Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating

Summary

Fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. After decellularization, this substrate can be used for the culture and study of other cardiac cells and cell-matrix interactions.

Abstract

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Introduction

All cells are located in vivo in a specialized microenvironment in which they can survive and carry out their specific functions. Within any given tissue, the cells are surrounded by an extracellular matrix composed of fibrillar and non-fibrillar proteins, and fundamental substances rich in glycosaminoglycans¹. The qualitative and quantitative changes in the matrix content influence cell biology, controlling processes such as cell proliferation, apoptosis, migration, or differentiation. Hence, efforts are invested in recreating this microenvironment for in vitro studies of cells from different tissues^2,3.

The myocardium consists of cardiomyocytes and an even larger quantity of fibroblasts that play a critical role in producing and maintaining the extracellular matrix within the myocardium⁴. Throughout life, the composition of the extracellular matrix can change in response to various normal and pathological factors. These modifications in extracellular matrix composition have a significant impact on the structure and biomechanical characteristics of the myocardium⁵. Accordingly, it should be advantageous for understanding cell-matrix interactions within the human myocardium if the microenvironment specific to different ages or pathological conditions was reproduced in vitro^6,7.

The method described here aims to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo.

Cardiovascular research presents specific challenges, including the difficulty in obtaining samples from living donors or patients and culturing human cardiac cells⁸. The method presented here addresses these challenges by enabling the acquisition of cardiac fibroblasts even from small bioptic fragments of human myocardium and culturing isolated cardiac cells in vitro on their native extracellular matrix typical of the human myocardium.

While current efforts focus on developing 3D scaffolds of biofartificial synthetic or natural polymers that mimic the biomechanical properties of normal myocardium⁹, they overlook the cell-matrix interactions and signaling that occur in both normal and pathological conditions. Since the cardiac ECM is synthesized by cardiac fibroblasts derived from the human heart, its composition is determined by the activity of these cells, which changes in response to various physiological and pathological conditions, thereby allowing the study of its specific influence on cardiac cell biology¹⁰.

The current protocol was specifically designed for human cardiac tissue, but its scientific basis should also apply to other organs, especially those with low regeneration potential, intense fibrosis, and scarring influencing overall structure and function, as well as limited sample numbers and sizes.

Protocol

Cardiac tissues were obtained from patients with end-stage heart failure due to ischemic cardiopathy who were undergoing heart transplantation. All specimens used for the experiments were collected with patient consent and without patient identifiers, following the protocols approved by the ethical committee of the University of Naples Federico II and in accordance with the principles outlined in the Declaration of Helsinki. The details of all the reagents and equipment used for the study are listed in the Table of Materials.

1. Experiment preparation

1. Prepare sterile tools/apparatus: One large pair of surgical scissors, two sets of fine forceps, two pairs of microdissecting scissors, 1 L sterile bottle, 500 mL sterile bottle, and a 250 mL sterile bottle.
2. Disinfect the 22 mm x 22 mm cover glasses with 70% ethanol for a few seconds. Aspirate the ethanol using a 10 mL serological pipette, let the cover glasses dry in the oven at 37 °C, and sterilize them in an autoclave.
3. Dissolve commercially available powdered salts in sterile double-distilled water. In a 1 L sterile bottle, add 0.35 g of sodium bicarbonate powder to prepare 1 L of Hank's balanced salt solution (HBSS) at pH 7.4. Sterilize the solution using 0.22 µm filter membranes under a sterile hood and store at 4 °C until use.
4. Weigh 0.1 g of potassium phosphate monobasic, 4.0 g of sodium chloride, 0.1 g of potassium chloride, and 0.575 g of sodium phosphate dibasic to prepare 500 mL of 1x phosphate-buffered saline (PBS). Dissolve the components in sterile double-distilled water and then check the pH value (7.4). Sterilize under a sterile hood by filtration and store at 4 °C until use.
5. Add 5 mL of fetal bovine serum (FBS) to 45 mL of HBSS to prepare a final volume of 50 mL of trypsin stop solution (TSS). Store at 4 °C until use.
6. Measure 223.75 mL of Dulbecco's modified Eagle medium (DMEM) supplemented with 25 mL of FBS (final concentration, 10%) and add 1.25 mL penicillin and streptomycin solution (pen/strep, final concentration, 0.5%) to prepare DMEM for fibroblast isolation and culture. Store at 4 °C until use.
7. Prepare 0.2% (w/v) gelatin solution by adding 0.2 g of gelatin from porcine skin powder to 100 mL of PBS.
   NOTE: The components and the respective concentrations for each solution utilized in the study are provided in Table 1.

2. Primitive culture of human cardiac fibroblasts by outgrowth from atrial myocardium fragments

NOTES: Steps 2.2-2.3 should be performed in sterile conditions.The protocol is validated for atrial samples approximately 0.3 cm in diameter, which is a typical dimension for biopsy specimens and allows the isolation of approximately 2 x 10⁶ fibroblasts.

1. Wash the freshly obtained sample of the atrium in a 100 mm glass plate with sterile HBSS, gently shaking it to remove the blood. Repeat this step three times, changing HBSS and the plate at each wash.
2. Place the sample in a 100 mm plate previously wetted with a small volume of HBSS and chop finely with crossed scalpels to about 2 mm cubes.
3. Place four small fragments of myocardium in a 35 mm plate, close enough to lie approximately at the angles of a 22 mm x 22 mm cover glass, place the sterile coverglass over the fragments, apply gentle pressure, and add 1.5 mL of DMEM.
4. Incubate the culture plates at 37 °C in 5% CO₂ for about 15 days or until cells reach 85% confluence, checking daily the outgrowth of cells at an inverted phase-contrast microscope and changing the culture medium every 3 days.

3. Subculture of human cardiac fibroblasts by warm trypsinization

NOTES: The steps reported below must be performed in sterile conditions. As the fibroblasts can migrate from the explanted tissue along the surface of the cover glass and culture plate, it is recommended to process both for the first subculture.

1. Lift the cover glass with fine, sterile forceps, place it upside-down in a new 35 mm plate, and rinse with sterile PBS.
2. Remove myocardium fragments, discard them, and rinse the 35 mm plate with outgrown cells with sterile PBS.
3. Discard the rinse and add 1 mL of 0.25% trypsin-EDTA (Ethylenediaminetetraacetic acid) to each plate. Incubate for 5 min at 37 °C with 5% CO₂. Confirm cell detachment at a microscope. The cells should be fully rounded up and detached, forming small clumps.
4. Block the trypsinization by adding 2 mL of TSS to each 35 mm plate and collect the suspension into a sterile 15 mL tube, centrifuge at 400 x g for 5 min at 4 °C.
5. Aspirate the supernatant using a 5 mL serological pipette, resuspend the pellet in 3 mL of DMEM and place cell suspension into a 60 mm plate.
6. Incubate such obtained cells in subculture 1 (or passage 1) in 3 mL of DMEM at 37 °C with 5% CO₂, changing the medium every three days until the cells reach 75% confluence.
7. Subculture the fibroblasts that reached 75% confluence, repeating warm trypsinization and splitting the cells 1:3 into new 60 mm plates up to two times (passages 2 and 3).

4. Extracellular matrix deposition

1. Prepare gelatin-coated dishes by adding 3 mL of sterile 0.2% gelatin solution to each 60 mm plate and incubating at 37 °C for 2 h; then aspirate the solution and add 3 mL of sterile PBS until use.
2. Detach the cardiac fibroblasts by warm trypsinization and subculture them at high density (15 x 10³ cells per cm²) on 60 mm gelatin-coated plates.
3. Culture the fibroblasts in a confluent state for up to 21 days, allowing for extracellular matrix synthesis and deposition. Replace 50% of the medium with fresh medium every 3 days.

5. Extracellular matrix decellularization

NOTE: Steps 5.3-5.4 should be performed with gentle shaking to dislodge only cells without detaching the extracellular matrix.

1. Prepare a decellularization solution containing 0.25% Triton X-100 and 10 mM NH₄OH in PBS (calculate the final volume, considering 1 mL for every 60 mm plate).
2. Remove the culture medium and rinse the plates with PBS. Discard the rinse.
3. Add 1 mL of decellularization solution and observe the plates at an inverted phase-contrast microscope; after 1-2 min, when the cells are no longer discernible, gently add 4 mL of PBS to dilute the decellularization solution.
4. Remove the diluted solution and gently rinse the plates with PBS; discard the rinse.
5. Add 1 mL of PBS and store the ECM-coated plates at 4 °C until further use.

Results

The outgrowth of fibroblasts from the small fragments of native myocardium placed in culture was observed within 3-5 days (Figure 1).

In the subsequent days, the number of fibroblasts continued to increase, possibly due to sustained outgrowth from the cardiac tissue specimen and the proliferation of migrated fibroblasts on the dish surface. It should not be expected that all myocardium fragments obtained by mechanical disaggregation with a scalpel yield the same n...

Discussion

The fibroblasts isolated from human heart samples were cultured to confluence for 21 days to synthesize and deposit the extracellular matrix, forming a cohesive layer firmly adherent to the surface of the culture plate. Subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of myocardium-specific extracellular matrix on other cells within the cardiac tissue.

The concept of using a natural and tissue-specific subs...

Materials

Name	Company	Catalog Number	Comments
1 L laboratory bottle	VWR	215-1595	Clean and autoclave before use
10 mL serological pipet	Falcon	357551	Sterile,  polystyrene
100 mm glass plate	VWR	391-0578	Clean and autoclave before use
100 mm plates	Falcon	351029	Treated, sterile cell culture dish
15 mL sterile tubes	Falcon	352097	Centrifuge sterile tubes, polypropylene
22 mm x 22 mm cover glasses	VWR	631-1570	Autoclave before use
25 mL serological pipet	Falcon	357525	Sterile,  polystyrene
250 mL laboratory bottle	VWR	215-1593	Clean and autoclave before use
35 mm plates	Falcon	353001	Treated, sterile cell culture dish
5 mL serological pipet	Falcon	357543	Sterile, polystyrene
50 mL sterile tubes	Falcon	352098	Centrifuge sterile tubes, polypropylene
500 mL laboratory bottle	VWR	215-1594	Clean and autoclave before use
60 mm plates	Falcon	353004	Treated, sterile cell culture dish
Ammonium hydroxide (NH4OH)	Sigma- Aldrich	338818	Liquid
Disposable scalpels	VWR	233-5526	Sterile and disposable
Dulbecco's Modified Eagle Medium (DMEM)	Sigma- Aldrich	D6429-500ml	Store at 2-8 °C; avoid exposure to light
Fetal Bovine Serum (FBS)	Sigma- Aldrich	F9665-500ml	Store at -20 °C. The serum should be aliquoted into smaller working volumes
Fine forceps	VWR	232-1317	Clean and autoclave before use
Gelatin from porcine skin	Sigma- Aldrich	G1890-100G	Commercial Powder
Hank's Balanced Salt Solution (HBSS)	Sigma- Aldrich	H1387-1L	Powder
Large surgical scissors	VWR	233-1211	Clean and autoclave before use
Microdissecting scissors	Sigma- Aldrich	S3146	Clean and autoclave before use
Penicillin and Streptomycin	Sigma- Aldrich	P4333-100ml	Store at -20°C. The solution  should be aliquoted into smaller working volumes
Potassium Chloride	Sigma- Aldrich	P9333	Powder
Potassium Phosphate Monobasic	Sigma- Aldrich	P5665	Powder
Sodium Chloride	Sigma- Aldrich	S7653	Powder
Sodium Phosphate Dibasic	Sigma- Aldrich	94046	Powder
Stericup Filters	Millipore	S2GPU05RE	Sterile and disposable 0.22 mm filter membranes
Triton X-100	Sigma- Aldrich	9002-93-1	Liquid
Trypsin-EDTA	Sigma- Aldrich	T4049-100ml	Store at -20 °C. It should be aliquoted into smaller working volumes