Abstract
Macromolecular crowding (MMC) accelerates matrix deposition through excluded volume effect (EVE). Herein, we ventured to identify the optimal decellularisation protocol of MMC enhanced fibroblast cultures as a new cell formed platform model. Human dermal fibroblasts (hDF), human lung fibroblasts (hLF), and human mammary fibroblasts (hMF)seeded at 50,000 cells/cm2 were cultured for 10 days without and with MMC (100 μg/mL carrageenan) and 100 μM L-ascorbic acid phosphate. Subsequently, the cultured cell layers were decellularised using various decellularisation protocols [i.e., ammonium hydroxide (NH4OH), sodium deoxycholate (DOC), SDS-EDTA mixed buffer, and nonident P40 (NP40)]. SDS-PAGE, hydroxyproline assay, sGAG assay, SEM, histological staining (i.e., picrosirius red stain and H&E), immunocytochemistry (i.e., collagen I, III and fibronectin), PicoGreen® assay. SDS-PAGE with complementary density and hydroxyproline analysis for assessing collagen deposition, and sGAG assay for total sGAG content assessment demonstrated significantly increased (p< 0.001) in the presence of MMC. SEM, histological and immunocytochemistry displayed enhanced ECM deposition, integrity, and maintenance of the matrix composition in the presence of MMC. PicoGreen® assay revealed efficient decellularisation with significant removal of DNA (p <0.001) in all matrices. MMC can be used effectively to accelerate ECM deposition by fibroblast from various tissue sources, to facilitate production of cell-derived matrix-rich constructs feasible as robust platform models.
