Lyophilized Progenitor Tenocyte Extracts: Sterilizable Cytotherapeutic Derivatives with Antioxidant Properties and Hyaluronan Hydrogel Functionalization Effects

Alexis Laurent; Alexandre Porcello; Annick Jeannerat; Cédric Peneveyre; Agathe Coeur; Philippe Abdel-Sayed; Corinne Scaletta; Murielle Michetti; Anthony de Buys Roessingh; Olivier Jordan; Eric Allémann; Wassim Raffoul; Nathalie Hirt-Burri; Lee Ann Applegate

doi:10.3390/antiox12010163

Lyophilized Progenitor Tenocyte Extracts: Sterilizable Cytotherapeutic Derivatives with Antioxidant Properties and Hyaluronan Hydrogel Functionalization Effects

Antioxidants (Basel). 2023 Jan 10;12(1):163. doi: 10.3390/antiox12010163.

Authors

Alexis Laurent^{1

2}, Alexandre Porcello^{3

4}, Annick Jeannerat², Cédric Peneveyre², Agathe Coeur^{3

4

5}, Philippe Abdel-Sayed^{1

6}, Corinne Scaletta¹, Murielle Michetti¹, Anthony de Buys Roessingh^{7

8}, Olivier Jordan^{3

4}, Eric Allémann^{3

4}, Wassim Raffoul^{8

9}, Nathalie Hirt-Burri¹, Lee Ann Applegate^{1

8

9

10

11}

Affiliations

¹ Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland.
² Applied Research Department, LAM Biotechnologies SA, CH-1066 Epalinges, Switzerland.
³ School of Pharmaceutical Sciences, University of Geneva, CH-1206 Geneva, Switzerland.
⁴ Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CH-1206 Geneva, Switzerland.
⁵ Engineering Cycle, Ecole de Biologie Industrielle, F-95885 Cergy, France.
⁶ DLL Bioengineering, Discovery Learning Program, STI School of Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
⁷ Children and Adolescent Surgery Service, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland.
⁸ Lausanne Burn Center, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland.
⁹ Plastic, Reconstructive, and Hand Surgery Service, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland.
¹⁰ Center for Applied Biotechnology and Molecular Medicine, University of Zurich, CH-8057 Zurich, Switzerland.
¹¹ Oxford OSCAR Suzhou Center, Oxford University, Suzhou 215123, China.

Abstract

Cultured primary progenitor tenocytes in lyophilized form were previously shown to possess intrinsic antioxidant properties and hyaluronan-based hydrogel viscosity-modulating effects in vitro. The aim of this study was to prepare and functionally characterize several stabilized (lyophilized) cell-free progenitor tenocyte extracts for inclusion in cytotherapy-inspired complex injectable preparations. Fractionation and sterilization methods were included in specific biotechnological manufacturing workflows of such extracts. Comparative and functional-oriented characterizations of the various extracts were performed using several orthogonal descriptive, colorimetric, rheological, mechanical, and proteomic readouts. Specifically, an optimal sugar-based (saccharose/dextran) excipient formula was retained to produce sterilizable cytotherapeutic derivatives with appropriate functions. It was shown that extracts containing soluble cell-derived fractions possessed conserved and significant antioxidant properties (TEAC) compared to the freshly harvested cellular starting materials. Progenitor tenocyte extracts submitted to sub-micron filtration (0.22 µm) and 60Co gamma irradiation terminal sterilization (5−50 kGy) were shown to retain significant antioxidant properties and hyaluronan-based hydrogel viscosity modulating effects. Hydrogel combination products displayed important efficacy-related characteristics (friction modulation, tendon bioadhesivity) with significant (p < 0.05) protective effects of the cellular extracts in oxidative environments. Overall, the present study sets forth robust control methodologies (antioxidant assays, H2O2-challenged rheological setups) for stabilized cell-free progenitor tenocyte extracts. Importantly, it was shown that highly sensitive phases of cytotherapeutic derivative manufacturing process development (purification, terminal sterilization) allowed for the conservation of critical biological extract attributes.

Keywords: antioxidants; cell-free extracts; cytotherapies; gamma irradiation; hyaluronic acid; hydrogel viscosity; progenitor tenocytes; rheology; sterilization; tendinopathies.

Grants and funding

LADE 20-411/SPEI