Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

ABSTRACT

PURPOSE: 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 AND MATERIALS 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. CONCLUSION: 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.