title: Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.
abstract: OBJECTIVE
  The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use.
  
  
  BACKGROUND
  MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases.
  
  
  METHODS
  In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use.
  
  
  RESULTS
  Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use.
  
  
  CONCLUSIONS
  We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.
authors.affiliation: Centro en red de Medicina Regenerativa y Terapia Celular de Castilla y León and Red Nacional de Terapia Celular (Tercel, ISCIII), Salamanca, Spain. smuntion@yahoo.es
authors.forename: S
authors.initials: S
authors.lastname: Muntión
authors.forename: F M
authors.initials: FM
authors.lastname: Sánchez-Guijo
authors.forename: S
authors.initials: S
authors.lastname: Carrancio
authors.forename: E
authors.initials: E
authors.lastname: Villarón
authors.forename: O
authors.initials: O
authors.lastname: López
authors.forename: M
authors.initials: M
authors.lastname: Diez-Campelo
authors.forename: J F
authors.initials: JF
authors.lastname: San Miguel
authors.forename: M C
authors.initials: MC
authors.lastname: del Cañizo
mesh_terms: D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007621:Karyotyping / Q000379:methods*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008677:Metaphase