Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

Podsumowanie

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 (<50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Streszczenie

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 µm 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 ± 11.9 vs. 7.89 ± 3.6%, p < 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 ± 15.0 vs. 6.87 ± 5.23 cells/mg, p < 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9–200 cells/mg) from minimal amounts of tissue (<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.

Wprowadzenie

A seminal technique that has paved the way to important insights into cardiomyocyte physiology is the isolation of living ventricular cardiomyocytes from intact hearts¹. Isolated cardiomyocytes can be used to study normal cellular structure and function, or the consequences of in vivo experiments; for example, to assess changes in cellular electrophysiology or excitation-contraction coupling in animal models of cardiac disease. Additionally, isolated cardiomyocytes can be used for cell culture, pharmacological interventions, gene transfer, tissue engineering, and many other applications. Therefore, efficient methods for cardiomyocyte isolation are of fundamental value to basic and translational cardiac research.

Cardiomyocytes from small mammals, such as rodents, and from larger mammals, such as pigs or dogs, are commonly isolated by coronary perfusion of the heart with solutions containing crude collagenases and/or proteases. This has been described as the “gold standard” method for cardiomyocyte isolation, resulting in yields of up to 70% of viable cells². The approach has also been used with human hearts, resulting in acceptable cardiomyocyte yields^3,4,5. However, because coronary perfusion is only feasible if the intact heart or a large myocardial wedge containing a coronary artery branch is available, most human cardiac specimens are not suited for this approach due to their small size and a lack of appropriate vasculature. Therefore, the isolation of human cardiomyocytes is challenging.

Human myocardial specimens mostly consist of tissue chunks of variable size (approximately 0.5 x 0.5 x 0.5 cm to 2 x 2 x 2 cm), obtained through endomyocardial biopsies⁶, septal myectomies⁷, VAD implantations⁸, or from explanted hearts⁹. The most common procedures for cardiomyocyte isolation start with mincing the tissue using scissors or a scalpel. Cell-to-cell contacts are then disrupted by immersion in calcium-free or low-calcium buffers. This is followed by multiple digestion steps with crude enzyme extracts or purified enzymes like proteases (e.g., trypsin), collagenase, hyaluronidase, or elastase, resulting in a disintegration of the extracellular matrix and liberation of cardiomyocytes. In a final, critical step, a physiological calcium concentration has to be carefully restored, or cellular damage can occur due to the calcium-paradox^10,11,12. This isolation approach is convenient but usually inefficient. For instance, one study found that nearly 1 g of myocardial tissue was required to obtain a sufficient number of cardiomyocytes suitable for subsequent experiments¹³. A possible reason for low yields is the relatively harsh method of mincing the tissue. This may particularly damage cardiomyocytes located at the chunk edges although these myocytes are most likely to be released by enzymatic digestion.

Another aspect that may influence isolation efficiency and quality of cells obtained from human specimens is the duration of tissue ischemia. Most protocols mention short transportation times to the laboratory as a prerequisite for good results. This restricts the study of human ventricular cardiomyocytes to laboratories with nearby cardiac surgery facilities. Together, these restrictions hamper the verification of important findings from animal models in human cardiomyocytes. Improved isolation protocols that allow for high cardiomyocyte yields from small amounts of tissue, preferably without serious damage after extended transportation times, are therefore desirable.

Described here is an isolation protocol based on the enzymatic digestion of thin myocardial tissue slices generated with a vibratome^14,15. We demonstrate that isolation from tissue slices is much more efficient than that from tissue chunks minced with scissors. The described method not only allows for high yields of viable human cardiomyocytes from small amounts of myocardial tissue but is also applicable to specimens stored or transported in cold cardioplegic solution for up to 36 h.

Protokół

All experiments with rats were approved by the Animal Care and Use Committee Mittelfranken, Bavaria, Germany. Collection and use of human cardiac tissue samples was approved by the Institutional Review Boards of the University of Erlangen-Nürnberg and the Ruhr-University Bochum. Studies were conducted according to Declaration of Helsinki guidelines. Patients gave their written informed consent prior to tissue collection.

Female Wistar rats (150–200 g) were commercially obtained, anesthetized by injecting 100 mg/kg of thiopental-sodium intraperitoneally, and euthanized by cervical dislocation followed by thoracotomy and excision of the heart. Human cardiac tissue samples were collected from the left-ventricular apical core during implantation of mechanical assist devices, from septal myectomy, from tetralogy of Fallot corrective surgery, or from the free left-ventricular wall of explanted hearts. The following protocol describes the isolation from human ventricular tissue. The isolation of rat cardiomyocytes was performed accordingly, but with different enzymes (see Table of Materials). A schematic workflow of the protocol is illustrated in Figure 1.

1. Preparation of buffers, solutions, and enzymes

1. Prepare buffers and solutions as listed in Table 1.
2. Warm up solutions 1, 2, 3 and the modified Tyrode’s solution to 37 °C. Store the cutting solution at 4 °C until use.
   NOTE: For 1–2 myocardial slices a total of approximately 15 mL, 8 mL, and 5 mL of solutions 1, 2, and 3 are required for the isolation in one 35 mm tissue culture dish. Scale up accordingly for simultaneous myocyte isolations in multiple dishes. Cutting solution can be frozen and kept at -20 ˚C for several months.
3. Weigh 1 mg of proteinase XXIV (see Table of Materials) into a prechilled 15 mL centrifuge tube and store on ice until use. This is for processing one sample. Scale up the amount accordingly. Do not mix the proteinase and collagenase.
4. Weigh 8 mg of collagenase CLSI (see Table of Materials) into a prechilled 15 mL centrifuge tube and store on ice until use. This is for processing one sample. Scale up the amount accordingly. Do not mix the proteinase and collagenase.
   CAUTION: Wear a face mask or work under a fume hood to avoid inhalation of enzyme powder.
   NOTE: Enzyme activity may vary in different lots. Therefore, the optimal concentration may differ and should be determined with each newly purchased enzyme¹⁶.

2. Storage and transport of myocardial tissue

1. Store and transport human cardiac samples in cooled, 4 ˚C cutting solution (Table 1).
2. Use the same solution for further tissue processing and vibratome slicing.
   NOTE: Biopsies and surgical heart samples should be transferred immediately to the cutting solution at 4 °C and can then be stored or transported at 4 °C for a maximum of 36 h before the application of this protocol.

3. Processing and slicing of the tissue

NOTE: The protocol for tissue slicing follows Fischer et al.¹⁵.

1. Trimming of the tissue block
   CAUTION: Human cardiac tissue is potentially infectious. Always use protective wear and follow safety regulations of your institution. Carefully handle used blades and discard in safety containers.
   1. Place the specimen into a 100 mm tissue culture dish filled with 20 mL cold cutting solution and keep on a cooled, 4 °C plate.
   2. Remove excess fibrotic tissue and epicardial fat with a scalpel. In case of a transmural specimen, remove trabeculae and tissue layers near the endocardium.
      NOTE: Fibrotic tissue is stiff and appears white. Fat is typically soft and appears white to yellow. Trabeculae and endocardial tissue layers can be identified from their loose tissue composition and a nonaligned fiber orientation compared to myocardium of the epicardial layers
   3. For optimal vibratome processing, cut rectangular tissue blocks of approximately 8 mm x 8 mm x 8 mm with a scalpel from a larger tissue specimen. For smaller biopsies skip this step and move to agarose embedding.
2. Embedding cardiac tissue into low-melting-point agarose
   1. Boil 400 mg of low-melting point agarose in 10 mL of cutting solution in a glass beaker.
      CAUTION: Wear gloves and safety glasses to avoid burns. Handle hot glassware only with heat protection wear.
   2. Fill a 10 mL syringe with the hot, dissolved agarose gel. Seal the syringe and allow the agarose to equilibrate in a 37 °C water bath for at least 15 min.
   3. Use forceps to place the trimmed cardiac specimen or biopsy into a clean 35 mm tissue culture dish with the epicardium facing down and remove excess fluid with a sterile swab.
   4. Pour the equilibrated agarose (step 3.2.2) over the tissue by emptying the syringe. Secure the tissue against movement with forceps while pouring the agarose. Make sure that the tissue is completely immersed in agarose.
   5. Immediately place the dish on ice and let the agarose solidify for 10 min.
3. Slicing the myocardium
   1. Mount a new razor blade to the blade holder of the vibratome and calibrate the vibratome by adjusting the z-deflection of the blade if possible.
      NOTE: This protocol uses a vibratome with an infrared-assisted calibration device to align the blade in a horizontal position with minimal z-deflection (measured deflection <0.1 µm).
   2. Use a scalpel to excise an agarose-tissue block that fits the specimen holder of the vibratome. To ensure stability, make sure the tissue is still sufficiently immersed in agarose (agarose margins ≥ 8 mm).
   3. Fix the agarose block to the specimen holder with a thin layer of cyanoacrylate glue and gentle pressure.
   4. Place the specimen holder with the tissue into the vibratome bath. Fill the bath with cutting solution and keep at 4–6 °C throughout the processing with crushed ice, filled in the outer cooling tank of the vibratome.
   5. Generate 300 µm thick slices with an advancing speed of ≤0.1 mm/s, an oscillating frequency of 80 Hz, a lateral amplitude of 1.5 mm, and a blade angle of 15°. When handling the slices, hold the agarose instead of the tissue itself to avoid tissue damage. Store the slices in the cutting solution at 4 °C for a maximum of 2 h, if necessary.
      NOTE: Cardiomyocytes are oriented in parallel to the epicardium. Therefore, it is important to cut in parallel to the epicardium to avoid excessive myocyte damage. It is recommended to discard the first 1–3 slices, as only uniform slices of constant thickness should be used for the isolation. For small tissue biopsies, however, only discard the first slice.
   6. Check cardiomyocyte alignment under a standard light microscope with a magnification of 40-100x.

4. Tissue digestion

1. Place the heat plate on the lab shaker and warm it to 37 °C. Start the lab shaker at 65 rpm.
2. Dissolve the proteinase (prepared in step 1.3) in 2 mL of solution 1 (step 1.1 and 1.2) and incubate at 37 °C until use. Do not mix the proteinase and collagenase.
3. Dissolve the collagenase (prepared in step 1.4) in 2 mL of solution 1 (step 1.1 and 1.2) and incubate at 37 °C until use. Do not mix the proteinase and collagenase.
   CAUTION: Wear protective eyewear and gloves, because dissolved enzymes can cause skin and eye injuries.
4. Add calcium chloride (CaCl₂) to the collagenase containing solution (prepared in step 4.3) to a final concentration of 5 µM.
5. Use forceps to transfer a tissue slice from the vibratome bath to a clean 60 mm tissue culture dish filled with 5 mL of prechilled cutting solution (step 1.1 and 1.2) and keep on ice.
6. Carefully remove the agarose from the myocardial tissue with blade or forceps.
   NOTE: Avoid excess tension and shear stress on the myocardial slices, because they can damage the cardiomyocytes.
7. To perform the initial wash, place a clean 35 mm tissue culture dish on the heat plate and fill it with 2 mL of prewarmed (37 °C) solution 1. Transfer 1–2 myocardial slices to the prepared dish with forceps. Aspirate the solution with a 1 mL pipette and perform the wash steps 2x to remove remnants of cutting solution.
   NOTE: Do not aspirate the cardiac slices. The solutions and the slices should remain at a constant temperature of 35 °C on the agitated heat plate. Adjust the heat plate temperature if necessary.
8. Remove solution 1 from the dish and add 2 mL of the proteinase solution (step 4.2). Incubate for 12 min on the heat plate at 65 rpm.
9. Wash 2x with 2 mL of prewarmed solution 1 (37 °C).
10. Remove solution 1 from the dish and add 2 mL of collagenase solution (steps 4.3 and 4.4). Incubate at least 30 min on the heat plate at 65 rpm.
11. Check for free individual myocytes at 30, 35, 40 min, etc., by placing the dish under a light microscope. Work quickly to avoid significant cooling of the solution.
    NOTE: The required digestion time may vary depending on the tissue constitution and the degree of fibrosis. As soon as the tissue gets visibly soft and dissociates readily when gently pulled, the optimal digestion time has been reached. If enough tissue is available, several slices can be digested in parallel with varying digestion times.
12. When the tissue is digested and individual myocytes are visible (step 4.11), wash 2x with 2 mL of prewarmed solution 2 (37 °C) and fill again with 2 mL.

5. Tissue dissociation

1. Dissociate the digested tissue slices with forceps by carefully pulling the fibers apart.
2. Carefully pipette several times with a single-use Pasteur pipette (opening diameter >2 mm).
   NOTE: The use of forceps and pipetting can induce mechanical stress and cause cardiomyocyte damage. However, it is a crucial step for separation of the cells. Use fine forceps to minimize cell damage and carefully dissociate the slices to smaller pieces.
3. Check for the liberated rod-shaped cardiomyocytes under the light microscope.

6. Reintroduction of physiological calcium concentration

1. Slowly increase the calcium concentration from 5 µM to 1.5 mM while agitating at 35 °C on the heat plate. Use 10 mM and 100 mM CaCl₂ stock solutions. Recommended steps: 20, 40, 80, 100, 150, 200, 400, 800, 1,200, 1,500 µM. Allow the cells to adapt to the increased calcium levels in 5 min incubation intervals between each step.
2. Remove undigested tissue chunks carefully with forceps or filter the cell suspension through a nylon mesh with 180 µm pore size at the end of the calcium increase.

7. Removal of mechanical uncoupling agent

1. Stop agitation and slowly remove one third of the solution (~700 µL) from the top with a 1,000 µL pipette. Avoid aspiration of the cardiomyocytes.
   NOTE: If undigested tissue was removed, the cardiomyocytes will accumulate in the center of the dish, which facilitates aspiration of cell-free solution. If not, transfer the cell solution to a 15 mL centrifuge tube and allow the cells to sediment for 10 min at 35 °C, then aspirate 700 µL of the supernatant and discard, resuspend the cells, transfer them back to a 35 mm tissue culture dish and proceed with step 7.2.
2. Add 700 µL of solution 3 to the cells, resume agitation on the heat plate and incubate for 10 min.
3. Repeat steps 7.1 and 7.2.
4. Transfer the solution to a 15 mL centrifuge tube and allow the cardiomyocytes to sediment for a minimum of 10 min and a maximum of 30 min at room temperature or spin at 50 x g for 1 min. Remove the supernatant completely and resuspend in modified Tyrode’s solution or the desired experimentation buffer.
   NOTE: Cardiomyocytes can be stored in modified Tyrode’s solution (Table 1) for several hours at 37 °C and 5% CO₂ before use.
5. Verify the cell quality with a standard light microscope at 40x and 200x magnification.
   NOTE: Around 30–50% of the cardiomyocytes should be rod-shaped, smooth without membrane blebs, and display clear cross-striations. Only 5–10% of the viable cells should show spontaneous contractions.

Wyniki

To verify isolation efficiency, the protocol was used with rat myocardium and the resulting number of viable myocytes was compared with the numbers obtained by isolation via coronary perfusion and by isolation from small tissue chunks (chunk isolation, Figure 2). Chunk isolation and isolation from tissue slices were performed from the same hearts. For the isolation via coronary perfusion, however, the whole heart was used. Coronary perfusion yielded predominantly rod-shaped and cross-striate...

Dyskusje

Although the isolation of living cardiomyocytes was established more than 40 years ago and is still a prerequisite for many experimental approaches in cardiac research, it remains a difficult technique with unpredictable outcomes. Cardiomyocyte isolation via perfusion of the coronary arteries with enzyme solution is commonly used for hearts of small animals and yields large numbers of viable cells. However, this requires a relatively complex system and expertise. Furthermore, most human tissue samples are not suited for ...

Materiały

Name	Company	Catalog Number	Comments
Chemicals
2,3-butanedionemonoxime	Carl Roth	3494.1	Purity>99%
Bovine serum albumin	Carl Roth	163.2
CaCl2	Carl Roth	5239.2
Creatine monohydrate	Alfa Aesar	B250009
Glucose	Merck	50-99-7
HEPES	Carl Roth	9105.3
KCl	Carl Roth	P017.1
KH2PO4	Carl Roth	3904.2
L-glutamic acid	Fluka Biochemica	49450
Low melting-point agarose	Carl Roth	6351.5
MgCl2 x 6H2O	Carl Roth	A537.1
MgSO4	Sigma Aldrich	M-7506
NaCl	Carl Roth	9265.1
NaHCO3	Carl Roth	8551.2
Paraformaldehyde	Sigma Aldrich	P6148
Taurine	Sigma Aldrich	T8691
Dyes
Di-8-ANEPPS	Thermo Fisher Scientific	D3167
Fluo-4 AM	Thermo Fisher Scientific	F14201
FluoVolt	Thermo Fisher Scientific	F10488
Enzymes
Collagenase CLS type I	Worthington	LS004196	Used for human tissue at 4 mg/mL (activity: 280 U/mg)
Collagenase CLS type II	Worthington	LS004176	Used for rat tissue at 1.5 mg/mL (activity 330 U/mg)
Protease XIV	Sigma Aldrich	P8038	Used for rat tissue at 0.5 mg/mL (activity ≥ 3.5 U/mg)
Proteinase XXIV	Sigma Aldrich	P5147	Used for human tissue at 0.5 mg/mL (activity: 7-14 U/mg)
Material
Cell analyzer (LSR Fortessa)	BD Bioscience	649225
Centrifuge tube, 15 mL	Corning	430790
Centrifuge tube, 50 mL	Corning	430829
Compact shaker	Edmund Bühler	KS-15 B control	Agitation direction: horizontal
Disposable plastic pasteur-pipettes	Carl Roth	EA65.1	For cell trituration use only pipettes with an inner tip diameter ≥2 mm
Forceps	FST	11271-30
Heatblock	VWR	BARN88880030
Nylon net filter, 180 µm	Merck	NY8H04700
TC Dish 100, Standard	Sarstedt	83.3902
TC Dish 35, Standard	Sarstedt	83.3900
TC Dish 60, Standard	Sarstedt	83.3901
Vibratome (VT1200S)	Leica	1491200S001	Includes VibroCheck for infrared-assisted correction of z-deflection