原代小鼠肝细胞培养方法的比较

目的: 比较3种不同培养条件下原代小鼠肝细胞的形态以及肝细胞功能,寻找适合于原代小鼠肝细胞体外培养的培养基配方。方法: 采用改良Seglen两步胶原酶灌注法分离原代小鼠肝细胞,糖原染色观察细胞纯度,在贴壁4 h后用基础培养基、基础培养基加DMSO(DMSO组)、基础培养基加DMSO和EGF(DMSO+EGF组)等3种不同的条件培养肝细胞。镜下观察肝细胞形态,用MTT法检测细胞活力,生化分析仪检测细胞培养上清液中乳酸脱氢酶(LDH)水平以及用ELISA法检测细胞培养上清液中白蛋白水平。结果: 成功分离小鼠原代肝细胞,肝细胞纯度达95%以上;原代肝细胞培养至96 h时,DMSO组仍能较好地维持细胞形态,基础培养基组的细胞活力比DMSO组和DMSO+EGF组分别降低了66.87%和67.16%(P<0.05),且基础培养基组的LDH水平均高于DMSO组和DMSO+EGF组(P<0.05)。此外DMSO组在96 h时仍能维持较高水平的白蛋白分泌,比基础培养基组和DMSO+EGF组分别增加了185%和24.2% (P<0.05)。结论: 小鼠原代肝细胞培养基中加入DMSO对于维持肝细胞的形态和功能有促进作用,本研究为体外原代肝细胞培养模型的建立和优化提供了实用的方法。

Comparison of different culture methods for mouse primary hepatocyte

OBJECTIVE: To identify a better maintenance medium for primary hepatocyte culture in vitro, we compare the cellular morphology and function of mouse primary hepatocytes under three different culture conditions. METHODS: Primary hepatocytes were isolated by using a modified two-step collagenase perfusion procedure as described by Seglen. The purity of hepatocytes were determined by PAS staining. After plating for 4 h, the medium was replaced with three different maintenance medium (base medium, base medium supplemented with DMSO, base medium supplemented with DMSO and EGF). The cellular morphology was observed under an inverted microscope. The cell viability was measured using the MTT assay, the concentrations of lactate dehydrogenase (LDH) was detected by automatic biochemical analyzer and the albumin level was examined using ELISA. RESULTS: Mouse primary hepatocytes were successfully isolated with purity more than 95%. When primary hepatocytes were cultured for 96 h, only the DMSO group had better cell morphology. Viability of cells in the base medium group was reduced by 66.87% and 67.16% compared with the DMSO and the DMSO+EGF groups (P<0.05), respectively. The LDH level of the base medium group was higher than the DMSO and the DMSO+EGF groups (P<0.05). In addition, cells in the DMSO group had high level of albumin secretion at 96h which was 185% and 24.2% higher than the base medium and the DMSO+EGF group (P<0.05), respectively. CONCLUSION: DMSO addition to maintenance medium supported primary hepatocytes to maintain its morphology and function. Our study provides an applicable method for optimization of primary hepatocytes in culture.