[Experimental study on adipose-derived stem cells amplified by silk fibroin/poly- L-lactic acid microcarriers in vitro].

ABSTRACT

OBJECTIVE: To investigate the effect of silk fibroin-poly- L-lactic acid (SF-PLLA) microcarriers on the expansion and differentiation of adipose-derived stem cells (ADSCs). METHODS: ADSCs were extracted from adipose tissue donated voluntarily by patients undergoing liposuction by enzymatic digestion. The 3rd generation ADSCs were inoculated on CultiSpher G and SF-PLLA microcarriers (set up as groups A and B, respectively), and cultured in the rotary cell culture system. ADSCs cultured in normal two-dimensional plane were used as the control group (group C). Scanning electron microscope was used to observe the microcarriers structure and cell growth. Live/Dead staining and confocal fluorescence microscope was used to observe the distribution and survival condition of cells on two microcarriers. DNA quantification was used to assess cell proliferation on two microcarriers. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect chondrogenesis, osteogenesis, and adipogenesis related gene expression of ADSCs in 3 groups cultured for 18 days. Flow cytometry was used to identify the MSCs surface markers of ADSCs in 3 groups cultured for 18 days, and differential experiments were made to identify differentiation ability of the harvested cells. RESULTS: ADSCs could be adhered to and efficiently amplified on the two microcarriers. After 18 days of cultivation, the total increment of ADSCs of the two microcarriers were similar ( P>0.05). qRT-PCR results showed that chondrogenesis related genes (aggrecan, cartilage oligomeric matrix protein, SOX9) were significantly up-regulated for ADSCs on SF-PLLA microcarriers and adipogenesis related genes (peroxisome proliferator-activated receptor γ, lipoprotein lipase, ADIPOQ) were significantly up-regulated for ADSCs on CultiSpher G microcarriers, all showing significant differences ( P<0.05). Flow cytometry and differentiation identification proved that the harvested cells of the two groups were still ADSCs. CONCLUSION: The ADSCs can be amplified by SF-PLLA microcarriers, and the chondrogenic differential ability of harvested cells was up-regulated while the adipogenic differential was down-regulated.