Laurent Alexis, Porcello Alexandre, Jeannerat Annick, Peneveyre Cédric, Coeur Agathe, Abdel-Sayed Philippe, Scaletta Corinne, Michetti Murielle, de Buys Roessingh Anthony, Jordan Olivier, Allémann Eric, Raffoul Wassim, Hirt-Burri Nathalie, Applegate Lee Ann
Regenerative Therapy Unit, Lausanne University Hospital, University of Lausanne, CH-1066 Epalinges, Switzerland.
Applied Research Department, LAM Biotechnologies SA, CH-1066 Epalinges, Switzerland.
Antioxidants (Basel). 2023 Jan 10;12(1):163. doi: 10.3390/antiox12010163.
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.
先前的研究表明，冻干形式的原代祖细胞肌腱细胞在体外具有内在的抗氧化特性和基于透明质酸的水凝胶粘度调节作用。本研究的目的是制备几种稳定化（冻干）的无细胞祖细胞肌腱细胞提取物，并对其功能进行表征，以用于受细胞疗法启发的复合注射制剂。此类提取物的特定生物技术制造工作流程中包括了分级分离和灭菌方法。使用多种正交的描述性、比色、流变学、力学和蛋白质组学读数对各种提取物进行了比较和功能导向的表征。具体而言，保留了一种最佳的基于糖的（蔗糖/葡聚糖）赋形剂配方，以生产具有适当功能的可灭菌细胞治疗衍生物。结果表明，与新鲜收获的细胞起始材料相比，含有可溶性细胞衍生部分的提取物具有保守且显著的抗氧化特性（TEAC）。经亚微米过滤（0.22 µm）和60Coγ射线辐照终端灭菌（5−50 kGy）的祖细胞肌腱细胞提取物显示出保留了显著的抗氧化特性和基于透明质酸的水凝胶粘度调节作用。水凝胶组合产品显示出重要的与功效相关的特性（摩擦调节、肌腱生物粘附性），细胞提取物在氧化环境中具有显著（p < 0.05）的保护作用。总体而言，本研究提出了用于稳定化无细胞祖细胞肌腱细胞提取物的稳健控制方法（抗氧化测定、H2O2挑战流变学设置）。重要的是，研究表明，细胞治疗衍生物制造工艺开发的高敏感阶段（纯化、终端灭菌）能够保留关键的生物提取物属性。
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