In Vitro Matrix Assembly Induced by Critical Assembly Concentration (CAC)

Abstract

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.

Quantifying extracellular matrix turnover in human lung scaffold cultures

“…These experiments revealed that the acellular scaffold altered the matrisome production by increasing the proportional amount of core ECM proteins, most notably for the proteoglycans. The proportional increase of proteoglycans in the scaffolds may be a consequence of the pre-existing and organized 3D ECM structure promoting either increased production of proteoglycans or proper integration of the produced proteoglycans into the existing ECM 49 . In the monolayer cultures failure to incorporate the synthesized matrisome proteins may lead to secretion into the cell media, which was not analyzed in this experiment.…”