Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood
Introduction
The cord blood (CB)-derived unrestricted somatic stem cell (USSC) is a non-hematopoietic multipotent cell displaying mesodermal, endodermal and neural differentiation capacities in vitro and in vivo (1., 2., 3., 4.). Together with the ability to migrate and accumulate to sites of injury and its response to growth factors (5,6), USSC is a promising candidate for future cellular therapy and clinical application in allogeneic settings, as well as for reprogramming for induced pluripotent stem cells (iPS) (7., 8., 9., 10., 11.).
Compliant clinical generation of cell drugs requires adherence to strict regulations to achieve high-product quality for downstream applications. Good manufacturing practice (GMP) is an essential part of the quality management system to ensure product safety and effectiveness. According to current European law, cell-based medical products should be propagated under principles of GMP when they are used in phase I studies (12).
We describe GMP-compliant generation of USSC during the pre-clinical phase to ensure consistent production, reliability and quality prior to clinical use. To conduct pre-clinical development of USSC, mononucleated cell (MNC) isolation from fresh CB, clinical-scale expansion, cryopreservation and thawing of USSC were performed in compliance with GMP standards. Release criteria for the product were defined by assessment of the immunophenotype, potency and genetic stability. Evaluation of sterility, pyrogens and endotoxins led to high safety and quality standards of the final cryopreserved and thawed products.
Section snippets
Collection of fresh CB and the processing environment
CB samples were obtained from donors meeting the criteria for hematopoietic banking according to the latest regulatory guidelines in Europe (Table I). Donated CB for USSC generation was used only if the initial cell count was not sufficient for hematopoietic banking.
Umbilical CB was collected by vein puncture in a blood collection bag (MSC1206DU; Macopharma, Langen, Germany) containing 29 mL citrate phosphate dextrose. Subsequent processing took place within 48 h under GMP conditions according to
Results
GMP-conforming generation and cultivation of USSC is mandatory for clinical applications. To ensure safety and reproducibility, the automated Sepax cell-processing system (Biosafe) was used for MNC separation from 98 fresh CB samples, similar to a methodology described previously (13).
The GMP-conforming Sepax-mediated MNC isolation of 98 CB samples (mean volume 84.61 ± 17.02 mL, range 51–136 mL) resulted in a mean recovery of 42.37 ± 10.09% NC (range 16.13–67.55%) and 63.03 ± 14.57% MNC (range
Discussion
Since its use as a hematopoietic stem cell transplant for many indications, such as leukemia and lymphoma, umbilical CB is no longer considered as biologic waste. The application of CB for non-hematopoietic indications, especially in regenerative fields, has undergone much investigation in recent years: the discovery of non-hematopoietic stem cells in CB (1) was met enthusiastically and led to it becoming a promising candidate in the fields of regenerative medicine, clinical research and
Acknowledgments
We gratefully acknowledge the Deutsche Forschungsgemeinschaft (DFG) for funding the research group FOR 717, including the project Ko2119/6-1, and the German José Carreras Leukemia Foundation for supporting us with grant DJCLS-R07/05v. We would like to thank A. Lefort and D. Stapelkamp for their technical support.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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