Application Of Different Blood Group Cord Blood Serum In Culturing For Stem Cells In Vitro

Mesenchymal stem cells (MSCs), as one of stem cells, have self-renew, boundless proliferative ability and exhibit multilineage differentiation potential towards diverse types of tissues including bone, cartilage, adipose et al. under the condition of definite induction. Therefore, Human MSCs are currently in focus regarding their clinical potential in cell therapy and tissue engineering. It is necessary to develop ex vivo culture systems to scale up the cell proliferation because the amount of cells from donors is insufficient for the clinical application. To date, fetal bovine serum (FBS) as the convinentional culture media supplement has been used for the isolation and cultivation of MSCs ex vivo or in vivo. However, FBS containing xenogeneic proteins carries an intrinsic risk of bacterial or viral contamination as well as a potential risk of prion diseases. So it is necessary to find the replacement of FBS in the proliferation and differentation of stem cells throughout long-term culturing. Of note, cord blood serum (CBS), an allogeneic source of placent, has lower immunogenicity and more distinct stimulating factors for human stem cells and it has been implicated for clinical therapy. In this work, the feasibility of using different blood group CBS substitute for FBS to expand human placenta-derived mesenchymal stem cells(hPDMSCs) was studied. Parallel experiments using FBS were also carried out for a comparative study and whether a distinct characteristic of hPDMSCs can be seen or not during the ex-vivo culture in CBS for the clinical-grade expansion of hPDMSCs.Method1. Isolation and Culture of hPDMSCs:The harvested pieces of placenta were washed and mechanically minced. Then the explant method was used to isolate and cultured MSC. 2. Preparation of cord blood serum:The blood was recovered in the absence of anticoagulants according to sterile principle and allowed to clot, then centrifuged and the supernatant was collected. Serum(15ml-25ml) was sterilized without bacterium after culturing and stored at-20℃until use.3. Cells expansion:hPDMSCs derived from different blood type group donors were cultured in medium containing different serum:10% ISA-FBS,10% TBD-FBS, 10% A-CBS,10%B-CBS,10%O-CBS and 10%AB-CBS.(1) At each passage, cells were plated at the same density and cultured for the same days. The ratio of proliferation was obtained through comparing absorbance after culturing with absorbance before planted, then drawed the growth curve of hPDMSCs at each passage.(2) hPDMSCs were plated at the density of 0.6×104 cells/mm2 on first passage, then replated at a dilution of 1:2. We calculated cumulative population doublings (CPD) and the generation time using the formula.4. Cell cycle of hPDMSCs:At passage 2,10, cells were stained with propidium iodide(PI) and analyzed with a flow cytometer following standard protocol.5. Characterization of surface markers and differentiation potential of hPDMSCs: To analyze expression of typical markers (CD31, CD45, CD73, CD105, CD166) with a flow cytometer following standard protocol. The differentiation potential of hPDMSCs was tested for the chondrogenic and endothelial differentiation after appropriate inductive stimulation at the third passage before ceasing culturing.Result1.Establishment of primary culture:The isolated cells demonstrated a spindle-shape or fibroblast-like morphology and can be passaged continuely.2.Charaeterization of by flowcytometry:hPDMSCs were positive for CD105, CD166 and were negative for CD31,CD34,CD45.3.Morphology and Growth Pattern:(1) Morphology of hPMSCs:CBS group cells appeared smaller, more spindle shaped with fewer cytoplasmic processes and longer term culture than did cells cultured with FBS. Starting at passage 3-passage 4, hPDMSCs cultured in CBS showed a distinct mesh-like growth pattern. No significantly different morphology of hPDMSCs were observed in light-microscope between different blood group CBS.(2)Proliferative activity of hPDMSCs:The TBD-FBS group maximum ratio of expansion, passages and CPD were the lowest and the passage of having significant difference with other serum groups were the earliest in all serum groups, Israel-FBS cells was second, and all CBS groups were the highest and latest(p<0.05). Calculating the generation time at each passage, in both cases, significantly extended generation time showed significantly reduced cells activity and cells expansion rate gradually stepped down(p<0.05). No significance between different blood group CBS(p>0.05).4.Cell cycle of hPDMSCs:At passage 10, most of hPDMSCs were in G0/G1 phase, and the percentage of CBS group cells was higger than FBS cells(p<0.05). These results suggest that most of cells were in stationary phase, which is the characteristic of stem cells and CBS cells were more primitive than FBS cells.5.Characterization of Surface Marker Expression and differentiation potential of hPDMACs cultured in CBS:No significant difference was observed in the expression of cell surface proteins of CD31, CD45, CD73, CD105, CD166 before and after culturing in CBS containing medium and the differentiation was confirmed.Conclusion1.CBS is more effective than FBS in culturing hPDMSCs.2.No significant difference was observed between different blood group CBS in culturing hPDMSCs derived from different blood type group donors.3.hPDMSCs quality and functionality were similar throughout culture in CBS.