itself, when moving it between the cutting tray and Petri dish to attach the plastic triangles. It is of utmost importance that these triangles are attached in the proper direction with respect to the direction of the myocardial fibers. Misalignment of the triangles will result in tissue damage.
In general, the stimulation threshold of newly cut slices is between 10-20 mA, and the current should be carefully increased. Overstimulation of the tissue by a current that is too high can lead to irreversible sample damage as well. Once stimulation has started, tissue agitation by rocking the cultivation chambers is necessary, and should never be halted for longer than 5 min to ensure adequate availability of oxygen and nutrients to the tissue.
Troubleshooting and modifications 
Hyper contracture 
Hyper contracture of the myocardial tissue sample is one of the main exclusion criteria for the discussed protocol. This can occur before and after the preparation of the myocardial slices. In cases where, before the preparation, the tissue felt stiff upon palpation, hypercontraction was observed in at least 70% of the preparations. Hyper contracture of the tissue sample may also occur upon insertion on the rocker, which likely depends on the quality of the tissue or the disease state of the patient. Hyper contracture can be seen as an increase of diastolic tone during the stimulation of the tissue, corresponding to the tonic shortening of damaged cardiomyocytes in cardiac ischemia. Hyper contracture during stimulation can be progressive, whereby the contraction exceeds the detection limit of 12 mN. However, the hyper contracture may also reverse spontaneously within 30-60 min, suggesting that the tissue can recover. To improve the recovery of the tissue, the preload settings should be readjusted (i.e., repeat step 8.3) after 1 h of incubation, as well as on days 2, 4, and 6 after preparation. Hyper contracture may be present both with and without the stimulated contraction of the samples. Yet, if no contraction is observed within 1 h, the sample should not be used. In this case, the tissue has hypercontracted to a degree from which it cannot recover or has been damaged otherwise. In general, the cultivation of human myocardial slices according to the presented protocol, has shown to have a success rate of 90%.
Expected contraction force changes 
Depending on a multitude of factors (e.g., patient, preparation, tissue damage), it is possible that myoslices that showed acceptable contraction initially, will undergo a progressive decline in contractile amplitudes during the first 24 h. In fact, this behavior can even be considered normal for slices obtained from human end-stage failing hearts. Readjusting the preload of the slices each day for the following 3 days has been found to alleviate this problem and improve the contraction. After 24 to 48 h, however, the contractility should start to increase again. Note that the contraction will not return to its initially observed level within the first days of cultivation.
Shortly after medium exchange, when the samples are returned to the incubator, the contraction typically shows markedly increased amplitudes. Opening and closing of the incubator contributes to this increase, as it causes the CO2 level to drop. This increases the pH inside the bicarbonate-buffered cultivation medium, which causes a positive inotropic response of the slices. Following the medium exchange, the contractile force of the slices often decreases for several hours until it reaches or surpasses its initial value. To prevent stimulation protocols being affected by medium exchange, stimulation protocols should not be executed until at least 1 h after medium exchange.
Limitations of the method 
An advantage of the present technique as compared to earlier cultivation methods for human myocardial tissue, is the possibility to apply (and modulate) pre- and afterload to the contracting tissue. However, it is difficult to quantify the applied load in terms of wall tension, although this load could be estimated from the spring constant and the slice dimensions. It is assumed that there is a positive relation between the viability of myocardial tissue and the contraction amplitude of tissue sample. Yet there currently is no method that allows viability testing of each individual slice before mounting it to the chambers. A colorimetric MTT staining can be performed on cut slices that are not used for cultivation, to determine the viability of the myocardial cells. In cells that are viable, NADPH will reduce the yellow MTT salt in purple formazan crystals. Another limitation is that currently, the nutrients in the cultivation medium are limited to basic ingredients. Hence, nutrients and circulating factors are different from the in vivo environment from which the tissue was obtained. However, medium can be supplemented or modified as needed.
Potential applications of the method 
There are several potential applications of the method both in terms of cardiotoxicity and drug testing, as well as in understanding the pathophysiology of heart disease. First, the system used in the discussed protocol can be used for drug and cardiotoxicity screenings. With the help of the programmable stimulation protocols, physiological changes can be analyzed in response to drug administration. In regard to the interpretation&#160;of refractory period, it needs to be considered that this is a functional parameter, which does not strictly reflect the effect of drugs on action potential duration. Second, the cultivation chambers can be utilized for various co-cultivation experiments of myocardium in combination with immune cells. By using this co-cultivation, the direct effects of immune cell secreted factors on the myocardial contraction can be assessed. Finally, non-transplant cardiomyopathy samples can also be cultivated, although these non-transplant tissue samples are generally smaller and therefore more difficult to process. Nevertheless, this may open up possibilities for the identification of novel therapeutic targets and the development of targeted medications. It is also conceivable to use the technique for patient specific tissue characterization, bringing us one step closer to personalized medicine. Furthermore, the presented method can be used for non-human myocardial tissue, examples of which are pig and rabbit. It has to be considered, that the high physiological heart rate of small mammals (mouse/rat) cannot be obtained, because the subsequent higher O2 requirement cannot be satisfied under the conditions of myocardial cultivation using the discussed set up. Thereby, a near-physiological environment is hard to mimic in these cases.

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

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 slices showed twitch forces varying between 1 mN and 3 mN upon stimulation. Systole is shown as a strong increase in contraction force, followed by diastole with an equally steep decrease of the contraction force.
Contraction of the myocardial slices was recorded by the cultivation software and saved in a designated file. Each of the generated raw data files were converted to a readable Axon binary file format (.abf) for easy analysis and quantification of the data. For the initial analysis, the .abf file was opened in an appropriate program. Approximately 5 min of contraction data was selected to establish the average contraction amplitude during this period. This was done for multiple time points in the recorded data file. Plotting these contraction values over time yielded a useful graph to compare contraction development in a control and experimental setting. To gain a more advanced insight into the performance of the generated slices, stimulation protocols were run. During these protocols, which take approximately 45 min, the stimulation parameters were altered to assess parameters of contraction coupling.
The current stimulation protocol consisted of four distinct sections: post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period (Figure 4, Table 4). During the post-pause-potentiation, stimulation is resumed after a brief halt of either 3, 12, 50, or 120 s. To determine the stimulation threshold, the stimulation current is increased in steps of 3 mA every 10 s, starting at 8 mA and increasing to 90 mA. With this test, the minimal stimulation current can be determined for each slice. This does not alter the general stimulation settings outside of the stimulation protocol. The force-frequency relation is assessed by a stepwise increase of the stimulation frequency (20, 30, 45, 60, 80, 100, 120, 150, 180, 210, and 240 BPM), while the respective duration of each step is shortened in parallel. Except for the first two frequency settings, this regimen yields between 20-40 contractions during each step. The refractory period of each slice is assessed by sending a premature stimulus (S2) after a normal stimulus (S1; 30 BPM). The S1-S2 interval is shortened every 10 s.
To demonstrate the potential of the presented cultivation system as a tool for testing pharmacological interventions, ex vivo human LV tissue slices were prepared from the same patient and subjected to pharmacological agents that influence the intracellular calcium ion (Ca2+) levels (n = 1) after 2 weeks of cultivation. The L-type Ca2+-channel antagonist nifedipine inhibits Ca2+ influx into the myocardial cells and therefore lowers the intracellular availability of Ca2+ and reduces the contractility17. Because of its vasodilator action, nifedipine is used as an anti-hypertensive drug. To demonstrate pharmacological differences of Ca2+-channel antagonists, calciseptine was investigated for comparison. Calciseptine is also an L-type Ca2+-channel antagonist, extracted from the Dendroaspis p. polylepis venom18. Therefore, it shares the negative inotropic action of nifedipine. However, calciseptine has different binding characteristics and is more potent compared to nifedipine19. In order to study the positive and negative modulations of Ca2+ availability, we also tested the calcium channel agonist Bay-K8644 (1,4-Dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)pyridine-3-carboxylic acid methyl ester)20.
Three slices were treated with nifedipine (125 nM), calciseptine (70.8 nM), and Bay-K8644 (417 nM) respectively, while the fourth slice received no drug (control). The contraction forces under general stimulation parameters (50 mA current, biphasic pulses 3 ms duration, 1 ms interval, and 30 BPM pacing rate) were compared before and after treatment. Furthermore, before the treatment, a stimulation protocol was run to assess the baseline values for post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. After 30 min of treatment, a second stimulation protocol was run. To eliminate inter-individual differences, contraction amplitude of each slice was normalized to its baseline level before treatment. The baseline (i.e., 100%) was determined by analyzing the last five contraction cycles before the start of the pre-treatment stimulation protocol.
When analyzing the contraction of the slices at general stimulation, it was observed that the contraction force of the slices treated with Ca2+ antagonists (nifedipine and calciseptine) decreased within 10 min post-treatment (Figure 5) and the effect was present up to 20 min post-treatment. In contrast, the voltage gated Ca2+ channel agonist Bay-K8644 increased the contraction force of the treated slice. The control slice did not show a noteworthy change. The contraction data generated during the stimulation protocols were analyzed in a similar way. Here, the data generated by the stimulation protocol performed before the treatment (pre-treatment) was compared to the post-treatment data of the same stimulation protocol.
As discussed before, the stimulation protocol started with the assessment of the post-pause-potentiation. During the pause, additional Ca2+ is taken up by the sarcoplasmic reticulum (SR), which is released upon first stimulation after the pause. Hence, post-pause-potentiation reflects intracellular Ca2+ release from the SR. As such, this parameter can be used to assess the relative contribution of Ca2+ released from the SR by the ryanodine receptor. To assess the potentiation of the slices after a stimulation pause, the strength of the first contraction after the pause was divided by the average contraction before the respective pause. The control slice did not show any noteworthy change (Figure 6A). It was observed that the inhibition of the L-type Ca2+ channels led to potentiation of the first contraction after a pause of at least 50 s (Figure 6B,D), reflecting a higher relative contribution of intracellular Ca2+ release to total contractility.&#160;The opposite effect was seen in the slice treated with Bay-K8644, which stimulates the entry of extracellular Ca2+ via the L-type Ca2+ channels (Figure 6C).
The force-frequency relation was assessed by successively increasing the pacing rate up to 180 BPM. For better visualization, the contraction force at different stimulation frequencies was normalized to the baseline contraction at 30 BPM within the same protocol (=100%). Data analysis showed that the treatment with calciseptine did not change the ability of the slice to follow the stimuli upon increase of the stimulation frequency when comparing the pre- and post-treatment data (Figure 7B). No change was observed in the control slice (Figure 7A). Contrary to calciseptine, nifedipine prevented an increase of contractility at higher pacing rates and reduced the maximum capture rate to 80 BPM (Figure 7D). The slice treated with the Ca2+ channel agonist Bay-K8644 showed an increased contraction force at very low stimulation frequencies (Figure 7C). However, at frequencies higher than 50 BPM, the contraction force appeared to be lower than during the pre-treatment condition. The stimulation threshold and the refractory period were also determined pre- and post-treatment. However, no differences were observed, and the data are therefore not shown.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63964/63964fig01.jpg" /> 
Figure 1: Overview of the required materials and the cultivation system. (A) An empty cultivation chamber connected to a green circuit board. The circuit board (1) measures the contraction with a sensor and transmits the data to the controller. (B) Eight filled chambers placed on the rocking main plate. Petri dish lids (35 mm) are used to cover the chambers. (C) The (surgical) tools needed to trim the transmural myocardial sample. Depicted are various tweezers, the blades needed for the vibratome, and a rubber patch to aid in trimming. (D) Four 0.9 mm x 70 mm 20 G needles that are fixed in a square position (0.9 cm x 0.9 cm) by a solid block of plastic. Using this construct prevents damage and movement of the sample when trimming and yields a tissue block with the required dimensions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63964/63964fig02.jpg" /> 
Figure 2: Processing of two LV samples. (A) Two myocardial tissue blocks (approximately 1 cm x 1 cm x 1 cm) embedded in solidified low-melt agarose in a 35 mm Petri dish. For demonstration purposes, porcine LV tissue was used. (B) The agarose block with the embedded samples was removed from the Petri dish, trimmed, and glued onto the cutting platform of the vibratome. Here, porcine LV tissue was used for demonstration purposes. Notice the uniform tissue color and the absence of white fibrotic tissue, indicated by the red arrow. (C) After slicing, the agarose is carefully removed, and plastic triangles (1) are connected perpendicular to the direction of the myocardial fibers. The red lines indicate the direction of the myocardial fibers. (D) Human LV tissue was prepared, which showed white fibrotic tissue within the slices. This does not necessarily lower the success rate of cultivation; however it is recommended to use slices that do not display fibrosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63964/63964fig03.jpg" /> 
Figure 3: The cultivation software. Depending on the number of channels used (maximum of eight), the contraction registration will be visualized in up to three designated windows (two visible in the figure). Each cultivation chamber is displayed as one contraction graph. In the settings window, stimulation parameters can be altered and tailored to the desired experimental situation. This window also allows to start or stop the stimulation protocol/schedule that is selected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63964/63964fig04.jpg" /> 
Figure 4: Read-out of the contraction of five myocardial slices during a typical stimulation protocol. In all panels, each individual slice is shown in the same color. (A) Post-pause-potentiation assesses the first contraction after a brief stimulation pause of 3, 12, 50, and 120 seconds respectively. (B) Determination of the stimulation threshold by increasing the stimulation current in steps of 3 mA every 10 s from 8 mA to 80 mA. (C) The force-frequency relation of each slice is assessed by stepwise increases of the stimulation frequency from 12 BPM to 240 BPM. The duration of stimulation periods becomes shorter at higher frequencies to keep the number of beats constant at each frequency. (D) To assess the refractory period, the slices are exposed to premature stimulations (S2) at decreasing intervals to the preceding stimulus (S1). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63964/63964fig05.jpg" /> 
Figure 5: Analysis of contraction force before and after treatment with L-type Ca2+ channel affecting agents. All data (n = 1) were normalized to the baseline contractility (mean of five separate beats before treatment (0)). L-type Ca2+ channel antagonists nifedipine (125 nM), calciseptine (70.8 nM), and L-type Ca2+ channel agonist Bay-K8644 (417 nM) were added to contracting human LV myocardial slices. The control slice received no treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63964/63964fig06.jpg" /> 
Figure 6: Post-pause-potentiation of the treated and control slices. Differences in post-pause-potentiation were observed (baseline vs post-treatment; n = 1). Here, the amplitude of the first contraction after a pause was normalized to the average contraction amplitude before the respective pause. Baseline was set as 100% and resembles the average contraction strength of the last five cycles before the first pause commences. The Y-axis displays the normalized first contraction after a pause of various durations. The X-axis shows the baseline and pause lengths. (A) Control slice without treatment. (B) Calciseptine treatment. (C) Bay-K8644 treatment. (D) Nifedipine treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63964/63964fig07.jpg" /> 
Figure 7: Analysis of the force frequency relation. Contraction data (n = 1) was normalized to the contraction force at 30 BPM within the respective protocol (= 100%). (A) The control slice was not treated with any substance; however, all other aspects of the cultivation were the same as those of the treated slices. (B) Calciseptine treatment of one LV myocardial slice. (C) Bay-K8644 treatment. (D) Nifedipine treatment. Data about the contraction force of this slice during stimulation frequencies above 80 BPM were omitted, as the contraction was not following the stimulation frequency. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/63964fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of the slicing buffer used for transport and during the slicing procedure. For the preparation of agarose, glucose is omitted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/Table1_SlicingBufferComposition_JHamers_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Composition of the 4% agarose gel. This glucose-free low-melt agarose gel is used for embedding of the tissue samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/Table2_AgaroseComposition_JHamers_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Preparation of the medium for cultivation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/Table3_MediumComposition_JHamers_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Details of the stimulation protocol. The stimulation protocol consists of four parts, which all can be altered to suit the needs of the project. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63964/Table4_StimulationProtocol_JHamers.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Preparation of Human Myocardial Tissue for Long-Term Cultivation

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

Nanyang Technological University

Research

JoVE Journal

Medicine

4.4K Views.  University Hospital, LMU Munich. Met behulp van dit protocol kunnen menselijke linkerventrikelweefselschijfjes ex vivo worden gekweekt in een biomimetische kamer. De toepassing van pre- en afterloads verbetert de gelijkenis van de fysiologische omgeving. Deze techniek maakt continue registratie van weefselcontractie mogelijk.Door de gebruiker gedefinieerde stimulatieprotocollen maken de beoordeling mogelijk van vitale contractieparameters zoals potentiatie na pauze, stimulatiedrempel, kracht-frequentierelatie en refra...

Video: Preparation of Human Myocardial Tissue for Long-Term Cultivation

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

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

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

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.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

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.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Biology

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

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

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.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Bioengineering

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

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.

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 ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

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

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. 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 and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

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.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

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

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

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.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

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 (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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. ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

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