造血干/祖细胞的体外扩增培养研究

The Investigation of Ex-vivo Expansion of Hematopoietic Stem/Progenitor Cells

利用旋转壁式生物反应器（Rotating wall vessel，RWV）体外培养脐带血干细胞，使其大量扩增。以满足临床应用对造血干／祖细胞的数量与质量要求。从脐带血分离得到的单个核细胞（Mononuclear cells,MNC）在T—flask中培养24h，之后接种到RWV反应器中。培养200h。每24h细胞计数，测量培养基的pH和渗透压变化；在144h和197h测CD34^＋细胞含量并做CFU—GM半固体培养。有核细胞（Nucleated cells，NC）与CD34^＋细胞在第197h，分别扩增了435．5±87．6倍和32．7±15．6倍，CFU—GM（Colony—forming unit—granuloeyte／maerophage）细胞扩增了21．7±4．9倍。整个培养过程中。RWV反应器中的pH和渗透压都保持在造血细胞最佳的扩增条件内，pH基本保持在7．2～7．4之间，渗透压基本保持在290-310mmol／kg之间。由于旋转壁式生物反应器（RWV）结构上的特殊性，可以保证细胞在悬浮流动的状态下生长，很好地模拟了脐带中的造血微环境。使脐带血造血干细胞在该反应器中短期内得到大量扩增。

Rotating wall vessel （RWV） was used for the ex-vivo expansion of umbilical cord blood stem cells to meet the requirement of clinical application in the aspect of quantity and quality of the stem cells. The mononuclear cells （MNCs） from umbilical cord blood were cultured in T-flasks for 24 h, and then inoculated in RWV to culture for 200 h. The nucleated-cell numbers, pH and osmolality of the culture medium were determined every 24 h. The CD34^＋ cells content was measured and CFU-GM culture was carried out at 144 h and 197 h. Nucleated cells （NC） and CD34^＋ cells had a 435. 5 ± 87. 6 fold expansion and a 32. 7 ± 15. 6 fold expansion respectively in 197 h, and CFU-GM （colony-forming unit-granulocyte/macrophage） cells had a 21. 7 ± 4. 9 fold expansion. In the whole course of culture, the pH and osmolality of the medium in the RWV were kept in the optimal hematopoietic stem cells＇ expansion conditions, pH was kept from 7.2 to 7.4, and the osmolality was kept from 290 mmol/kg to 310 mmol/kg. Owing to its structural particularity, the RWV could ensure cells to grow in the suspension state, could simulate the micro-environment of umbilical cord blood, and thus could make the hematopoietic stem cells expand largely in the RWV in short time.