The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors
The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors
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DOI：
10.1038/sj.bmt.1701088
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发表时间：
1998-02-01
影响因子：
4.8
通讯作者：
Wernet, P
中科院分区：
文献类型：
--
作者：
Kögler, G;Callejas, J;Wernet, P
假设移植所需的有核细胞数为2×10(7)/公斤体重，每单位脐带血的有核细胞数为10+/-5×10(8)(n=1828,1997年7月)，杜塞尔多夫脐带血银行储存的脐血单位中分别有100%、65%和25%的有核细胞足以移植10公斤、35公斤和50-70公斤的病人。因此，在成人中使用CB存在潜在的限制，然而，通过在植入的不同阶段重要的细胞的体外扩增可以克服这一限制。因此，对干细胞因子、Flt3-L、IL-3、促红细胞生成素和GM-CSF的四种组合以及三种培养基扩增造血祖细胞的能力进行了评估。扩增的先决条件是CD34(+)细胞、集落形成细胞(CFC)和长期培养启动细胞(LTC-IC)的回收率显著提高，方法是用等渗白蛋白/葡聚糖溶液解冻冷冻保存的CB单位。4种细胞因子组合在H5100培养液中培养CD34(+)的脐血细胞，7d内均能促进细胞总数(43~356倍)和CFC49~462倍的扩增，而混合集落形成的早期祖细胞(CFU-GEMM)仅经SCF、Flt3-L结合IL-3扩增(94.3+/-62.4倍)。H5100培养液或添加SCF、Flt3-L和IL-3的无血清培养液对所有祖细胞类型的扩增均优于20%FCS/RPMi-1640培养液，对总细胞、CFU-GM、BFU-E/CFU-E和LTC-IC的扩增效果相似(第7天最高分别为6.7+/-3.4倍和5.5+/-0.5倍)。但在培养第7天，无血清培养组CFU-GEMM的扩增倍数(176.9+/-81.7倍)显著高于H5100培养基组(83.5+/-26.2倍)，并且只有在无血清条件下，CFU-GEMM在组织培养中才能维持14天以上。这些结果表明，忠诚的祖先也是如此。由于CFU-GEMM和LTC-IC较不成熟，可以在不耗尽增殖潜能的情况下同时大量扩增。
Assuming a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Dusseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50-70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34(+) cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34(+) CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L pins IL-3 (94.3 +/- 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 +/- 3.4-fold and 5.5 +/- 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 +/- 81.7-fold) than H5100 medium (83.5 +/- 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well. as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.