微载体技术高密度培养MRC-5细胞初探

Pilot study on microcarrier-based high density cultivation of MRC-5 cells

目的 通过探索MRC-5细胞的微载体培养条件,以期建立MRC-5细胞的高密度培养方法.方法 用3种不同培养基（MEM、M199、DMEM）分别培养3.0×104/mL的MRC-5细胞,观察它们的细胞增殖及传代能力；在Spinner培养系统中,用1.0 g/L Cytopore 2和3.0 g/L Cytodex 3分别培养密度为3.70× 105/mL和2.98×105/mL的MRC-5细胞,观察两种载体对细胞生长代谢和细胞密度的影响,筛选出最适宜的培养基及微载体.使用5 L CelliGen310生物反应器对筛选获得的培养基及微载体进行MRC-5细胞的灌流培养初步摸索.结果 MRC-5细胞在MEM、M199、DMEM培养基中培养96 h细胞分别增殖了7.0、4.4和3.0倍；在MEM培养基中连续传代至5次以上,细胞增殖稳定,1：3传代72 h形成致密单层,而在M199和DMEM培养基中连续传代均未能获得理想的细胞形态.经96～120 h的培养后,使用1.0 g/L Cytopore 2培养的MRC-5细胞密度达到2.20× 106/mL高于使用3.0 g/L Cytodex 3的1.12×106/mL,且前者葡萄糖和乳酸代谢较后者更为活跃.首选MEM和Cytopore 2作为MRC-5细胞的灌流培养体系.在5L生物反应器中以1.0 g/L Cytopore 2和2.5LMEM培养基培养MRC-5细胞至144h,细胞密度达到1.54×106/mL.结论 初步建立的MEM Cytopore 2微载体细胞培养方法能够获得高密度、活性良好的MRC-5传代细胞.

Objective To create a high density MRC-5 cell culture method by exploring the microcarrierbased cell culture conditions.Methods MRC-5 cells （3.0×104/mL） were cultured in three different media （MEM,M199,DMEM） and their proliferation and passage capacity were observed.In Spinner culture system,MRC-5 cells were cultured at the density of 3.70×105/mL and 2.98×105/mL with 1.0 g/L Cytopore 2 and 3.0 g/L Cytodex 3 microcarriers,respectively.The effect of the two microcarriers on cell growth,metabolism and cell density was recorded to screen out the most appropriate medium and microcarrier.The 5 L CelliGen310 bioreactor was used to explore the MRC-5 perfusion culture condition by the established medium and microcarrier.Results After 96 h cultured in MEM,M199 and DMEM media,MRC-5 cells were proliferated 7.0,4.4,and 3.0 times,respectively.MRC-5 cells cultured in MEM medium generated more than 5 passages and formed dense monolayer after 72 h with passage ratio 1：3,while MRC-5 cells in M199 and DMEM media failed to obtain the ideal cell morphology during the passage.After 96-120 h cultured in Spinner system,the concentration of MRC-5 cells in 1.0 g/L Cytopore2 was higher than that in 3.0 g/L Cydodex3 （2.20×106/mL vs 1.12×106/mL）,with more active metabolism of glucose and lactic acid.MEM and Cytopore2 were selected as the MRC-5 perfusion culture system.The concentration of MRC-5 cells reached 1.54×106/mL when cultured with 1.0 g/L Cytopore2 and 2.5 L MEM in 5 L bioreactor for 144 h.Conclusions The initial establishment of cell culture method based on Cytopore 2 microcarrier and MEM medium can obtain high density MRC-5 passage cells with good viability.