PROCESSING AND CRYOPRESERVATION OF PLACENTAL UMBILICAL-CORD BLOOD FORUNRELATED BONE-MARROW RECONSTITUTION

Clinical evidence of hematopoietic restoration with placental/umbilica l cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In t he unrelated setting, human leukocyte antigen (HLA)-matched donors mus t be obtained for candidate patients and, hence, large panels of froze n HLA-typed PCB units must be established, The large volume of unproce ssed units, consisting mostly of red blood cells, plasma, and cryopres ervation medium, poses a serious difficulty in this effort because sto rage space in liquid nitrogen is limited and costly. We report here th at almost all the hematopoietic colony-forming cells present in PCB un its can be recovered in a uniform volume of 20 ml by using rouleaux fo rmation induced by hydroxyethyl starch and centrifugation to reduce th e bulk of erythrocytes and plasma and, thus, concentrate leukocytes. T his method multiplies the number of units that can be stored in the sa me freezer space as much as 10-fold depending on the format of the sto rage system. We have also investigated the proportion of functional st em/progenitor cells initially present that are actually available to t he recipient when thawed cryopreserved PCB units are infused. Progenit or cell viability is measurably decreased when thawed cells, still sus pended in hypertonic cryopreservative solutions, are rapidly mixed wit h large volumes of isotonic Solutions or plasma. The osmotic damage in flicted by the severe solute concentration gradient, however, can be a verted by a simple 2-fold dilution after thawing, providing almost tot al recovery of viable hematopoietic progenitor cells.