Preparation of Human Myocardial Tissue for Long-Term Cultivation

Summary

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.

Abstract

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 µm 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.

Introduction

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 myocytes^1,2,3. Ex vivo and primary cell cultivations have shown to be of great value, especially for genetic studies and drug development^4,5,6. 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 results^7,8,9,10,11.

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 groups^{2,12,13,14,15}. 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 myocardium^7,10,11. 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 approaches^2,12,13,15. Here, we give a detailed step-by-step description of the preparation of human tissue and provide solutions for frequently encountered cultivation problems.

Protocol

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

1. Obtain human tissue from patients undergoing heart transplantation or cardiac surgery.
2. Before procuring the tissue, prepare 2 L of cardioplegic solution (further referred to as slicing buffer (Table 1)).
3. 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 °C) slicing buffer. Keep at 4 °C.
   ​NOTE: The slicing buffer used in this protocol allows for cold storage (4 °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 ≤ 24 h have proven to be optimal.

2. Preparing agarose and the vibratome

1. Make sure sufficient cultivation chambers are prepared and sterilized (Figure 1A).
   1. 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 °C for 10 min.
   2. Let the chambers and graphite electrodes air-dry under a laminar flow hood.
   3. 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's instructions.
   4. Place a 35 mm Petri dish lid on top of the chamber to prevent contamination.
2. Set up the Myodish cultivation system (see Table of Materials) in an incubator at 37 °C and 5% CO₂ by connecting the system to a computer (Figure 1B).
3. Prepare the 4% low-melt agarose in slicing buffer (without glucose; Table 2). The agarose can be stored at 4 °C for 6 months.
   1. On the day of the experiment, melt a stock volume of agarose solution using a water bath set at 80 °C. Depending on the quantity, melting takes approximately 30 min.
   2. 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 °C water bath for 20 min, to prevent the agarose from solidifying, and to equilibrate its temperature to prevent hyperthermia damage of the sample.
4. 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 °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's cutting tray and/or cooling plate at this point in the protocol.
5. Clean the vibratome'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.
6. 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's cutting tray and cooling plate.
7. 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.

3. Trimming and embedding the samples

1. Transfer the tissue sample to a 100 mm Petri dish filled with cold slicing buffer. Keep the dish on a cooling plate at 4 °C (connected to cooler or placed on ice).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
   ​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.

4. Placing the samples on cutting tray

1. 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.
2. With a pipette, place and distribute 60 µL of glue in and around the center of the cutting platform.
3. 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.
4. Place the sample platform in its designated position in the cutting tray of the vibratome, filled with slicing buffer.

5. Starting the vibratome

1. Set vibration amplitude to 1 mm and the initial cutting speed to 0.07 mm/s. Set the thickness of the slice to 300 µm to cut slices.
2. 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.
   ​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.

6. Medium and incubator preparation during slicing procedure

1. Fill each cultivation chamber with 2.4 mL of complete cultivation medium (Table 3).
2. Place the cultivation chambers filled with medium on the cultivation system in the incubator set at 37 °C, 5% CO₂, 21% O₂, and a humidity of 80%. Equilibrate the medium for at least 20 min.
3. Connect the cultivation system with a computer and start the corresponding software program.
4. 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.
5. Check the electrode indicators of the software to verify that the electrodes of the cultivation chambers are working correctly.
   ​NOTE: Action is needed whenever the channel indicator in the cultivation software turns red. In this case, the bipolar pulse charges are not balanced.

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.

1. 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.
2. 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.
3. 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.
4. Attach two plastic triangles to a sample using glue in order to anchor the tissue inside the cultivation chambers.
   1. Place 1 µL 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.
5. 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'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.

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.

1. 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.
2. 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!
3. Set the preload tension after placement of the dish onto the rocker.
   1. 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.
   2. 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.
      ​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's instructions. The recording software permits consideration of the individual chamber calibration so that forces will uniformly be displayed in µN. 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.

9. Changing the medium

1. Prepare cultivation medium according to the recipe in Table 3. Shortly before use of the medium, add 50 µL of β-mercaptoethanol (50 mM) to a 50 mL tube. Store at 4 °C.
2. 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.
3. Pre-warm the fresh medium in a water bath or hot-air incubator at 37 °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 °C (> 35 °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.
4. 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.
5. Place the cover of the chamber back and place the cultivation chamber back into its respective position.

Results

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 ...

Discussion

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...

Materials

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