Abstract
Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS-derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27− free CS cultures produced a significantly reduced yield and a 10-fold drop in c-kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research-grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent-like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma-irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP-complying CPCs culture.
Highlights
During the last decade, fast-paced advancements in regenerative medicine are offering novel promising therapeutic tools such as cardiac cell therapy, which is emerging as an effective personalized treatment for end-stage heart failure (HF) patients [1,2,3,4]
Explant-derived cells (EDCs) from three different patients were plated in CS-growth medium (CGM) with or without B27, and CSs were obtained
Primary and secondary CSs yields in B27À compared with B27+ were significantly reduced to 32.8 Æ 6.9% and 46.2 Æ 12.1% respectively (Fig. 1A), whereas the average diameter of the spheres after 7 days of culture was slightly higher in B27À samples: 135.5 Æ 4.6% in CSs and 139.8 Æ 10.1% in IICSs (Fig. 1A)
Summary
Fast-paced advancements in regenerative medicine are offering novel promising therapeutic tools such as cardiac cell therapy, which is emerging as an effective personalized treatment for end-stage heart failure (HF) patients [1,2,3,4]. One of the most important issues involved is the use of supplements of xenogeneic origin for human cell culture [12]. Effective alternatives with the same wide acceptability are still missing, and it is very often impossible to exclude its use without significant changes in cell culture efficiency [12]. In this case, all possible precautions must be implemented, in terms of FBS origin, preparation and precautionary pathogen inactivation (see EMA ‘Note for Guidance on the Use of Bovine Serum in the Manufacture of Human Medicinal Products’ at www.ema.europa.eu). The same infective and immunogenic issues and risks obviously exist for any other xenogenic and/ or artificial media additive, which has to be GMP compliant
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