The Influnce Of Different-month-old Fetal Umbilical Cord And Different Culture Methods On Isolating Mesenchymal Stem Cells

Objective: UC-MSC (Mesenchymal Stem Cells derived from human Umbilical Cord )as a new source of seed cells attracted more and more people's attention. Previous studies shows UC-MSC were not only With proliferative capacity, but also has the ability to multi-cell(neuron-like cells, Astrocytes, fat cells and Less oligodendrocyte )differentiation . Moreover, a wide variety of sources, drawing easy, particular in cell transplantation, gene therapy and tissue engineering a wide range of potential applications. However, we have learned from the past four years that isolation mesenchymal stem cells from umbilical cord were not easy. Particularly , how fast and efficient, obtained the MSC from the umbilical cord, to meet the foundation and future of clinical research is an issue which urgent need to resolve Therefore, we study the impact of different gestational age fetal umbilical cord and different training methods for the isolation of mesenchymal stem cells.Methods: 1. Human Mesenchymal stem cells culture from umbilical cord : method of tissue block: first we take the full-term umbilical cords 35cases and the small-moths umbilical cords 33 cases. Them were washed with PBS and umbilical arteries and vein were stripped off. Then emaining parts of umbilical cord were diced into small fragments of 1mm in diameter and plated in growth medium consisting of low glucose-Dulbecco's Modified Essential Media (LG-DMEM), 20%FBS, 2mL-glutamine (L- Glu), 100units/ml peniciline, and 100 ug/ml streptomycin.3-7 days after primary culture, great number of cells came out of fragments and became adherent to the flasks. When the cells reached 80% convergence.they were detached with 0.02% trypsin and were passaged. Collected the third generation of human umbilical cord mesenchymal stem cells (HUMSCs) in a plastic tube contained culture medium and preserved them in -196 nitrogen canister for further utilization. Method of Collagenase- Pancreatin digestion : we take the full-term umbilical cords 17cases and the small-moths umbilical cords 15 cases. Get the umbilical cord into super clean bench washed umbilical arteries and vein with PBS, then they were diced into small fragments of 1mm in diameter. Put the fragment into 1% Collagenase IV continue agitate and digest for 30 min at 37℃,later add 1% Pancreatin to the mixture agitate and digest for another 30 min at the same temperature. add the low glucose-Dulbecco's Modified Essential Media (LG-DMEM) include 10%FBS to end digestion, use cells sifter to filter Centrifug the filtrate including cells, inoculate the cells into T5 plastic culture flask ,after 3 or 4 days changed the culture medium ,washed off the unadherenced cells . changed the culture medium until the cells reached 80% covergence 3. Digest the stem cells adherent, add 0.20%typsin/0.02%EDTA solution ,after most of the cells became round ,add the low glucose-Dulbecco's Modified Essential Media (LG-DMEM) include 10%FBS to end digestion, then make adherent cells become suspension, 1000rmp Centrifug for 10 min, drop the supernatant, washing with the PBS,At the room temperature 1000rmp Centrifug for 10 min . Trypan blue assessment cell activity . Cells Counting. Flow cytometry analysis by FACS caliber flow cytometry with mouse monoclonal phycoerythrin (PE),fluorescein isothiocyanate (FITC) labeled antibodies, which includes PE-CD13,FITC-CD14,APC-CD29,FITC-CD31,FITC-CD34,PE-CD38,PE-CD44,PERCP-CD45,APC-CD90,FITC-CD105,PE-CD133,PE-CD166,HLA-DR。Mouse PE,FITC,PERCP,APCconjugated IgG1 were used as controls.Results: 1. Tissue primary culture : method of tissue block The full-term cases about 7 days after primary culture, adherent cells came out of fragments, forming a uniform radiating appearance. a long fusiform shape.after about 10 days ,they first reach 80-90% convergence They were detached and passaged at a ratio of 1:2-1:3. Cells became adherent and formed a compact, disordered appearance. About 30 minutes after vaccination began adherent cells, 6-8 hours adherent completely, The small-month group adherent cells came out of fragments about 4 days about five days to achieve 80% convergence the passage character was similar to the full-term group. 2 Method of Collagenase- Pancreatin digestion :on the second day we can see some various shape adherence cells .among the Fusiform shape cells we can find some colonies coformed from cobblestone cells. They may be endothelial cells derived from allantoic veins. The fusiform shape cells was MSCs, they similar to the fibroblast, with high capability of proliferation. 1 week later the adherenced cells formed colony , most of cells was fusiform shape cells. there only was a bit of endothelial cells derived from allantoic veins. They can reach 80% covergence after 2 weeks, the growth of the endothelial cells will be refrained obviously. After digest and passage we can get purificate cells . 3.Test results of flow cytometry analysis: The cells derived from small-month group expressed putative marker of MSCs, which included CD13,CD29,CD44,CD90,CD105. but not hematopoietic and endothelial cell markers, such as CD14,CD31CD34CD45.they also did not express CD106,CD117,and HLA-DR, The test result was consist with full-term group and the same as bone marrow,cord blood,fetal lung and the other tissues。CD13, CD29, CD44, CD90 expression more than 97% shows cellular components relatively uniform. 4.Statistical analysis showed that:The small-month group significantly earlier the full-term group in the time interval of first come out of fragment,the cell account reach 10~6 and reach 80-90% convergence, The small-month group has higher achievement ratio than full-term group statistically significant. (P < 0.05 ),the passage character was similar between the two group. there is no significant difference( P >0.05 ). The tissue block has higher achievement ratio than the Collagenase Pancreatin digestion group, statistically significant (P < 0.05 ).Conclusions: The small-month group was shorter significantly than the full-term group in the time interval during the primary culture. You may get the required cells in a short period of time. there is no difference in passage character in diferrent months aged UC-MSC. The UC-MSC was easy to amplification and purification in vitro ,and with good proliferation ability, The flow cytometry analysis test result was consist with full-term group and the same as bone marrow,cord blood,fetal lung and the other tissues。...