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 …

Representative 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

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