The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors

Abstract

Summary:Cord blood (CB) is an alternative source of marrow repopulating cells for the treatment of pediatric patients. Over the past 7 years, the results of CB transplantation for pediatric Assuming a threshold of 2 × 10 7 nucleated cells (NC)/kg body weight required for transplantation and 10 ± 5 × 10 8 diseases have been promising. Over 500 unrelated and related cord blood transplants have been performed. + CB cells were cultured with the four cytokine combinations in H5100 medium, all combi-10 ± 5 × 10 8 nucleated cells (NC) stored in the CB Bank Düsseldorf, 100%, 65% and 25% contain sufficient NC to nations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, engraft patients of 10 kg, 35 kg and 50-70 kg, respectively, when a threshold of 2 × 10 7 NC/kg body weight required early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with for transplantation is assumed. Therefore, ex vivo expansion of stem/progenitor cells from human cord blood prior to SCF, Flt3-L plus IL-3 (94.3 ± 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L transplantation would be an attractive approach to obtain the quantity and quality of CB cells required for successful plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were simi-(ie rapid, complete and sustained) long-term hematopoietic reconstitution after transplantation in adults. larly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximumAlthough ex vivo expansion of CB cells has already been described, [10][11][12][13][14][15][16][17][18][19][20][21][22] in few reports all progenitor cell compartat day 7: 6.7 ± 3.4-fold and 5.5 ± 0.5-fold, respectively). However, the serum-free medium promoted a signifiments (ie LTC-IC) have been studied, to determine whether expanded CB cells still contain cells with long-term repopcantly higher expansion of CFU-GEMM (176.9 ± 81.7-fold) than H5100 medium (83.5 ± 26.2-fold) at day 7 and ulating potential. 10,18,19 This point is, however, of critical relevance for clinical application. Recently, a long-term only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results expansion system was described, which allows a substantial amplification of primitive hematopoietic progenitors demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be subincluding LTC-IC after 20 weeks in serum-containing liquid culture in the presence of Flt3-L and thrombopoiestantially amplified at the same time without exhausting the proliferative potential.tin. 19 However, for a clinically relevant expansion, a shorter culture period would be preferable. Thus, the establishment Keywords: cord blood; ex vivo expansion; Flt3-ligand of a short-term large-scale system for the simultaneous expansion of the various stem/progenitor cell populations involved in the different phases of ...

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood

“…27 Another group of investigators studied the effect of different thawing methods on the ex vivo expansion of primitive and committed progenitors from CB. 28 These investigators thawed CB samples using a solution containing dextran and albumin as described by Rubinstein et al 13 or FCS and RPMI-1640 and found that the former protocol significantly enhances the recovery of CD34 ϩ cells, CFC and LTC-IC. Moreover, they expanded the thawed CD34 ϩ cells in the presence of IL-3, IL-6, SCF, epo, granulocytecolony stimulating factor (G-CSF) and showed a 20-to 40-fold CFC and a two-fold LTC-IC expansion after 7 to 14 days.…”

“…[27][28][29][30][31] In this regard, Querol et al 27 determined optimal conditions for CB thawing and expansion with a system using Teflon bags. These investigators evaluated the impact of thawing and CD34 ϩ cell selection on the expansion of frozen CB cells.…”

Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia

“…[16][17][18][19][20] Previous results of our group 19 and others have demonstrated that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be amplified from CD34 ϩ enriched CB samples in suspension cultures within 7-14 days without exhausting the proliferative potential and, thus, it appeared possible to improve the hematopoietic recovery after CB transplantation by simultaneous application of such ex vivo amplified cells from a part of the whole cord blood generated 10 days prior to transplant date.…”

Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells

“…5 Therefore, much effort has focused on the in vitro expansion of cord blood cells. [19][20][21][22][23] However, the optimal culture conditions for clinical use have not been defined as yet. For successful clinical use of ex vivo expanded material several prerequisites need to be fulfilled: to minimize the risk of contamination, extensive manipulation during the culture period should be avoided.…”