Collection and cryopreservation of human stem and progenitor cells for bone marrow transplantation

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Abstract
Bone marrow collection was undertaken from human organ donors (Group 1; n = 7) to develop a closed‐system single‐step technique for stem and progenitor cell enrichment, using the Cobe 2997 continuous‐flow blood‐cell separator. The effects of programmed freezing, storage in liquid nitrogen, and thawing were then defined using these grafts. Once standardised, this method was extended to autografting following cryopreservation of a comparable fraction (Group 2; n = 8) and then to allogeneic transplantation after ex vivo exposure to the lytic monoclonal antibody, Campath‐1 IgM and human complement, but without cryopreservation (Group 3; n = 9). The median number of mononuclear cells harvested was 5.0 × 106/mL (n = 24), and this was not significantly different in the three groups. The ex vivo graft, composing marrow rich anticoagulated whole blood, was recirculated in the separator at a flow rate of 60 mL/minute, with a centrifuge speed of 1,100 r.p.m., and the mononuclear cell fractions were collected at the rate of 1.5 mL/minute. The average procedure time from formation of the interface in the single disposable channel to achievement of the final volume was 90 minutes. The mean recovery of the mononuclear cells was 101.4% (SD 38.0) and the GM:CFUc was 91% (SD 43.86). These figures were not significantly influenced by subsequent cryopreservation (Group 1; n = 7 and Group 2; n = 8) or following exposure to the monoclonal antibody, Campath‐1 IgM (Group 3; n = 9). The Cobe Model 2997 continuous‐flow blood fraction separator is ideally suited for the ex vivo enrichment of human bone marrow stem and progenitor cells in a volume that makes handling practical by a technique that is rapid and readily applicable to both autografting and allogeneic transplantation programmes.