Abstract
BackgroundHuman deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear.MethodsWe isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB.ResultsWe optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB.ConclusionThe manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.
Highlights
Human deciduous pulp stem cells have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models
Establishment of a standard operating procedure (SOP) for the manufacture and quality control of Human deciduous pulp stem cells (hDPSCs) production using colony-forming unitfibroblast (CFU-F)-derived multicolonies under XF conditions The basic procedure of the present isolation and expansion of hDPSCs was similar to the original procedure [21, 22], but the difference was propagation to secure xenogeneic-free and serum-free (XF/SF) conditions compared to the original procedure
Remnant dental pulp tissues of deciduous teeth extracted from healthy donors were treated with a XF tissue digestion regent, and the single-cell suspension was seeded into a regular T-75 cm2 plastic culture flask with a commercially available XF medium
Summary
Human deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Large-scale ex vivo growth and a steady supply of clinical-grade MSCs is required for the wide range of clinical applications in human MSC-based tissue engineering and regenerative medicine. The production of clinical-grade human MSCs for patient treatment cannot be achieved using the simple laboratory-scale procedure, including the isolation and expansion of MSC for basic and translational studies. The ex vivo standard procedure for clinical-grade MSCs should be employed to produce clinical-grade MSC products with a defined quality and safety as well as an ensured steady supply [9, 10]. To produce final products with a defined quality and safety that are clinically utilized for patients, a validated standard operating procedure (SOP) needs to be established for the whole manufacturing and quality control process to produce clinical-grade MSCs [11]. MSC products have been commercially approved for treating patients with refractory graft-versus-host disease (GVHD) in Korea, Canada, New Zealand, and Japan [12, 13]
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