title: Ex vivo expansion of megakaryocyte progenitors from cryopreserved umbilical cord blood. A potential source of megakaryocytes for transplantation.
abstract: OBJECTIVE
  Umbilical cord blood (CB) provides an alternative source of hematopoietic progenitor cells for transplantation; however, prolonged thrombocytopenia remains a major obstacle due to the low numbers of megakaryocyte progenitor (Mk-prog) cells and their subsequent delayed engraftment. In this study, we improved techniques for enrichment, cryopreservation, and ex vivo expansion of Mk-prog cells from CB.
  
  
  METHODS
  CB mononuclear cells (MNC) were isolated and Mk-prog enriched by sedimentation on gelatin followed by centrifugation with Ficoll-Hypaque and cryopreserved. The capacity of MNC to produce Mk-prog cells, assessment of CD34(+) and Mk-prog expansion in liquid culture, and analysis of the cell populations by flow cytometry were studied in cryopreserved separated CB and compared to whole CB and freshly separated samples.
  
  
  RESULTS
  Excellent viability of greater than 85% was maintained after cryopreservation of separated CB. The number of colony-forming Mk-prog, myeloid, and erythroid progenitor cells did not decrease with cryopreservation. Flow cytometric analysis of cryopreserved cells revealed significant removal of the residual red blood cells while maintaining complete recovery of CD34(+), CD41(+) (Mk), myeloid, and T and B cells compared to noncryopreserved CB cells. There was no difference in the ability of separated cryopreserved MNC CB cells to be expanded in short-term liquid cultures.
  
  
  CONCLUSIONS
  The conditions defined here for cryopreservation of gelatin/Ficoll-Hypaque separated CB, followed by ex vivo expansion of MNC, allowed complete recovery of proliferating CD41(+), CD34(+), Mk-prog cells, and other hematopoietic progenitors. Mk-prog cell expansion just before the scheduled transplantation is easily applicable by this technically simple and economical procedure that requires only an aliquot of red cell cell-depleted MNC to be separated from the CB unit before cryopreservation.
authors.affiliation: The Hematology Institute, Tel-Aviv Sourasky Medical Center, The Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel. mpick@tasmc.health.gov.il
authors.forename: Marjorie
authors.initials: M
authors.lastname: Pick
authors.forename: Amiram
authors.initials: A
authors.lastname: Eldor
authors.forename: Dan
authors.initials: D
authors.lastname: Grisaru
authors.forename: Axel R
authors.initials: AR
authors.lastname: Zander
authors.forename: Michael
authors.initials: M
authors.lastname: Shenhav
authors.forename: Varda R
authors.initials: VR
authors.lastname: Deutsch
mesh_terms: D001793:Blood Preservation; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured / Q000166:cytology; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D003973:Diatrizoate; D005312:Fetal Blood / Q000166:cytology*; D005362:Ficoll; D005434:Flow Cytometry; D005780:Gelatin; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology*; D006801:Humans; D007231:Infant, Newborn; D008533:Megakaryocytes / Q000166:cytology*; D033581:Stem Cell Transplantation
chemical_list: D003973:Diatrizoate; D005362:Ficoll; D005780:Gelatin