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

Summary

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.

Abstract

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.

Introduction

The extracellular matrix (ECM) provides not only structural support but is also important for biologic cell and tissue function¹. In the heart, the ECM participates in the regulation of pathophysiologic responses such as fibrosis, inflammation, angiogenesis, cardiomyocyte contractile function and viability, and resident progenitor cell fate. In addition to its primary components - fibrous glycoproteins, glycosaminoglycans, and proteoglycans - it contains a host of secreted growth factors, cytokines, and membranous vesicles containing nucleic acids and proteins^2,3.

It has recently become clear that acellular ECM preparations are not only invaluable for studying cell-matrix interactions, but also for potential therapeutic cell-based applications. The importance of providing an adequate environment to therapeutic cell products or engineered tissues is now widely acknowledged. Attempts have been made to combine cell suspensions or active compounds with defined biopolymeric hydrogels^4,5,6 or with protein cocktails secreted by murine sarcoma cells (i.e., Matrigel, Geltrex)⁷. However, the former have limited bioactivity, the latter are problematic in GMP-grade processes, and both lack the tissue-specific bioactivity of cardiac ECM (cECM)^{8,9,10,11,12,13}.

Decellularization of the myocardium has previously been performed by perfusion of the whole heart via the coronary vasculature^14,15. While this is possible in animal hearts, intact human hearts are rarely available. Therefore, an immersion process that allows for handling tissue samples obtained in the operating room was favored. Our "3-step" protocol contains 3 separate incubation steps namely lysis, solubilization, and DNA removal. It yields human myocardial ECM with largely preserved protein and glycosaminoglycan composition^16,17. These cECM slices allow for in vitro studies of cell-matrix interactions but are poorly suited for potential human-scale therapeutic applications. The manufacturing process was then extended to produce either lyophilized cECM microparticles or a cECM hydrogel¹⁸.

This protocol allows for the decellularization of human myocardium obtained from surgical samples, preserving the major components of myocardial extracellular matrix (ECM) and their biologic activity. This protocol is recommended when human cardiac ECM with preserved tissue-specific bioactivity is required for experimental studies of cell-matrix interactions, or when a suitable environment is needed for cell-based myocardial regeneration approaches. In principle, it is also possible to adapt this protocol to GMP-grade conditions, so that the use of processed cECM should be feasible in future therapeutic applications.

Protocol

The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Charité Medical University. All patients provided written, informed consent for use of heart tissue for experimental studies.

1. Preparation of Human Myocardium for Sectioning

1. Obtain left ventricular myocardium (size varies depending on the type of surgery) directly from the operating room and transport in cold PBS in a sterile container.
2. Remove all fat tissue with a sterile scalpel with a no. 10 blade under sterile conditions.
3. Cut the myocardium in cubes of approximately 1 x 1 x 1 cm using a scalpel.
4. Place the cubes in sterile 50 mL tubes.
   NOTE: To pause, perform step 1.5, otherwise continue with step 2.2. Avoid repeated freeze-thaw cycles to prevent protein degradation and a decrease in tissue quality.
5. Store the tubes containing the cubes at -80 °C. The tissue can be stored for several months.

2. Sectioning of the Myocardium Cubes into Slices

1. Take the tube(s) containing the prepared cubes out of the freezer.
2. Transport on ice to the cryostat.
3. Set the object and chamber temperature to -15 °C.
   NOTE: The right temperature is crucial to guarantee perfect sectioning.
4. Chill empty 50 mL tubes and stamps in the chamber or on dry ice.
5. Add a layer (approximately 0.5 - 1.0 mL) of cryosection medium onto the cold stamp and let it freeze until it is white and solid.
6. Add a second layer of cryosection medium and place a myocardium cube onto this layer.
7. Make sure the cryosection medium and myocardium are completely frozen before you continue.
   NOTE: When the sample and cryosection medium are not frozen the sectioning will be impaired. Completely frozen cryosection medium turns from fluid and transparent to solid, white, and non-transparent. The myocardial cubes need at least 30 min up to 1 h to completely freeze if continuing immediately from step 1.4. When the tissue is frozen it should not deform when touched/handled with tweezers.
8. Insert stamp with frozen myocardium into the holder and tighten all screws.
9. Trim the surface of the tissue until an even sectioning surface is obtained.
10. Set sectioning thickness to 300 µm.
11. Section the frozen myocardium with automatic sectioning. This guarantees uniform cut sections. Approximately 30 - 40 sections can be sliced from one cube.
12. Place each slice after sectioning loosely in a precooled 50 mL tube.
    NOTE: Do not press the slices or pack tightly. Approximately 40 slices can fit in one tube.
13. Close the filled tubes and place on ice.
    NOTE: To pause go on with step 2.14, otherwise continue with step 3.2.
14. Store myocardium slices at -80 °C. The slices can be stored for several months.

3. Decellularization of Human Myocardium Slices

1. Take the necessary number of 50 mL tubes containing myocardium sections out of the -80 °C freezer and transport on ice.
2. Transfer the frozen sections of all 50 mL tubes from step 3.1 into one sterile beaker. Approximately 40 slices are added to the beaker per 50 mL tube. The beaker should be three times the volume of the 50 mL tubes; for example, for decellularization of one 50 mL tube use a 150 mL beaker.
3. Add 50 mL lysis solution (10 mM Tris, 0.1% w/v EDTA, pH 7.4 in H₂O) per 50 mL tube with myocardium slices.
4. Shake the myocardium slices in a sterile beaker at 100 - 150 rpm continuously for 2 h at room temperature.
5. Strain the liquid with the tissue slices through a coarse strainer. Transfer the tissue slices with a blunt tweezer to a new sterile beaker. Add 50 mL of 0.5% SDS in PBS per initial 50 mL tube of myocardium slices (e.g., use 100 mL SDS solution when two 50 mL tubes of myocardial sections are being decellularized).
6. Shake continuously for 6 h at 100 - 150 rpm at room temperature.
7. Strain the liquid with the tissue slices through a coarse strainer. Transfer the tissue slices with a blunt tweezer to a new sterile beaker and wash 3 times with 50 mL PBS for 10 min each while shaking at 150 rpm. Next, wash overnight in PBS with 1% Penicillin/Streptomycin and 1% Nystatin (PBS-P/S-N) at 100-150 rpm and 4 °C while shaking.
   NOTE: Thorough washing is crucial to completely remove any residual SDS. Remaining traces of SDS are toxic when the ECM is used in combination with cells.
8. Remove the washing solution and add 25 mL preheated FBS (37 °C) with 1% Penicillin/Streptomycin and 1% Nystatin to the decellularized slices per initial 50 mL tube of myocardium slices.
9. Incubate for 3 h at 37 °C. In this step, any remaining DNA is removed from the matrix due to the intrinsic DNase activity of the FBS.
10. Transfer the slices to a new sterile beaker and wash 3 times with 50 mL PBS for 10 min each while shaking at 150 rpm.
    NOTE: The ECM slices can be stored in PBS-P/S-N at 4 °C for several days. However, it is recommended to proceed immediately.

4. Processing the Decellularized cECM to a Powder

1. Place the ECM slices loosely in a new sterile 6-well plate. Freeze the ECM at -80 °C and lyophilize for 2 days in a lyophilizer.
   NOTE: More than one slice can be placed per well. Arrange the slices to completely cover the surface of the well. Overlapping the slices does not affect the subsequent pulverization success.
2. For the pulverization of the ECM slice, use specific milling tubes (see Table of Materials) containing 1.4 mm ceramic beads.Fill the tubes loosely with lyophilized ECM using sterile blunt tweezers. For optimal results, a total weight of ECM between 10 - 20 mg is recommended.
   NOTE: If the ECM is packed too tightly in the milling tubes the pulverization process will be negatively affected.
3. Snap-freeze the tubes in liquid nitrogen.
   CAUTION: Liquid nitrogen is extremely low temperature, wear appropriate personal protection.
4. Insert the frozen tubes into the milling machine and pulverize the ECM.
   1. Set a duration of 30 s and a maximum speed and start the device.
   2. After finishing, take out the tubes and snap-freeze again in liquid nitrogen.
   3. Repeat five times.
5. Wash the microparticles from the tubes by adding 1 mL ddH₂O per tube and shake or vortex for complete solubilizing. Filter the heterogeneous solution through a 200 µm mesh into a 50 mL tube.
6. Freeze the tube in -80 °C or liquid nitrogen.
7. Replace the standard lid of the tube with a 0.22 µm filter. This filter lid prevents the powder from flowing out of the tube but allows air circulation for water evaporation.
8. Insert the tubes in the lyophilizer and lyophilize again to remove the water from the step (step 4.5)
   NOTE: Lyophilization at this step can take up to 3 days.
9. Store powder at -80 °C until further processing or continue with step 5.1.

5. Enzyme Based Homogenizing of cECM Micro-Particles

1. Dissolve porcine pepsin in 0.01M HCl (pH 2.0) to a concentration of 1 mg/mL. Place on a rotary shaker at room temperature until it is completely dissolved.
2. Dissolve the lyophilized human ECM powder (Step 4.8) in the pepsin solution to a final concentration of 10 mg/mL. Mix thoroughly through pipetting. The total volume depends on the amount of ECM solution needed. For example, dissolve 10 mg of ECM powder in 1 mL pepsin solution.
   NOTE: Dissolving particles can be supported through gentle vortexing (1,000 - 1,500 RPM). Remaining lumps of microparticles will negatively affect the digestion.
3. Transfer the solution to 2 mL tubes with a volume of 1 mL per tube.
4. Place the tubes in a tube shaker for 48 h at 27 °C and 1,200 rpm.
5. Take the tubes out of the shaker and place on ice or a pre-cooled tube holder.
6. Mix 1/9 of the volume of ECM solution of cold 10x PBS (pH 7.4) with 1/10 of the volume of ECM solution of cold 0.1M NaOH in a precooled tube and place on ice. This mixture will neutralize the digested ECM solution and irreversibly inactivate the pepsin.
7. Add the ECM solution to the neutralization mixture and mix thoroughly through pipetting or vortexing.
8. Set the ECM to the desired concentration with 1x PBS (pH 7.4). The pH of the solution can be checked with pH paper.
   NOTE: Example calculation for 10 mL digested ECM solution:
   Add 10 mg porcine pepsin to 10 mL HCl (pH 2.0).
   Add 100 mg ECM powder to pepsin solution.
   NOTE: Example calculation for neutralizing 10 mL digested ECM solution and setting the concentration to 8 mg/mL:
   Vol_Final = c_start x Vol_start / c_Final = 10 mg/mL x 10 mL / 8 mg/mL = 12.5 mL
   Vol_10xPBS = Vol_start / 9 = 1/9 from 10 mL = 1.11 mL 10x PBS
   Vol_NaOH = Vol_start / 10 = 1/10 from 10 mL = 1 mL
   Vol_1xPBS = Vol_Final - Vol_start - Vol_10xPBS - Vol_NaOH = 12.5 mL - 10 mL - .11 mL - 1 mL = 0.39 mL

Results

The 3-step protocol for decellularization of human myocardium presented here results in near-complete removal of cellular material, while preserving the key ECM components and the fibrillar structure of ECM. After decellularization, the gross removal of cells from the tissue is evident by the change in color (Figure 1A). Histological analysis with H&E and Masson Trichrome stains revealed the complete absence of residual intact cells (...

Discussion

When preparing human myocardial ECM, the goal is to achieve the following: removal of relevant immunogenic cellular material, preservation of ECM integrity and bioactivity, sterility, non-toxicity of the end-product, GMP-process compatibility, and suitability of the product for a given application in terms of handling. By combining our 3-step decellularization protocol with further processing into microparticles or self-assembling hydrogel, human cardiac ECM material is obtained that possesses specific biological activit...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Balance	DR Precisa, Dietikon, Switzerland	Precisa XR 205SM
Blades Nr.10 Skalpell Nr.3	InstrumenteNRW, Erftstadt, Germany	SK-10-004
Cell culture plates (6-well)	Greiner, Frickenhausen, Germany	657160
Cryostat CM	Leica, Wetzlar, Germany	3050S
EDTA	Carl Roth, Karlsruhe, Germany	8043.3
Eppendorf reaction tubes (1.5 or 2 ml)	Greiner, Frickenhausen, Germany	616201, 623201
Falcon 15ml, 50ml	Greiner, Frickenhausen, Germany	188271, 227270
Fetal Bovine Serum (FBS)	Biochrome, Berlin, Germany	S 0115
Freeze Dry System	Labconco, Kansas City, USA	7670520
Freezer (-80°C)	Thermo Scientific, Waltham, MA, USA	Forma 900 Series
HCl	Carl Roth, Karlsruhe, Germany	281.1
Microtome Blades Type 819	Leica, Wetzlar, Germany	14035838925
Minilys Homogeniser	PEQLAB Biotechnologie GmbH, Erlangen, Germany	91-PCSM
NaOH	Carl Roth, Karlsruhe, Germany	K021.1
Nystatin	PAN Biotech, Aidenbach, Germany	P06-07800
PBS	Thermo Scientific, Waltham, MA, USA	14190-094
Penicillin/streptomycin	Life Technologies, Darmstadt, Germany	15140122
Pepsin	Sigma-Aldrich, Taufkirchen, Germany	P6887-1G
Precellys Keramik-Kit 1.4 mm	Peqlab Biotechnolgie, Erlangen, Germany	91-PCS-CK14
Rotamax 120 Plate shaker	Heidolph, Schwabach, Germany	544-41200-00
SDS	Carl Roth, Karlsruhe, Germany	CN30.3
Stereo microscope	Leica, Wetzlar, Germany	M125
Steriflip-GP, 0,22 µm	Merck Millipore, Darmstadt, Germany	SCGP00525
Stuart analogue rocker & roller mixers	Sigma-Aldrich, Taufkirchen, Germany	Z675113-1EA
Tissue Tek O.C.T compound	Hartenstein, Wurzburg, Germany	TTEK
Transfusion set 200µm	Sarstedt, Nümbrecht, Germany	798.200.500
TRIS	Carl Roth, Karlsruhe, Germany	5429.3
vedena Skalpellgriff Fig. 3, Standard, 125 mm	Medical Highlights, Rohrdorf, Germany	CV102-003
Vortex-Genie2	Scientific Industry, New York, USA	SI-0256