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

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

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。

Objective To investigate the influence of three different culture mediums and different initial cell concen- trations 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 thrombopoi- etin （TPO）. Initial cell concentrations were 5 × 10^3/ml, 10^4/ml or 10^5/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 significandy higher than that of X-VIVO10 and IMDM/10% FBS group. By the day 14, （11 000 ±577.35） folds cell amplifica- tion could be achieved from the initial inoculating density of 10^4/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 10^4/ml of CD34 ＋ initial inoculating density was preferred for amplification of platelets.