title: Loss of the ability to generate large burst-forming unit-like megakaryocytic colonies from thawed cord blood in semisolid cultures after short term suspension culture.
abstract: OBJECTIVE
  Total colony-forming cells from thawed cord blood units (CBUs) include megakaryocytic colony-forming units (CFU-Mks), which survive the freezing process. The aim of this study was to evaluate whether different megakaryocytic progenitors from unseparated CBUs survive the freezing process and a short-term liquid culture.
  
  
  METHODS
  Thawed samples of CBUs were cultured in liquid medium. During the cultures, serial samples were drawn to assess the growth of different megakaryocytic progenitors in a semisolid collagen medium with identical cytokines as in the liquid medium. Megakaryocytic cells were detected using immunohistochemistry and flow cytometry.
  
  
  RESULTS
  In suspension culture, the megakaryocytic progenitors almost completely lost the ability to generate large (burst-forming unit-like, BFU-like) megakaryocytic colonies in semisolid cultures (large colonies, median count per chamber d0: 7.25 vs. d7: 1.5; P < 0.0001), whereas the number of small colonies (median count per chamber d0: 7.25 vs. d7: 16.0; P = 0.0505) peaked at day seven. Further 7-day culture in suspension resulted in the decline of small colonies as well (d7: 16.0 vs. d14: 5.75; P = 0.0088). Total CFU-Mk count declined from 23.3 (range 12.5-34.0) at d0 to 7.25 (range 1.0-13.5) at d14 (P < 0.0001).
  
  
  CONCLUSIONS
  Immediately post-thaw, CBUs possess an ability to generate large BFU-like megakaryocytic colonies, whereas the colonies were not detectable in most CBUs in semisolid culture after a short suspension culture. Small CFU-Mks were observed throughout the cultures. It may be that the BFU-Mk colonies matured and acquired CFU-Mk behaviour.
authors.affiliation: Department of Advanced Therapies and Product Development, Finnish Red Cross Blood Service, Helsinki, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Eskola
authors.forename: S
authors.initials: S
authors.lastname: Bäckman
authors.forename: S
authors.initials: S
authors.lastname: Möttönen
authors.forename: R
authors.initials: R
authors.lastname: Kekomäki
mesh_terms: D001793:Blood Preservation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology
keywords: colony-forming unit; cord blood; megakaryocyte; platelet