title: Polyunsaturated fatty acids confer cryoresistance on megakaryocytes generated from cord blood and also enhance megakaryocyte production from cryopreserved cord blood cells.
abstract: OBJECTIVE
  Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated.
  
  
  METHODS
  MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34(+) cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5-3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2-5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples.
  
  
  CONCLUSIONS
  Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.
authors.affiliation: Stem Cell Laboratory, National Center for Cell Science, Ganeshkhind, Pune, India.
authors.forename: Namrata C
authors.initials: NC
authors.lastname: Shabrani
authors.forename: Nikhat Firdaus Q
authors.initials: NF
authors.lastname: Khan
authors.forename: Vaijayanti P
authors.initials: VP
authors.lastname: Kale
authors.forename: Lalita S
authors.initials: LS
authors.lastname: Limaye
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000737:chemistry; D017209:Apoptosis; D016718:Arachidonic Acid / Q000494:pharmacology*; D001793:Blood Preservation / Q000379:methods; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects*; D005615:Freezing; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D013926:Thrombopoietin / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry; D017540:Tunica Media / Q000737:chemistry
chemical_list: D018952:Antigens, CD34; D003451:Cryoprotective Agents; D014157:Transcription Factors; D004281:Docosahexaenoic Acids; D016718:Arachidonic Acid; D013926:Thrombopoietin; D004121:Dimethyl Sulfoxide