The ex vivo expansion capacity of normal human bone marrow cellsis dependent on experimental conditions: role of the cell concentration, serum and CD34+ cell selection in stroma-free cultures

Poloni, Antonella; Giarratana, Marie‐Catherine; Firat, Hüseyin; Kobari, Ladan; Gorin, Norbert Claude; Douay, Luc

doi:10.1007/s00282-997-0049-9

Cited by 32 publications

(22 citation statements)

References 24 publications

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“…Increasing this initial concentration induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion. These results confirm the observations of Shapiro et al 21 concerning late precursors and our own 9 for early progenitor/stem cells. The limited productivity of hematopoietic cell cultures initiated at high cell concentration may be partly due to the consumption of metabolic substrates in the medium, aggravated by the suboptimal weekly feeding protocol.…”

Section: Discussionsupporting

confidence: 83%

“…CD34 + cells were selected by the Isolex TM 50 procedure (Baxter Fenwal Division, Deerfield, IL, USA) as previously described. 9 The mean CD34 + cell purity in the subsequent experiments was 93 Ϯ 3% (range: 78 to 99%).…”

Section: Bone Marrow (Bm) Cell Preparationmentioning

confidence: 99%

“…Using tissue culture flasks (25 cm 2 T flasks; Falcon, Heidelberg, Germany), CD34 + cells were seeded in 4 ml of complete LTCM according to our previously published procedure. 9,10 Fresh medium containing cytokines (4 ml) was added to each flask on day 6. On day l4, non-adherent cells were collected and mixed with adherent cells which were recovered with a rubber scraper.…”

Section: Liquid Culturesmentioning

confidence: 99%

“…In a previous study, we showed that the successful expansion of hematopoietic cells requires the establishment of well-controlled, reproducible and reliable culture conditions, notably with regard to the cell concentration in the inoculum, the composition of the culture medium 9 and the combination of stimulatory cytokines. 10 These previously defined experimental conditions were applied here to a study of the expansion of CD34 + cells from normal bone marrow in gas-permeable polypropylene bags suitable for large-scale use.…”

mentioning

confidence: 99%

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Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

Giarratana

Kobari

Nguyen

et al. 1998

Bone Marrow Transplant

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Summary:The aim of this study was to evaluate the ex vivo expansion of normal CD34 + cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-l, G-CSF ؎ MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 × 10 3 to 1 × 10 5 CD34 + cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39 ؎ 0.5% in expanded cultures derived from fractions initiated at 5 × 10 3 , 10 4 or 10 5 cells/ml and 45 ؎ 6% in cultured cells obtained from starting fractions containing 5 × 10 4 cells/ml, as compared to 58 ؎ 4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H 2 O 2 , although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45 ؎ 4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.

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Section: Discussionsupporting

confidence: 83%

Section: Bone Marrow (Bm) Cell Preparationmentioning

confidence: 99%

Section: Liquid Culturesmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

Giarratana

Kobari

Nguyen

et al. 1998

Bone Marrow Transplant

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“…Firstly, low concentrations of progenitors expand better in liquid culture conditions due to more optimal nutritional conditions. In a previous study of the effects of CD34 + cell concentration on the expansion potential of the cells, 17 we observed an inverse correlation between number of cells per ml of culture medium and final expansion rate. However, the high expansion capacity of CFU-GM and CFU-MK from cells separated by immunoadsorption in the present experiments cannot be explained by the concentrations of these cells in the 10-day liquid cultures, as numbers of colonies if not of BFU-E were similar using the two separation techniques.…”

Section: Discussionmentioning

confidence: 54%

Comparison of CD34+ bone marrow cells purified by immunomagnetic and immunoadsorption cell separation techniques

Firat

Kobari

et al. 1998

Bone Marrow Transplant

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Summary:We tested two positive selection techniques for separation of CD34 + cells from bone marrow and analyzed the yields of CD34 + cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34 + cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 ؎ 14%) than for the immunomagnetic technique (44 ؎ 27%). After separation, CD34 + cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments. Keywords: CD34 antigen; cell separation; ex vivo expansion; long-term culture-initiating cells Hematopoietic stem/progenitor cells specifically express the CD34 surface antigen and this property provides a basis for the purification of stem cells from different sources. The several techniques commercially available today to purify CD34 + cells, which have major applications both in fundamental research and in clinical stem cell transplantation, differ by the procedures and anti-CD34 antibodies employed. 1 Our objective was to compare the phenotypic profile and clonogeneic capacity before and after expansion of cells selected by immunoadsorption 2 or using an immunomagnetic system. 3 These two commercial systems are, to date, the most widely employed in a variety of clinical settings. Until now, all teams who compared different selection techniques used different samples for each technique. 4,5 Thus, in the present study, the same bone marrow sample was used simultaneously for both CD34 + cell separation methods while, in addition, the expansion capacity of the selected cells was investigated in liquid cultures. Materials and methods Bone marrow (BM) cell preparationBM cells were obtained from patients undergoing hip surgery, all of whom gave informed consent for BM cell donation. Light-density mononuclear cells (MNCs) were separated on a Ficoll-Isopaque density gradient (1.077 g/ml; Seromed, Biochrom, Berlin, Germany). MNCs were washed twice and the cell concentration was adjusted to 0.5-1 × 10 8 /ml in Ca-and Mg-free PBS (Gibco, Life Technologies, Paisley, UK) supplemented with 1% bovine serum albumin (BSA) pro...

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Retroviral Transduction and Engraftment Ability of Primate Hematopoietic Progenitor and Stem Cells Transduced Under Serum-Free versus Serum-Containing Conditions

et al. 2002

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The ability to efficiently transduce hematopoietic stem and progenitor cells under serum-free conditions would be desirable for safety and standardization of clinical gene therapy protocols. Using rhesus macaques, we studied the transduction efficiency and engraftment ability of CD34-enriched SCF/G-CSF mobilized progenitor cells (PBSC) transduced with standard amphotropic marking vectors under serum-free and serum-containing conditions. Supernatants were collected from producer cells 16 hours after serum-free medium or medium containing 10% fetal calf serum was added. Vector titers were approximately two- to threefold higher when producer cells were cultured in serum-containing medium. However, retroviral transduction of rhesus CFU-GM was improved using serum-free vector-containing medium. For analysis of engraftment with transduced cells, three macaques had CD34+ peripheral blood stem cells split into two fractions for transduction. One fraction was transduced using serum-free vector-containing medium, and the other fraction was transduced using standard serum-containing medium. The two fractions were re-infused simultaneously following total body irradiation. In all three animals, there was equivalent marking from both vectors for 7-9 months post-transplantation. These data are encouraging regarding the removal of serum-containing medium from clinical hematopoietic cell transduction protocols, given the lack of a detrimental effect on transduction and engraftment with transduced cells.

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The ex vivo expansion capacity of normal human bone marrow cellsis dependent on experimental conditions: role of the cell concentration, serum and CD34+ cell selection in stroma-free cultures

Cited by 32 publications

References 24 publications

Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for large-scale clinical applications

Comparison of CD34+ bone marrow cells purified by immunomagnetic and immunoadsorption cell separation techniques

Retroviral Transduction and Engraftment Ability of Primate Hematopoietic Progenitor and Stem Cells Transduced Under Serum-Free versus Serum-Containing Conditions

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