Isolation and culture of human amniotic mesenchymal stem cells: proper digestion time and concentrations of trypsin and collagenase

doi:10.3969/j.issn.2095-4344.2014.06.020

Abstract

Abstract:

BACKGROUND: Extraction methods of human amniotic mesenchymal stem cells are inconsistent in the number of cells.

OBJECTIVE: To explore the optimal method to in vitro isolate and culture human amniotic mesenchymal stem cells.

METHODS: Under sterile conditions, full-term cesarean fetal amniotic membrane was cut into pieces, then to isolate human amniotic mesenchymal stem cells by seven methods in four experiments. In experiment 1, human amniotic mesenchymal stem cells were isolated by the following three methods: (1) 0.05 g/L trypsin digestion for 10 minutes followed by 0.75 g/L collagenase digestion for 60 minutes; (2) 0.75 g/L collagenase I for 120 minutes; (3) co-digestion with 0.05 g/L trypsin and 0.75 g/L collagenase for 60 minutes. In experiment 2, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes followed by 0.75 g/L collagenase digestion for 30 minutes. In experiment 3, the samples were digested by two methods: (1) 0.05 g/L trypsin digestion for 30 minutes×2, followed by 0.75 g/L collagenase digestion for 60 minutes; (2) 0.05 g/L trypsin digestion for 40 minutes×2, followed by 0.75 g/L collagenase digestion for 60 minutes. In experiment 4, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes×2, followed by 1 g/L collagenase digestion for 60 minutes. Following morphology observation under a microscope, we studied the most suitable method for isolating human amniotic mesenchymal stem cells.

RESULTS AND CONCLUSION: Digestion with 0.05 g/L trypsin for 30 minutes twice followed by 1 g/L of collagenase digestion of 60 minutes was the most suitable isolation and culture condition in vitro. Cells became elongated fusiform or star-shaped with rich cytoplasm, and nuclei were round with 1-3 nuts. We can harvest the most number of human amniotic mesenchymal stem cells using the method described in experiment 4.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, mesenchymal stem cells, amnion, trypsin, collagenases

CLC Number:

R394.2

Zhang Hui-juan, Cong Shan, Liang Mei-ping, Liu Jun-ping, Huang Li-gang, Song Jin, Cao Gui-fang . Isolation and culture of human amniotic mesenchymal stem cells: proper digestion time and concentrations of trypsin and collagenase[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(6): 944-949.

Figures/Tables 2

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