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

doi:10.3390/antiox12010163

. 2023 Jan 10;12(1):163.

doi: 10.3390/antiox12010163.

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

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.

PMID: 36671025
PMCID: PMC9854832
DOI: 10.3390/antiox12010163

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

Alexis Laurent et al. Antioxidants (Basel). 2023.

. 2023 Jan 10;12(1):163.

doi: 10.3390/antiox12010163.

Authors

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.

PMID: 36671025
PMCID: PMC9854832
DOI: 10.3390/antiox12010163

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.

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Conflict of interest statement

Authors A.L., A.J. and C.P. were employed by LAM Biotechnologies SA during the course of the study. The remaining authors declare no conflicts of interest.

Figures

**Figure A1**
Schematic and illustrated technical overview of the main steps of the present study. The retained approach enabled a translational assessment of the cellular extracts under consideration, ranging from formulation options to ex vivo investigation of HA-containing combination product efficacy-related parameters. Steps 1 and 2 enabled the identification of optimally stable and sterilizable cellular derivatives. Step 3 enabled the characterization of the intrinsic properties and effects of the lyophilized cellular extracts. Step 4 enabled the study of the effects of the cellular extracts in a hydrogel environment with simulated oxidative stress within H₂O₂ challenge assays (i.e., mimicking accelerated degradation conditions or a pathological in vivo environment). Steps 5 and 6 enabled the validation of the technical applicability of the extracts and combination products for potential therapeutic management of tendinopathies, based on formulation, preliminary safety, and efficacy-related parameters. FRAP, ferric reducing antioxidant power; HA, hyaluronic acid; TEAC, Trolox equivalent antioxidant capacity.

**Figure A2**
Schematic workflow of the preliminary formulation study, which enabled the selection of the optimal lyoprotectant formula for all of the assays of the main study. Placebo samples contained no cellular derivatives. LYO-PLA, lyophilized placebo sample; LYO-LYS, lyophilized lysate fraction; LYO-MEM, lyophilized membrane fraction; LYO-SN, lyophilized soluble fraction; LYO-WC, lyophilized whole-cell fraction.

**Figure A3**
Step-by-step presentation of the experimental plan of the main study, with reference to the questions addressed by the various assays and reference to the obtained experimental results (i.e., for each assay). A distinction is made between the functional attributes of the cellular extracts in lyophilizate form (i.e., intrinsic antioxidant activity) and the functional attributes of the cellular extracts reconstituted in HA (i.e., mainly the viscosity modulating functions, mediated by oxidative stress). References to the corresponding experimental results are provided (i.e., in blue font; figures, and tables). η*, complex viscosity; BSA, bovine serum albumin; FRAP, ferric reducing antioxidant power; HA, hyaluronic acid; LYO-WC, lyophilized whole-cell fraction; TEAC; Trolox equivalent antioxidant capacity.

**Figure 1**
Comparative assessment of the TEAC values and hydrogel viscosity modulating properties of various doses of progenitor tenocyte whole cell samples (i.e., lyophilizates reconstituted in aqueous solvent), before and after submicron filtration and γ-irradiation, respectively. TEAC dose-response of reconstituted non-irradiated (A) or γ-irradiated (31 kGy, (B)) whole cell samples containing 1.5 to 7.5 million cell equivalents/vial before and after 0.22 µm filtration, with the corresponding placebo controls. Results notably outlined a strong response (i.e., relative increase) of the γ-irradiated samples in TEAC measurements compared respectively to the same non-irradiated samples ((B) vs. (A)). Complex viscosity η* of reconstituted (i.e., in a hydrogel of HA 2.2–2.4 MDa MW at 1% in H₂O:PBS 1:1) non-irradiated (C) or γ-irradiated (31 kGy, (D)) whole cell samples containing 1.5 to 7.5 million cell equivalents/vial, with the corresponding placebo controls. Each sample was analyzed following the addition of H₂O₂ (i.e., challenge item) or PBS (i.e., internal non-challenged controls) and incubation for 1 h at 37 °C. Very significant statistical differences (i.e., ** or 0.001 < p value < 0.01) or extremely significant statistical differences (i.e., *** or 0.0001 < p value < 0.001; **** or p value < 0.0001) were found between the presented mean values. HA, hyaluronic acid; kGy, kiloGray; LYO-WC, lyophilized whole cell fraction; MW, molecular weight; PBS, phosphate buffered saline; TEAC, Trolox equivalent antioxidant capacity.

**Figure 2**
TEAC values of various non-irradiated (A) and γ-irradiated (i.e., irradiation dose of 31 kGy, (B)) progenitor tenocyte extracts, before and after 0.22 µm filtration, respectively. Results outlined a strong response (i.e., relative increase) of the γ-irradiated samples in TEAC measurements compared respectively to the same non-irradiated samples ((B) vs. (A)). Significant statistical differences (i.e., * or p value < 0.05), very significant statistical differences (i.e., ** or 0.001 < p value < 0.01), or extremely significant statistical differences (i.e., **** or p value < 0.0001) were found between the presented mean values. kGy, kiloGray; LYO-PLA, lyophilized placebo sample; LYO-LYS, lyophilized lysate fraction; LYO-MEM, lyophilized membrane fraction; LYO-SN, lyophilized soluble fraction; LYO-WC, lyophilized whole-cell fraction.

**Figure 3**
Complex viscosity η* of reconstituted (i.e., in a hydrogel of HA 2.2–2.4 MDa MW at 1% in H₂O:PBS 1:1) non-irradiated (A) or γ-irradiated (5 kGy, (B); 25 kGy, (C); 50 kGy, (D)) progenitor tenocyte extracts, with the placebo controls. Each sample was analyzed following H₂O₂ challenge or PBS addition (i.e., internal non-challenged controls). Very significant (i.e., ** or 0.001 < p value < 0.01) or extremely significant (i.e., **** or p value < 0.0001) differences were found between the sample mean values. HA, hyaluronic acid; kGy, kiloGray; LYO-PLA, lyophilized placebo sample; LYO-LYS, lyophilized lysate fraction; LYO-MEM, lyophilized membrane fraction; LYO-SN, lyophilized soluble fraction; LYO-WC, lyophilized whole-cell fraction; MDa, megaDaltons; MW, molecular weight; Pa·s, Pascal seconds; PBS, phosphate-buffered saline.

**Figure 4**
Photographic records of whole-cell lyophilizates after three months of storage at 4 °C (A1), after three months of storage at 37 °C and 90% relative humidity (A2), after three months of storage at −80 °C and 48 h at ambient temperature (A3), and after γ-irradiation at 50 kGy (A4). Complex viscosity η* value evolution at various timepoints after non-irradiated (B) and γ-irradiated (31 kGy, (C)) whole-cell lyophilizate reconstitution in HA-based hydrogels (2.2–2.4 MDa MW) within an oxidative challenge assay. Extremely significant (i.e., **** or p value < 0.0001) differences were found between each challenged sample and the corresponding unchallenged control. HA, hyaluronic acid; kGy, kiloGray; LYO-WC, lyophilized whole-cell fraction; MDa, megadalton; MW, molecular weight; Pa·s, Pascal seconds.

**Figure 5**
Rheological study of the behavior of various hydrogel samples under oxidative challenge. Complex viscosity η* of whole-cell samples resuspended in 1.0–1.25 MDa MW HA-based hydrogel (A) or in 2.2–2.4 MDa MW HA-based hydrogel (B) and challenged with various concentrations of H₂O₂. (C) Complex viscosity η* of samples containing a constant quantity of BSA suspended in a 2.2–2.4 MDa MW HA-based hydrogel and challenged with various concentrations of H₂O₂. (D) Complex viscosity η* of samples containing various quantities of BSA suspended in a 2.2–2.4 MDa MW HA-based hydrogel and challenged with a constant quantity of H₂O₂ (i.e., 30% *w/w*). Very significant (i.e., ** or 0.001 < p value < 0.01) or extremely significant (i.e., **** or p value < 0.0001) were found between mean values. BSA, bovine serum albumin; HA, hyaluronic acid; LYO-WC, lyophilized whole-cell fraction; MDa, megaDalton; MW, molecular weight; Pa·s, Pascal seconds.

**Figure 6**
Results of the in vitro friction modulation capacity determination assay. Mean kinetic friction forces were determined without any lubricating agent between the base plate and the sliding bloc (i.e., “control” sample), with PBS or linear HA as a simple lubricant, or with the various non-irradiated and gamma-irradiated (i.e., 31 kGy) lyophilizates resuspended in HA before analysis (i.e., combination product samples). Extremely significant (i.e., **** or p value < 0.0001) differences were found between mean values for the control and both reference conditions. HA, hyaluronic acid; kGy, kiloGray; LYO-PLA, lyophilized placebo sample; LYO-LYS, lyophilized lysate fraction; LYO-MEM, lyophilized membrane fraction; LYO-SN, lyophilized soluble fraction; LYO-WC, lyophilized whole-cell fraction; N, Newton; PBS, phosphate-buffered saline.

**Figure 7**
Results of the ex vivo bioadhesion assays for hydrogel combination product samples containing non-irradiated or gamma-irradiated (i.e., 31 kGy) cellular extracts. Panels (A,B) express different parameters of the same force profiles (i.e., analysis of unchallenged samples). Similarly, panels (C,D) express different parameters of the same force profiles (i.e., analysis of H₂O₂-challenged samples). (A) Mean force of adhesion values of the various hydrogel samples compared to PBS. (B) Mean work of adhesion values of the same hydrogel samples compared to PBS. (C) Mean force of adhesion values of the various H₂O₂-challenged hydrogel combination product samples compared to unchallenged PBS and unchallenged linear HA controls. (D) Mean work of adhesion values of the same H₂O₂-challenged hydrogel samples compared to unchallenged PBS and unchallenged linear HA controls. Significant statistical differences (i.e., * or p value < 0.05), very significant statistical differences (i.e., ** or 0.001 < p value < 0.01), or extremely significant statistical differences (i.e., **** or p value < 0.0001) were found between the presented mean values. HA, hyaluronic acid; kGy, kiloGray; LYO-LYS, lyophilized lysate fraction; LYO-MEM, lyophilized membrane fraction; LYO-PLA, lyophilized placebo sample; LYO-SN, lyophilized soluble fraction; LYO-WC, lyophilized whole-cell fraction; N, Newton; PBS, phosphate-buffered saline.

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References

1. Grognuz A., Scaletta C., Farron A., Raffoul W., Applegate L.A. Human fetal progenitor tenocytes for regenerative medicine. Cell Transpl. 2016;25:463–479. doi: 10.3727/096368915X688515. - DOI - PubMed
1. Grognuz A., Scaletta C., Farron A., Pioletti D.P., Raffoul W., Applegate L.A. Stability enhancement using hyaluronic acid gels for delivery of human fetal progenitor tenocytes. Cell Med. 2016;8:87–97. doi: 10.3727/215517916X690486. - DOI - PMC - PubMed
1. Laurent A., Abdel-Sayed P., Grognuz A., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A.S., Raffoul W., Kronen P., Nuss K., et al. Industrial development of standardized fetal progenitor cell therapy for tendon regenerative medicine: Preliminary safety in xenogeneic transplantation. Biomedicines. 2021;9:380. doi: 10.3390/biomedicines9040380. - DOI - PMC - PubMed
1. Laurent A., Porcello A., Fernandez P.G., Jeannerat A., Peneveyre C., Abdel-Sayed P., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A., et al. Combination of hyaluronan and lyophilized progenitor cell derivatives: Stabilization of functional hydrogel products for therapeutic management of tendinous tissue disorders. Pharmaceutics. 2021;13:2196. doi: 10.3390/pharmaceutics13122196. - DOI - PMC - PubMed
1. Pearce K.F., Hildebrandt M., Greinix H., Scheding S., Koehl U., Worel N., Apperley J., Edinger M., Hauser A., Mischak-Weissinger E., et al. Regulation of advanced therapy medicinal products in Europe and the role of academia. Cytotherapy. 2014;16:289–297. doi: 10.1016/j.jcyt.2013.08.003. - DOI - PubMed

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[1] Grognuz A., Scaletta C., Farron A., Raffoul W., Applegate L.A. Human fetal progenitor tenocytes for regenerative medicine. Cell Transpl. 2016;25:463–479. doi: 10.3727/096368915X688515. - DOI - PubMed

[2] Grognuz A., Scaletta C., Farron A., Raffoul W., Applegate L.A. Human fetal progenitor tenocytes for regenerative medicine. Cell Transpl. 2016;25:463–479. doi: 10.3727/096368915X688515. - DOI - PubMed

[3] Grognuz A., Scaletta C., Farron A., Pioletti D.P., Raffoul W., Applegate L.A. Stability enhancement using hyaluronic acid gels for delivery of human fetal progenitor tenocytes. Cell Med. 2016;8:87–97. doi: 10.3727/215517916X690486. - DOI - PMC - PubMed

[4] Grognuz A., Scaletta C., Farron A., Pioletti D.P., Raffoul W., Applegate L.A. Stability enhancement using hyaluronic acid gels for delivery of human fetal progenitor tenocytes. Cell Med. 2016;8:87–97. doi: 10.3727/215517916X690486. - DOI - PMC - PubMed

[5] Laurent A., Abdel-Sayed P., Grognuz A., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A.S., Raffoul W., Kronen P., Nuss K., et al. Industrial development of standardized fetal progenitor cell therapy for tendon regenerative medicine: Preliminary safety in xenogeneic transplantation. Biomedicines. 2021;9:380. doi: 10.3390/biomedicines9040380. - DOI - PMC - PubMed

[6] Laurent A., Abdel-Sayed P., Grognuz A., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A.S., Raffoul W., Kronen P., Nuss K., et al. Industrial development of standardized fetal progenitor cell therapy for tendon regenerative medicine: Preliminary safety in xenogeneic transplantation. Biomedicines. 2021;9:380. doi: 10.3390/biomedicines9040380. - DOI - PMC - PubMed

[7] Laurent A., Porcello A., Fernandez P.G., Jeannerat A., Peneveyre C., Abdel-Sayed P., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A., et al. Combination of hyaluronan and lyophilized progenitor cell derivatives: Stabilization of functional hydrogel products for therapeutic management of tendinous tissue disorders. Pharmaceutics. 2021;13:2196. doi: 10.3390/pharmaceutics13122196. - DOI - PMC - PubMed

[8] Laurent A., Porcello A., Fernandez P.G., Jeannerat A., Peneveyre C., Abdel-Sayed P., Scaletta C., Hirt-Burri N., Michetti M., de Buys Roessingh A., et al. Combination of hyaluronan and lyophilized progenitor cell derivatives: Stabilization of functional hydrogel products for therapeutic management of tendinous tissue disorders. Pharmaceutics. 2021;13:2196. doi: 10.3390/pharmaceutics13122196. - DOI - PMC - PubMed

[9] Pearce K.F., Hildebrandt M., Greinix H., Scheding S., Koehl U., Worel N., Apperley J., Edinger M., Hauser A., Mischak-Weissinger E., et al. Regulation of advanced therapy medicinal products in Europe and the role of academia. Cytotherapy. 2014;16:289–297. doi: 10.1016/j.jcyt.2013.08.003. - DOI - PubMed

[10] Pearce K.F., Hildebrandt M., Greinix H., Scheding S., Koehl U., Worel N., Apperley J., Edinger M., Hauser A., Mischak-Weissinger E., et al. Regulation of advanced therapy medicinal products in Europe and the role of academia. Cytotherapy. 2014;16:289–297. doi: 10.1016/j.jcyt.2013.08.003. - DOI - PubMed

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

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

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Abstract

Conflict of interest statement

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