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.
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.
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 tissues2,3.
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 vitro6,7.
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.
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…
The authors have nothing to disclose.
None.
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 |
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