培养条件对体外诱导造血干/祖细胞向血小板定向增殖与分化的影响

目的观察3种不同培养基以及细胞接种密度对体外诱导造血干/祖细胞向血小板方向增殖和分化的影响。方法用添加干细胞因子(SCF)和促血小板生成素(TPO)等细胞因子的无血清培养基(StemSpan(SFEM和X-VIVO10)或IMDM/10%FBS来扩增脐血CD34+细胞向血小板定向分化,比较不同培养基的培养效果;CD34+细胞接种浓度为5×103/ml、104/ml和105/ml,比较不同接种浓度对扩增效果的影响。结果培养d 14、d 24和d 29时,StemSpan(SFEM培养基体系中细胞分别扩增了(11 000±577.35)、(196 666.67±14 529.66)和(176 666.67±8 819.17)倍,显著高于X-VIVO10和IMDM/10%FBS组,其中在d 24和d 29时,巨核系细胞所占比例分别是(54.57±2.32)%和(69.4±2.02)%,显著高于X-VIVO10和IMDM/10%FBS组。培养14 d后初始接种浓度为104/ml的CD34+细胞在StemSpan(SFEM培养基体系中扩增(11 000±577.35)倍,显著高于初始接种浓度为5×103/ml和105/ml组。结论和X-VIVO10和IMDM/10%FBS相比,StemSpan(SPEM培养基最有利于脐血CD34+细胞体外向血小板定向扩增和分化;104/ml的CD34+细胞接种密度最有利于细胞扩增和分化。

Influence of culture conditions on proliferation and differentiation of umbilical cord blood CD34~+ cell s into platelets in vitro

Objective To investigate the influence of three different culture mediums and different initial cell concentrations on proliferation and differentiation of cultured platelets from human umbilical cord blood CD34+ cells in vitro.Methods The CD34+ cells from human umbilical cord blood were amplified in serum-free medium(StemSpan SFEM or X-VIVO10) or IMDM + 10% FBS supplemented with cytokine cocktail including stem cell factor(SCF) and thrombopoietin(TPO).Initial cell concentrations were 5×103/ml,104/ml or 105/ml.Results At the 14,24 and 29 day,the cells cultured in StemSpan SPEM medium were amplified(11 000±577.35),(196 666.67±14 529.66) and(176 666.67±8 819.17) folds in which MK cells accounted for over(54.57±2.32)% and(69.4±2.02)%,which were significantly higher than that of X-VIVO10 and IMDM/10% FBS group.By the day 14,(11 000±577.35) folds cell amplification could be achieved from the initial inoculating density of 104/ml in StemSpan SFEM culture medium,significantly higher than the other groups.Conclusion StemSpan SPEM medium is the most suitable medium for the induction of platelets from unbilical cord blood CD34+ cells in vitro comparing with X-VIVO10 and IMDM/10% FBS.And 104/ml of CD34+ initial inoculating density was preferred for amplification of platelets.