原代小鼠肝细胞培养方法的比较

doi:10.3969/j.issn.1004-616x.2016.02.009

Abstract

Abstract: 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.

Key words: mouse, primary hepatocyte culture, dimethyl sulfoxide, epidermal growth factor

CLC Number:

R114

WANG Shan, BAI Qing, WANG Fangping, LI Huiyao, CHEN Shen, HE Zhini, WANG Qing, CHEN Wen, CHEN Liping. Comparison of different culture methods for mouse primary hepatocyte[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(2): 125-130.

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Comparison of different culture methods for mouse primary hepatocyte

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