Factorial designs combined with the steepest ascent method to optimize serum-free media for ex vivo expansion of human hematopoietic progenitor cells

The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, a serum-free, stroma-free and cytokine-containing culture system for CD34 + and colony-forming cell (CFC) expansion was systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results show that the optimal compositions of the serum substitutes and the cytokine cocktail were BIT2 (1.5 g/l BSA, 4.39 μg/ml insulin, 60 μg/ml transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/ml TPO, 4.09 ng/ml IL-3, 15 ng/ml SCF, 6.73 ng/ml FL, 0.78 ng/ml IL-6, 3.17 ng/ml G-CSF, and 1.30 ng/ml GM-CSF) in the Iscove's modified Dulbecco's medium, respectively. After one-week culture, the increases in the total number of white blood cells (WBC), CD34 + cells and CFC were 64-, 27- and 22-fold, respectively. Its expansion ability of CD34 + cells and CFC was comparable to that of X-vivo 20, Stemline, and Stemspan commercial media. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.

[1]  J. Hows,et al.  Cytokine Expansion Culture of Cord Blood CD34+ Cells Induces Marked and Sustained Changes in Adhesion Receptor and CXCR4 Expressions , 2003, Stem cells.

[2]  E. Papoutsakis,et al.  Hematopoietic cell culture therapies (Part I): Cell culture considerations. , 1996, Trends in biotechnology.

[3]  K. Moore,et al.  In vitro maintenance of highly purified, transplantable hematopoietic stem cells. , 1997, Blood.

[4]  W M Miller,et al.  Hematopoietic cell culture therapies (Part II): Clinical aspects and applications. , 1996, Trends in biotechnology.

[5]  D Barnes,et al.  Methods for growth of cultured cells in serum-free medium. , 1980, Analytical biochemistry.

[6]  C. Shih,et al.  A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. , 2000, Blood.

[7]  W. Miller,et al.  Evaluation of Cytokines for Expansion of the Megakaryocyte and Granulocyte Lineages , 1997, Stem cells.

[8]  P. Zandstra,et al.  Advances in hematopoietic stem cell culture. , 1998, Current opinion in biotechnology.

[9]  H. Mayani,et al.  In vitro proliferation, expansion, and differentiation of a CD34+ cell-enriched hematopoietic cell population from human umbilical cord blood in response to recombinant cytokines. , 2002, Archives of medical research.

[10]  Alan K. Smith,et al.  Alternatives to animal sera for human bone marrow cell expansion: human serum and serum-free media. , 1998, Journal of hematotherapy.

[11]  Shiaw-Min Hwang,et al.  Factorial designs combined with the steepest ascent method to optimize serum-free media for CHO cells. , 2001, Enzyme and microbial technology.

[12]  B. Lord,et al.  CD34+AC133+ Cells Isolated from Cord Blood are Highly Enriched in Long‐Term Culture‐Initiating Cells, NOD/SCID‐Repopulating Cells and Dendritic Cell Progenitors , 1998, Stem cells.

[13]  D. Sutherland,et al.  The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. , 1996, Journal of hematotherapy.

[14]  I McNiece,et al.  Ex vivo expansion of hematopoietic progenitor cells and mature cells. , 2001, Experimental hematology.

[15]  J M Piret,et al.  Cytokine manipulation of primitive human hematopoietic cell self-renewal. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Jer-Yiing Houng,et al.  Search method for the optimal medium for the production of lactase by Kluyveromyces fragilis , 1992 .

[17]  Chi-Hsien Liu,et al.  Medium optimization for L-phenylalanine production by a tryptophan auxotroph of Corynebacterium glutamicum , 1994, Biotechnology Letters.

[18]  C. Shih,et al.  Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. , 1999, Blood.

[19]  H. W. Lee,et al.  Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. , 2001, Experimental hematology.

[20]  L. Nilsson,et al.  Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells. , 1999, Blood.

[21]  Marija J. Norusis,et al.  SPSS for Windows Base System User''s Guide , 1992 .

[22]  O. Ottmann,et al.  Interleukin 3 Improves the Ex Vivo Expansion of Primitive Human Cord Blood Progenitor Cells and Maintains the Engraftment Potential of SCID Repopulating Cells , 2001, Stem cells.

[23]  G. Dravid,et al.  Ex Vivo Expansion of Stem Cells from Umbilical Cord Blood: Expression of Cell Adhesion Molecules , 2002, Stem cells.

[24]  Y. Tzeng,et al.  Use of response surface methodology to optimize culture medium for production of lovastatin by Monascus ruber , 2002 .

[25]  J. Kearney,et al.  AC133, a novel marker for human hematopoietic stem and progenitor cells. , 1997, Blood.

[26]  G. Gilmore,et al.  Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. , 2000, Experimental hematology.

[27]  M. Bhatia.  AC133 expression in human stem cells , 2001, Leukemia.