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Chinese Journal of Biotechnology ; (12): 622-627, 2005.
Article in Chinese | WPRIM | ID: wpr-305192


To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.

Bioreactors , Cell Culture Techniques , Methods , Cells, Cultured , Fetal Blood , Cell Biology , Hematopoietic Stem Cells , Cell Biology , Humans
Chinese Journal of Biotechnology ; (12): 587-592, 2003.
Article in Chinese | WPRIM | ID: wpr-259143


Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than the static culture systems for hematopoietic cell proliferation. The biocompatibility of cord blood MNC to different types of materials used in bioreactors was also tested. The results showed that glass, stainless steel 316L and polytetraflouroethylene (PTFE) supported the growth of hematopoietic cells well. A higher cell density was reached in stirred bioreactors with controlled pH and DO than static culture. These findings suggested that the controlled large-scale culture could be used to overcome the clinical shortage of hematopoietic cells.

Antigens, CD34 , Metabolism , Bioreactors , Cell Culture Techniques , Methods , Erythroid Precursor Cells , Cell Biology , Fetal Blood , Cell Biology , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Humans , Polytetrafluoroethylene , Stainless Steel