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

Zhang Hui-juan; Cong Shan; Liang Mei-ping; Liu Jun-ping; Huang Li-gang; Song Jin; Cao Gui-fang

doi:10.3969/j.issn.2095-4344.2014.06.020

Abstract：BACKGROUND:Extraction methods of human amniotic mesenchymal stem cells are inconsistent in the number of cells. <br> OBJECTIVE:To explore the optimal method to in vitro isolate and culture human amniotic mesenchymal stem cells. <br> METHODS:Under sterile conditions, ful-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 fol owing three methods:(1) 0.05 g/L trypsin digestion for 10 minutes fol owed by 0.75 g/L col agenase digestion for 60 minutes;(2) 0.75 g/L col agenase I for 120 minutes;(3) co-digestion with 0.05 g/L trypsin and 0.75 g/L col agenase for 60 minutes. In experiment 2, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes fol owed by 0.75 g/L col agenase 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, fol owed by 0.75 g/L col agenase digestion for 60 minutes;(2) 0.05 g/L trypsin digestion for 40 minutes×2, fol owed by 0.75 g/L col agenase digestion for 60 minutes. In experiment 4, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes×2, fol owed by 1 g/L col agenase digestion for 60 minutes. Fol owing morphology observation under a microscope, we studied the most suitable method for isolating human amniotic mesenchymal stem cells. <br> RESULTS AND CONCLUSION:Digestion with 0.05 g/L trypsin for 30 minutes twice fol owed by 1 g/L of col agenase 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.