title: Immunosuppressive capabilities of mesenchymal stromal cells are maintained under hypoxic growth conditions and after gamma irradiation.
abstract: OBJECTIVE
  The discovery of regenerative and immunosuppressive capacities of mesenchymal stromal cells (MSCs) raises hope for patients with tissue-damaging or severe, treatment-refractory autoimmune disorders. We previously presented a method to expand human MSCs in a bioreactor under standardized Good Manufacturing Practice conditions. Now we characterized the impact of critical treatment conditions on MSCs with respect to immunosuppressive capabilities and proliferation.
  
  
  METHODS
  MSC proliferation and survival after γ irradiation were determined by 5-carboxyfluorescein diacetate N-succinimidyl ester and annexinV/4',6-diamidino-2-phenylindole (DAPI) staining, respectively. T-cell proliferation assays were used to assess the effect of γ irradiation, passaging, cryopreservation, post-thaw equilibration time and hypoxia on T-cell suppressive capacities of MSCs. Quantitative polymerase chain reaction and β-galactosidase staining served as tools to investigate differences between immunosuppressive and non-immunosuppressive MSCs.
  
  
  RESULTS
  γ irradiation of MSCs abrogated their proliferation while vitality and T-cell inhibitory capacity were preserved. Passaging and long cryopreservation time decreased the T-cell suppressive function of MSCs, and postthaw equilibration time of 5 days restored this capability. Hypoxic culture markedly increased MSC proliferation without affecting their T-cell-suppressive capacity and phenotype. Furthermore, T-cell suppressive MSCs showed higher CXCL12 expression and less β-galactosidase staining than non-suppressive MSCs.
  
  
  CONCLUSIONS
  We demonstrate that γ irradiation is an effective strategy to abrogate MSC proliferation without impairing the cells' immunosuppressive function. Hypoxia significantly enhanced MSC expansion, allowing for transplantation of MSCs with low passage number. In summary, our optimized MSC expansion protocol successfully addressed the issues of safety and preservation of immunosuppressive MSC function after ex vivo expansion for therapeutic purposes.
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Nold
authors.affiliation: Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University Giessen, Giessen, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Hackstein
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.
authors.forename: Tabea
authors.initials: T
authors.lastname: Riedlinger
authors.affiliation: Department for Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kasper
authors.affiliation: Department for Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
authors.forename: Anne
authors.initials: A
authors.lastname: Neumann
authors.affiliation: Institute of Molecular Biology and Tumor Research, Philipps University Marburg, Marburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Mernberger
authors.affiliation: Department of Orthopedics and Rheumatology, Philipps University Marburg, Marburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Fölsch
authors.affiliation: Department of Orthopedics and Rheumatology, Philipps University Marburg, Marburg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Schmitt
authors.affiliation: Department of Orthopedics and Rheumatology, Philipps University Marburg, Marburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fuchs-Winkelmann
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Barckhausen
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Killer
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Neubauer
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany. Electronic address: Brendelc@staff.uni-marburg.de.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Brendel
mesh_terms: D000328:Adult; D015687:Cell Hypoxia / Q000502:physiology*; D049109:Cell Proliferation / Q000528:radiation effects*; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000096:biosynthesis; D015925:Cryopreservation; D005452:Fluoresceins; D005720:Gamma Rays / Q000009:adverse effects*; D006801:Humans; D007165:Immunosuppression Therapy / Q000379:methods; D007166:Immunosuppressive Agents / Q000528:radiation effects; D008213:Lymphocyte Activation / Q000276:immunology / Q000528:radiation effects; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000528:radiation effects*; D008875:Middle Aged; D013601:T-Lymphocytes / Q000276:immunology; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D005452:Fluoresceins; D007166:Immunosuppressive Agents; C084577:5-carboxyfluorescein diacetate; D001616:beta-Galactosidase
keywords: MSC; bioreactor; cryopreservation; equilibration; hypoxia; immunosuppressive capacities; irradiation; mesenchymal stromal cells; passaging; senescence