表达重组人红细胞生成素细胞株的高密度、高表达培养

用生物反应器培养能表达重组人红细胞生成素的工程细胞，使其达到高密度高表达。方法：先将工程细胞在含有５％胎牛血清的 ＤＭＥＭ－Ｆ１２培养基中培养，待细胞总数达到 ２×１０~８个以上时，接种到５ Ｌ生物反应器中。用含有血清的培养基培养 ７ｄ后转为无血清ＣＨＯ专用培养基，继续培养 ３０ ｄ。在整个培养过程中，采用流加的方式连续培养，根据情况随时测定培养基中葡萄糖浓度，使其保持高于０．５ｇ／Ｌ，同时监测氨及乳酸盐浓度，避免浓度过高。每日采样测定收获液中红细胞生成素含量与活性，培养结束后，以０．２５％胰酶消化载体，待细胞全部脱落后计数细胞总量。 结果：细胞密度达 ６×１０~６个／ｍｌ培养液，表达量约 ３０ ０００ ＩＵ／ｍｌ左右，且表达的红细胞生成素具有较高的生物比活性。结论：在较为适当的培养条件下，利用生物反应器可使工程细胞的培养达到高密度、高表达。

High Density and High Expression Culture of Recombinant Human Erythropoietin Expressing Cell Line

Purpose: Bioreactor was used for the culture of recombinant erythropoietin expressing CHO cell line, in order to realize high density and high expression culture. Methods: Expressing cell line was first culured in flasks with DMEM-F12 medium containing 5% serum. When the total number of the cells reached about 2 × 10~8, they were inoculated into the 5 liters bioreactor. After 7 days culture with medium containing serum, and the serum- free medium was utilized to continue the culture for another 30 days. Based on the growth situation, continuous culture was performed by Per fusion method. Glucose concentration was kept above 0. 5 g/L. Lactate and ammonia were also measured at the same time to avoid their accumulation. Sample was taken everyday for the analysis of EPO expression in the harvest medium. When the culture was over, 0. 25 % trypsin was used to digest the carriers, and the total cell number was counted after the cells dropped from the carriers. Results: The cell density reached 6 ×10~6/ml medium, the expression level was about 30 000 IU/ml, and the expressed EPO had a relatively high biological specific activity. Conclusion: Under adequate culture conditions, high density and high expression culture of the recombinant cell line could be realized by using bioreactor.