Serum and supplement optimization for EU GMP ‐compliance in cardiospheres cell culture

Cardiac progenitor cells ( CPC s) isolated as cardiospheres ( CS s) and CS ‐derived cells ( CDC s) are a promising tool for cardiac cell therapy in heart failure patients, having CDC s already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices ( GMP s) 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 E uropean GMP s were investigated in CS s and CDC s 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 (a HS ) and two different commercially available GMP AB HS s were compared with standard research‐grade FBS . CPC s from all HS s 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 FBS s (gi FBS s) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPC s yield/phenotype. In conclusion, the use of HS s for the isolation and expansion of CS s/ CDC s has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected gi FBS allows successful EU GMP ‐complying CPC s culture.