title: Non-enzymatic dissociation of human mesenchymal stromal cells improves chemokine-dependent migration and maintains immunosuppressive function.
abstract: OBJECTIVE
  Human bone marrow-derived mesenchymal stromal cells (MSC) can suppress inflammation; therefore their therapeutic potential is being explored in clinical trials. Poor engraftment of infused MSC limits their therapeutic utility; this may be caused by MSC processing before infusion, in particular the method of their detachment from culture.
  
  
  METHODS
  Enzymatic methods of detaching MSC (Accutase and TrypLE) were compared with non-enzymatic methods (Cell Dissociation Buffer [CDB], ethylenediamine tetra-acetic acid and scraping) for their effect on MSC viability, chemokine receptor expression, multi-potency, immunomodulation and chemokine-dependent migration.
  
  
  RESULTS
  TrypLE detachment preserved MSC viability and tri-lineage potential compared with non-enzymatic methods; however, this resulted in near complete loss of surface chemokine receptor expression. Of the non-enzymatic methods, CDB detachment preserved the highest viability while retaining significant tri-lineage differentiation potential. Once re-plated, CDB-detached MSC regained their original morphology and reached confluence, unlike with the use of other non-enzymatic methods. Viability was significantly reduced with the use of ethylenediamine tetra-acetic acid and further reduced with the use of cell scraping. Addition of 1% serum during CDB detachment led to higher MSC numbers entering autophagy and increased MSC recovery after re-plating. TrypLE and CDB-detached MSC suppressed CD3(+)CD4(+)CD25(-) T-cell proliferation, although TrypLE-detached MSC exhibited superior suppression at 1:20 ratio. CDB detachment retained surface chemokine receptor expression and consequently increased migration to CCL22, CXCL12 and CCL4, in contrast with TrypLE-detached MSC.
  
  
  CONCLUSIONS
  This study demonstrates that non-enzymatic detachment of MSC with the use of CDB minimizes the negative impact on cell viability, multipotency and immunomodulation while retaining chemokine-dependent migration, which may be of importance in MSC delivery and engraftment in sites of injury.
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom. Electronic address: garga@adf.bham.ac.uk.
authors.forename: Abhilok
authors.initials: A
authors.lastname: Garg
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Diarmaid D
authors.initials: DD
authors.lastname: Houlihan
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Victoria
authors.initials: V
authors.lastname: Aldridge
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Shankar
authors.initials: S
authors.lastname: Suresh
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ka Kit
authors.initials: KK
authors.lastname: Li
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: King
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Rupesh
authors.initials: R
authors.lastname: Sutaria
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Janine
authors.initials: J
authors.lastname: Fear
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Ricky H
authors.initials: RH
authors.lastname: Bhogal
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Patricia F
authors.initials: PF
authors.lastname: Lalor
authors.affiliation: Centre for Liver Research and National Institute for Health Research, Birmingham Liver Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom. Electronic address: p.n.newsome@bham.ac.uk.
authors.forename: Philip N
authors.initials: PN
authors.lastname: Newsome
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018925:Chemokines / Q000096:biosynthesis; D017364:Collagenases / Q000494:pharmacology*; D005029:Ethylenediamines / Q000494:pharmacology*; D006801:Humans; D007165:Immunosuppression Therapy; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D010447:Peptide Hydrolases / Q000494:pharmacology*
chemical_list: D018925:Chemokines; D005029:Ethylenediamines; C575294:accutase; C031234:ethylenediamine; D010447:Peptide Hydrolases; D017364:Collagenases
keywords: chemokine receptors; differentiation; immune suppression; mesenchymal stromal cells; migration