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Research

EFFICIENT DECELLULARIZATION OF EXTRACELLULAR MATRIX-RICH CELL-DERIVED MATRICES

The European Orthopaedic Research Society (EORS) 2018 Meeting, PART 1, Galway, Ireland, September 2018.



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.


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