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Clinical-grade cell purification from thawed cord blood: an example of translational research

Bone Marrow Transplantation volume 32, pages 965–966 (2003)Cite this article

Cord blood (CB) is a useful source of hemopoietic stem cells (HSCs) for transplantation. However, its use is hampered by the limited number of available cells, which in turn can cause delayed engraftment.1 Therefore, protocols for graft manipulation have been developed, with the aim of expanding the number of HSCs. In our previous studies, we described a static serum-free system for ex vivo expansion of purified CB CD34+ cells, including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11, which was able to induce significant and sustained ex vivo expansion of primitive CB HSCs.2 In order to evaluate the safety and therapeutic potential of expanded CB HSCs, we designed a clinical protocol based on transplantation of ex vivo expanded, highly purified CD34+ cells into pediatric patients. The translation of bench-based techniques into clinical procedures did not merely consist of replacing research reagents and materials with clinical-grade products, but also in selecting a suitable cell purification system. Specifically, we needed to adapt the use of a closed, clinical-grade automated device designed to process large-volume, fresh leukapheresis products (CliniMACS, Miltenyi Biotech, Gladbach, Germany), to the selection of CD34+ cells from small-volume thawed CB samples. Moreover, the new procedure required validation on CB units cryopreserved either as whole blood (unfractionated) or after removal of a large proportion of red cells (volume reduction), which are the two processing methods in use in CB banking.

To this end, we processed 10 cryopreserved and thawed CB samples (five unfractionated and five volume reduced). Units were selected randomly from routine CB donations, which were close to, but did not reach the minimum volume (60 ml) or number of NC (800 × 106) required for banking, or were discarded for clinical reasons. CB units were collected after maternal informed consent. Volume reduction was carried out with an automated blood component separation device (Compomat G-4, Fresenius, Schweinfurt, Germany; CB was aseptically transferred into a top-and-bottom Compoflex bag (Fresenius), centrifuged at 2900 g for 12 min and a 39 ml buffy coat was collected. The median (and range) numbers of NC and CD34+ cells before cryopreservation were 603 × 106 (289–1270) and 2.5 × 106 (1.5–6.9), respectively. Both the unfractionated and the volume-reduced CB units were cryopreserved with our standard procedure (10% final DMSO concentration), thawed according to Rubinstein et al,3 labeled with CD34 monoclonal antibody conjugated to superparamagnetic particles (CD34 CliniMACS reagent, Miltenyi Biotech) and selected using the CliniMACS system, adjusting the manufacturer's instructions to the limited number of NC and CD34+ cells contained in each sample. According to this adjustment, we maintained the concentration of NC before selection between 3 and 5 × 106/ml. Moreover, the immunoselection was performed using a new low-capacity tubing set with a small separation column (CliniMACS Tubing set 150). The analysis of the CD34+ cell-enriched fraction was performed by flow cytometry with an anti-CD34 monoclonal antibody (hpca-2; Becton Dickinson, BD, San Jose, CA, USA). The percentage of CD34+ cells was evaluated on the gate of the CD45+ cells.

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References

  1. Gluckman E . Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28: 1197–1205.

    Article  CAS  Google Scholar 

  2. Lazzari L, Lucchi S, Rebulla P et al. Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system. Br J Haematol 2001; 112: 397–404.

    Article  CAS  Google Scholar 

  3. Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995; 92: 10119–10122.

    Article  CAS  Google Scholar 

  4. McNiece I, Stoney GB, Kern BP et al. CD34+ cell selection from frozen cord blood products using the Isolex 300i and CliniMACS CD34 selection devices. J Hematother 1998; 7: 457–461.

    Article  CAS  Google Scholar 

  5. Querol S, Capmany G, Azqueta C et al. Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex-vivo expansion protocols. Transfusion 2000; 40: 625–631.

    Article  CAS  Google Scholar 

  6. Eichler H, Beck C, Shröder B et al. Nonobese diabetic-severe combined immunodeficient mice transplantation of volume-reduced and thawed umbilical cord blood transplants following closed-system immunomagnetic cell selection. Transfusion 2002; 42: 1285–1292.

    Article  Google Scholar 

  7. Blundell MP, Demaison C, Brouns G et al. Quality of repopulation in nonobese diabetic severe combined immunodeficient mice engrafted with expanded cord blood CD34+ cells. Blood 1999; 94: 3269–3276.

    CAS  Google Scholar 

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  1. Cell Factory ‘Franco Calori’, Centro Trasfusionale e di Immunologia dei Trapianti, IRCCS Ospedale Maggiore Policlinico, Milan, Italy

    R Giordano, L Lazzari, T Montemurro, L Lecchi, L Porretti & P Rebulla

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  1. R Giordano
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  2. L Lazzari
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  3. T Montemurro
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  5. L Porretti
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  6. P Rebulla
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Giordano, R., Lazzari, L., Montemurro, T. et al. Clinical-grade cell purification from thawed cord blood: an example of translational research. Bone Marrow Transplant 32, 965–966 (2003). https://doi.org/10.1038/sj.bmt.1704263

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