Objective To investigate the preparation method of umbilical cord blood plasma derived platelet lysate (UCB-PL) and to develop a heparin and animal origin-free culture medium for umbilical cord mesenchymal stem cells (UCMSCs).Methods Platelet rich plasma (PRP) was separated from umbilical cord blood plasma by centrifugation,and platelets were concentrated via high-speed centrifugation of PRP. UCB-PL was prepared through three cycles of freeze-thaw at-80 ℃/37 ℃. The total protein content of UCB-PL was measured using a BCA assay,and the main growth factors in UCB-PL and fibrinogen in the UCB-PL medium were detected through ELISA assay. A heparin-free UCMSCs medium was developed by depleting fibrinogen in the UCB-PL medium using a natural sedimentation method. UCMSCs were cultured in the UCB-PL medium,and their morphology,proliferation quantity,cell viability,cell surface markers expression,colony formation ability,and differentiation abilities were assessed. Results A platelet concentrate was obtained from cord blood plasma with a recovery rate of (50.68±7.28)％. When UCMSCs were cultured in 5％,10％,20％ UCB-PL medium without fibrinogen depletion treatment,the medium coagulated. However,after fibrinogen depletion treatment,the medium did not coagulate,and the fibrinogen content decreased by 85.26％,85.90％,and 93.71％,respectively and significantly. The cell count of UCMSCs in the 20％ UCB-PL group was the highest,and there was no significant difference in cell viability among the groups. UCMSCs exhibited spindle-shaped morphology,with parallel or spiral stripes adhering to the dishes. The positive cells surface marker antigen proportion of CD45+/CD34+/CD14+/CD19+were 0.09％,while the proportion of CD73+,CD90+,and CD105+positive cells were 98.33％,99.56％,and 96.37％,respectively. After induction,Oil red O,Alizarin red S,and Alician blue staining showed that UCMSCs exhibited the adipogenesis,osteogenesis,and chondrogenesis differentiate ability. Crystal violet staining indicated a good colony forming ability of UCMSCs. Major growth factors in UCB-PL were measured by ELISA assay,with transforming growth factor beta 1 (TGF-β1),platelet derived growth factor AB (PDGF-AB),insulin like growth factor 1 (IGF-1),and basic fibroblast growth factor (bFGF) being (19.65±1.55) ng/ml,(3.81±0.35) ng/ml,(21.25±3.84) ng/ml and (39.69±5.31) pg/ml,respectively. The total protein content of UCB-PL was (1130.51±164.96) μg/ml. When stored at-80 ℃ for 90 days,the contents of IGF-1,PDGF-AB,and TGF-β1 in UCB-PL were not significantly changed;while that of bFGF decreased significantly. bFGF,IGF-1,and TGF-β1 stored at-20 ℃ for 90 days showed no significant changes while PDGF-AB significantly decreased compared with the condition of 30 days storage. The cytokine content significantly decreased when stored at 4 ℃ and room temperature (RT) for 30 days.Conclusion UCB-PL prepared from cord blood plasma can be a substitute for fetal bovine serum (FBS) to UCMSCs culture. Cryopreservation of UCB-PL after preparation is recommended. An animal origin-free anticoagulant culture system can be established by depleting fibrinogen in the UCB-PL culture medium.
维普
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