Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34⁺ Cells for Clinical...

We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34⁺ stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34⁺ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34⁺ -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34⁺ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34⁺ cell immunoselection and formulation. The assessment of CD34⁺ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 10⁶  ± 36.8 × 10⁶ viable CD34⁺ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 10⁶  ± 8.1 × 10⁶ viable CD34⁺ cells. CD34⁺ identity, genetic stability, and telomere length were consistent with those of basal CD34⁺ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34⁺ -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34⁺ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.