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Harvesting Prevascularized Smooth Muscle Cell Sheets from Common Polystyrene Culture Dishes

Penelitian - Scientists report a novel cell sheet technology based on a coculture system in which SMCs are cocultured with EPCs on common polystyrene dishes. The method is simple, cost-effective and highly repeatable.

The most common strategy in tissue engineering is based on the incorporation of seed cells into biodegradable scaffolds. However, inflammatory responses and pathological fibrosis may occur upon the degradation of these scaffolds. Low cellularity within the scaffolds is also a limitation of this approach.

Penelitian Harvesting Prevascularized Smooth Muscle Cell Sheets from Common Polystyrene Culture Dishes

Cell sheet engineering has recently emerged as a promising strategy for scaffold-free tissue engineering. This method involves enzyme-free detachment of cells and their extracellular matrix (ECM) from the culture surface. Cell sheets are directly transplantable and three-dimensional engineered tissues can be fabricated with various kinds of cell sheets depending on the histological structure.

The primary method of harvesting cell sheets is the use of polystyrene culture surfaces coated with the temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm), which has proven to be effective for various cells, such as cardiomyocytes, smooth muscle cells (SMCs), and hepatocytes.

However, this technique has potential limitations by nature. The cell culture needs to be carefully performed as soon as possible to avoid cell detachment. Second, the detachment process requires over 40 min at a lower temperature that may alter gene expression or cellular function.

Fabricating PIPAAm-coated surfaces requires specialized facilities and materials, which are not easily available in most laboratories. Although temperature-responsive culture dishes are now commercially available, they are extremely expensive, which restricts their widespread use.

Other less invasive cell harvesting methods have been researched to improve cell sheet engineering, such as light-induced, electrochemistry-induced, pH change-induced and non-proteolytic enzyme methods. However, these methods may have the risk of inducing cell damage during harvest and require complicated equipment or techniques.

The main principle of cell sheet engineering is that cells seeded on the surface grow and strongly adhere to each other, after which the cultured cells can be detached from the surface with a minimally invasive method, keeping ECM and cell junctions intact. Endothelial progenitor cells (EPCs) are precursor cells of vascular endothelial cells (ECs) and can promote the neovascularization of engineered tissues.

Fang Chen of the Shanghai Jiao Tong University and team cocultured SMCs and EPCs on polystyrene culture dishes. After the cells were 90% confluent, trypsinization was performed, resulting in production of fragmented cell sheets, as opposed to single-cell suspensions.

“We found that an intact and highly viable cell sheet could be harvested using mechanical methods when SMCs and EPCs were cocultured on common polystyrene dishes at a ratio of 6:1 for 5 to 6 days; the method is simple, cost-effective and highly repeatable,” Chen said.

The cocultured cell sheet contained capillary-like networks and could secrete a variety of angiogenic factors. In vivo studies proved that the cocultured cell sheets were more favorable for the fabrication of vascularized smooth muscle tissues compared to single SMC sheets. This study provides a promising avenue for smooth muscle tissue engineering.

“This suggested cocultured SMCs and EPCs secreted abundant adhesion molecules and formed strong ECM. Thus, it is feasible to harvest cell sheets using mechanical methods by continuing the cell culture to the point when increased adherence between cells exceeds the adhesion between cells and the culture surface,” Chen said.

Journal : Zhiming Jia et al. Harvesting prevascularized smooth muscle cell sheets from common polystyrene culture dishes, PLOS ONE, September 26, 2018, DOI:10.1371/journal.pone.0204677



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