[Optimalization for preparation and expansion of ex vivo human umbilical blood cells for transplantation].
This work was aimed at finding optimal conditions for isolation and storage of hematopoietic cells from umbilical cord blood and checking the usefulness of ex vivo expansion in a serum-free medium. Mononuclear cells were isolated either by density gradient centrifugation (Ficoll-Histopaque or Gradisol L) or by hypoosmotic lysis of erythrocytes and granulocytes in ammonium chloride. Toxicity of metabolic dyes (Hoechst 33342, Rhodamine 123, Pyronine Y) used in flow cytofluorimetry and the effects of freezing in dry ice or in a mechanical freezer with and without a methanol bath were compared. The influence of ex vivo expansion of cells in a serum-free medium on their proliferative potential and apoptosis was investigated. In comparison with ammonium chloride, density gradient centrifugation both in Ficoll-Histopaque and Gradisol L offered superior and consistent results as to cell yield, vitality and proliferative potential. Hoechst 33342 demonstrated the highest toxicity with CD34+ cells, disqualifying this dye for transplantation purposes. Mechanical freezing with prior methanol bath was found to be the safest method of storage. The concentration of TGF beta released by cells subjected to expansion ex vivo in a serum-free medium was markedly elevated and correlated with the percentage of apoptotic cells. Furthermore, the proliferative potential of cells deteriorated as a result of expansion.
Zakładu Biochemii i Chemii Pomorskiej Akademii Medycznej w Szczecine, al. Powstańców Wlkp. 72, 70-111 Szczecin.
Wioletta
W
Mikołajek
D001793:Blood Preservation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods; D005312:Fetal Blood / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation / Q000295:instrumentation / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents / Q000633:toxicity
D007202:Indicators and Reagents