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Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells

Authors

  • Pascale Duchez,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
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  • Jean Chevaleyre,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
    2. CNRS, Université Bordeaux, Bordeaux, France
    3. Cryo-Save Labs, Niel, Belgium
    4. Fat Stem Laboratories, Buggenhout, Belgium
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  • Philippe Brunet de la Grange,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
    2. CNRS, Université Bordeaux, Bordeaux, France
    3. Cryo-Save Labs, Niel, Belgium
    4. Fat Stem Laboratories, Buggenhout, Belgium
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  • Marija Vlaski,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
    2. CNRS, Université Bordeaux, Bordeaux, France
    3. Cryo-Save Labs, Niel, Belgium
    4. Fat Stem Laboratories, Buggenhout, Belgium
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  • Jean-Michel Boiron,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
    2. CNRS, Université Bordeaux, Bordeaux, France
    3. Cryo-Save Labs, Niel, Belgium
    4. Fat Stem Laboratories, Buggenhout, Belgium
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  • Guy Wouters,

    1. Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
    2. CNRS, Université Bordeaux, Bordeaux, France
    3. Cryo-Save Labs, Niel, Belgium
    4. Fat Stem Laboratories, Buggenhout, Belgium
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  • Zoran Ivanovic

    Corresponding author
    1. CNRS, Université Bordeaux, Bordeaux, France
    2. Cryo-Save Labs, Niel, Belgium
    3. Fat Stem Laboratories, Buggenhout, Belgium
    • Etablissement Français du Sang Aquitaine–Limousin, Bordeaux, France
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  • This work was supported by a grant from Agence de la Biomédecine (France)—Appel d'Offres Recherche “Recherche et Greffe” 2009, Cryo-Save AG (2007 contract) as well as by research funds of Aquitaine-Limousin Branch of French Blood Institute (EFS-AL).

Address reprint requests to: Zoran Ivanovic, MD, PhD, Etablissement Français du Sang Aquitaine–Limousin, Place Amélie Raba Léon, BP24, 33035 Bordeaux Cedex, France; e-mail: zoran.ivanovic@efs.sante.fr.

Abstract

Background

Our ex vivo expansion procedure starting from cord blood (CB) CD34+ cells enabled expansion of committed progenitors (CPs) without a negative impact on hematopoietic stem cells (HSCs) exhibiting both short- and long-term repopulating capacity. Upgraded to clinical scale (Macopharma HP01 in the presence of stem cell factor, FLT3-L [100 ng/mL each], granulocyte–colony-stimulating factor [10 ng/mL], and thrombopoietin [20 ng/mL]), it is being used for an ongoing clinical trial (adult allogeneic context) yielding promising preliminary results. Transplantation of ex vivo expanded CB cells is becoming a reality, while the issue of expanded cells' cryopreservation emerges as an option that allows the conservation of the product for transportation and future use. Here, we investigated whether it is possible to maintain the functional HSC and CP properties after freezing and thawing of expanded cells.

Study Design and Methods

We compared cryopreservation efficiency of the ex vivo expanded CB cells using the standard protocol (freezing solution human serum albumin (HSA)-dimethyl sulfoxide [DMSO]) with the newly designed protocol based on an enriched freezing solution (HP01-DMSO) with respect to the viability index, number of CD34+ and total cells, and recovery of CPs (colony-forming units) and HSCs (NOG/Scid/gamma–null mice engraftment).

Results

Cryopreservation and thawing of expanded CB cells using the “standard” procedure (HSA-DMSO) reduced recovery of the CPs (40%) and HSCs (drastically decreasing engraftment capacity). HP01-based protocol resulted in improvement of preservation of both CPs (>60%) and HSCs (nonaltered engraftment capacities).

Conclusion

Functional maintenance of the expanded graft by cryopreservation is feasible in conditions compatible with human cell therapy requirements.

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