Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine.

Annick Jeannerat,Wassim Raffoul,Anthony De Buys Roessingh,Diego Chiappe,Florence Armand,Romain Hamelin,Lee Ann Applegate,Cédric Peneveyre,Nathalie Hirt-Burri,Alexis Laurent,Corinne Scaletta

doi:10.3390/cells10112872

Abstract

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products.

Highlights

Human fetal progenitor tenocytes produced in vitro under defined multi-tiered cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine [1,2,3,4,5]
To confirm that the Human fetal progenitor tenocytes (hFPT) responded to cell culture transition from normoxia to hypoxia, a timecourse assay of HIF-1α induction was performed and monitored by Western blot analysis
In normoxia (i.e., T0 timepoint), no HIF-1α was detected in the cell lysate, while a strong induction of the protein of interest was already observed after 3 h and up to 9 h in hypoxic conditions (Figure 1, Table S1)

Summary

Introduction

Human fetal progenitor tenocytes (hFPT) produced in vitro under defined multi-tiered cell bank systems have recently been characterized (i.e., in vitro and in preclinical models) and qualified as potential therapeutic cell sources in tendon regenerative medicine [1,2,3,4,5]. Within the establishment of scalable hFPT (i.e., cellular API) manufacturing processes, multiple parameters require appropriate technical optimization and validation phases in order to comply with the current systematic requirements of a risk-based, process-oriented, and quality-driven production approach [5]. The adequate selection of consumable options and process parameters are paramount to ensuring the consistency in both quality and safety of the manufactured cells, to be used as intermediary or bulk raw materials (e.g., for further derivation of lysates or sub-cellular vesicles) or as an API in its final form for standardized transplant development (e.g., extemporaneous thawing and seeding of viable hFPTs on suitable implantable scaffolds)

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