Phenotypic instability of mesenchymal stromal cells isolated and expanded in different growth media

Abstract

Background & Aim Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are an attractive candidate for use in regenerative therapies and are being widely investigated for the treatment of a broad spectrum of diseases. Despite this, the efficacy of treatment is unproven and interpretation of trial data is difficult, in part due to the heterogeneity of MSC preparations as a result of variable manufacturing processes. MSCs are currently isolated in basal media containing fetal bovine serum (FBS), platelet lysate or other supplements but little is understood about the impact of changes in media composition on the biology, therapeutic action or potency of MSCs. The aim of this study was to analyse the influence of widely used culture media on the growth parameters, differentiation potential and surface marker expression of human BM-MSCs. Methods, Results & Conclusion Whole bone marrow obtained from the iliac crest of a 24-year-old healthy donor was plated in a standard basal medium with 5 different supplements: (1) a proprietary serum-free supplement, (2) 5% human platelet lysate, (3) 10% FBS, screened and proven to support MSC isolation and expansion (4) 10% screened FBS and 1 ng/ml recombinant human basic fibroblast growth factor (FGF2) and (5) 10% unscreened FBS and 1 ng/ml FGF2. The impact of media composition on growth kinetics, morphology, differentiation propensity and surface immunophenotype was assessed. The latter involved the BD Lyoplate flow cytometry screening panel of 242 monoclonal antibodies. MSC growth kinetics were highly influenced by expansion medium, with the greatest proliferation rate observed in MSCs cultured in the serum-free supplemented medium. Cell morphology varied greatly between cell preparations. Adipogenesis and osteogenesis were maintained in all culture conditions, but the degree of differentiation varied between conditions. Chondrogenesis was most starkly effected by changes in expansion medium and was only observed in serum-containing media. Analysis of surface marker expression revealed significant variation in response to culture conditions, with 70 of the 242 markers differentially expressed between cell preparations. These data suggest that changes in the composition of the culture media – for example switching batches of FBS – have profound effects on key biological attributes of BM-MSCs and on their surface marker expression profiles. This demonstrates the critical need for harmonisation of media formulation to aid interpretation of clinical results.