Comparison of reproductive activity of mesenchymal stem cells from human amniotic membrane stored for different time domain

Background: In previous studies, mesenchymal stem cells are harvested from fresh amniotic membrane. With regard to domain and time limitations, how to keep amniotic membrane is a practical problem. Objective: To compare the proliferative capacity of amniotic mesenchymal stem cells collected from human amniotic membrane at different preservation time. Design, time and setting: The in vitro control cell experiment was performed at the Department of Bioengineering, College of Basic Medical Sciences, Zhengzhou University from January 2007 to February 2008. Materials: Totally 12 samples of fresh amniotic membrane were harvested from healthy puerperants, who were pregnant for 38 weeks and negative for examinations, including 5 items of hepatitis B, HCV, HIV and syphilis. Methods: Each fresh amniotic membrane, was sterilely divided into three groups Group A (fresh for one hour), Group B (at 4 °C for 24 to 36 hours) and Group C (at 4 °C for 48 to 60 hours). Samples in the three groups were cut into pieces, and then digested in trypsin and collagenase. After filtering, mononuclear cells were collected, and incubated at the density of 2 × 10⁸ L^-1 in DMEM/F12 medium containing fetal bovine serum and basic fibroblast growth factor. Cells were purified and amplified by adherence. Main outcome measures: Cell morphous was observed with an inverted microscope to draw growth curve. At the third passage, cells were identified by immunohistochemistry and flow cytometry. Results: After digestion, samples in the three groups contained varied single cell components, showing round, rhombus, short-spindle and polygon, with a good refraction. A mass of endothelial cells was detected. 1-3 days after inoculation, cells were in latency phase in each group. 4-7 days later, cells in the Groups A and B entered logarithmic growth phase, actively proliferated and cell number significantly increased, but cells in the Group C were still in latency phase. 7 days later, cells in the Groups A and B had been confluent, but cells in the Group C began to significantly increase. Cells in each group were positive for neuron enolase, neurofilament protein and nestin, and negative for glial fibrillary acidic protein. Cells in each group were positive for CD29 and CD44. Conclusion: Mesenchymal stem cells with reproductive activity can be isolated from human amniotic membrane stored within 60 hours. Moreover, mesenchymal stem cells from amniotic membrane stored within 36 hours have a rapid proliferation.