Effect of Alpha-Ketoglutarate on Growth and Metabolism of Cells Cultured on Three-Dimensional Cryogel Matrix

Abstract

Alpha- ketoglutarate (α-KG) is a well-known intermediate of Kreb's cycle and thus one of the important candidates in the role of cellular metabolism. The aim was to investigate the effect of α-KG on the growth and proliferation of different cells (fibroblasts and chondrocytes). Further change in the consumption of glucose and release of ammonia in the media, upon the addition of α-KG was also examined. NIH3T3 and chondrocytes were seeded on two different macroporous cryogel matrices synthesized by poly (hydroxyethylmethaacrylate)-gelatin (HG) and alginate-gelatin (AG). Higher proliferation was observed for both the cell lines in 2-D as well as 3-D where α-KG was added to the media compared to the controls. It was evident that the chondrocytes were metabolically more active in the case of α-KG containing samples as early extracellular matrix (ECM) accumulation was observed. In comparison to the 6-8 weeks duration required by the chondrocytes for ECM accumulation in normal in-vitro culture conditions, α-KG containing samples showed an earlier accumulation within 3 weeks. In order to further validate the results, scanning electron microscopic (SEM) analysis was performed showed high ECM deposition and cells embedded in the matrix. Homogenous distribution of cells on both the synthesized matrix was reported using 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) staining. The results clearly showed reduction in ammonia concentration in α-KG containing samples thus leading to decreased ammonia toxicity that builds up during long cell culture conditions thereby enhancing the proliferation and metabolic activity. Thus α-KG can be used potentially for long batch-cultures for the production of vaccines or antibody as well as can play an important role as a bioactive molecule for in vitro neo-cartilage generation.