Abstract
BackgroundDevelopment of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.MethodsDPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.ResultsSimilar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions.ConclusionsThese findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.
Highlights
Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant conditions
Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for Dental pulp stem cell (DPSC) under all expansion conditions. Alveolar bone marrow mesenchymal stem cell (aBMMSC) showed lower cumulative Population doubling (PD) compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7
In the context of establishing standardized conditions for safe and efficient clinical-grade expansion of oral Mesenchymal stem cell (MSC), bridging the gap between research models and clinical applications in the orofacial region, the present study aimed to identify the critical “stemness” properties influenced by the culture “micro-milieu” that might have an impact on the MSC clinical performance
Summary
Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. A recent definition designates MSCs as cell-based medicinal products (CBMPs); that is, medicinal products presented as having properties for, or used in or administered to, human beings with a view to treating, preventing or diagnosing a disease in which the pharmacological, immunological or metabolic actions are carried out by cells or tissues [7]. These products have to be prepared in compliance with good manufacturing practices (cGMP) that ensure consistent production and controlled quality standards appropriate to their intended use, as described in EU Regulation 2003/94/EC [8]. To specify procedures for proper handling under cGMP conditions, current research focuses on evaluating the effects that expansion procedures might have on the MSC “stemness” characteristics, their phenotypic and genetic stability, the efficacy in regenerating the target tissues and the permitted population doublings (PDs) before senescence emerges to establish reliable characterization methodologies for the accurate assessment of each of these parameters [9]
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