title: Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells.
abstract: OBJECTIVE
  Mesenchymal stromal cells (MSC) can be expanded in vitro for clinical use and have been evaluated in clinical trials as immunosuppressants and to heal damaged tissues. We investigated the impact of freezing and prolonged storage on cell viability, proliferation and immunosuppression in vitro.
  
  
  METHODS
  MSC were expanded from bone marrow (BM) of healthy subjects according to standard protocols in the presence of fetal calf serum (FCS). The immunosuppressive potential of MSC was analyzed in mixed lymphocyte cultures (MLC) and after stimulation with phytohemagglutinin (PHA).
  
  
  RESULTS
  Expansion of frozen mononuclear cells (MNC) diminished MSC yield after expansion compared with plating of fresh MNC. MSC derived from frozen MNC were also less immunosuppressive. MSC harvested at various passages after expansion in vivo suppressed lymphocyte proliferation equally. Pooling of MSC from several donors generated higher and more stable suppression in both MLC and after PHA stimulation. After passage 1, plating at lower densities in 20% FCS increased cell expansion of MSC up to 25-fold compared with standard conditions.
  
  
  CONCLUSIONS
  MNC should not be frozen prior to MSC expansion. Decreased replating density and increased FCS levels generate higher numbers of MSC. Freezing of ex vivo-expanded MSC for >30 months did not affect cell viability or the ability to suppress lymphocyte proliferation. For effective immunosuppression in vitro MSC should be stored for less than 6 months and pooled from two or three donors.
authors.affiliation: Karolinska Institutet, Stockholm, Sweden. hakan.samuelsson@ki.se
authors.forename: H
authors.initials: H
authors.lastname: Samuelsson
authors.forename: O
authors.initials: O
authors.lastname: Ringdén
authors.forename: H
authors.initials: H
authors.lastname: Lönnies
authors.forename: K
authors.initials: K
authors.lastname: Le Blanc
mesh_terms: D001853:Bone Marrow; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D005615:Freezing; D006648:Histocompatibility; D006801:Humans; D007165:Immunosuppression Therapy; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000502:physiology; D007959:Lymphocyte Culture Test, Mixed; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D010835:Phytohemagglutinins / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology* / Q000502:physiology; D013997:Time Factors
chemical_list: D010835:Phytohemagglutinins