Combi stopper

The strains used in this study are listed in Table 1. S. piezotolerans WP3 was cultured microaerobically in 2216E broth (Difco, United States) at 20°C. To investigate the growth of WP3 and derived mutants under HHP, each culture was grown in 2216E media at atmospheric pressure to stationary phase and then diluted to an optical density of 0.01 at 600 nm (Cary 60, UV-Vis, Agilent Technologies). When necessary, 4 mM nitrate and 20 mM lactate were added to the media. For the HHP growth experiments, the cells were cultivated in 2.5 ml disposable syringes without air. Then, a combi stopper (B. Braun, Melsungen, Germany) was used to replace the needle to insulate the media. The prepared syringes were placed inside stainless-steel high-pressure vessels (Feiyu Science and Technology Exploitation Co., Ltd., Nantong, China), and the hydrostatic pressure was applied using a water pump (Top Industrie, France).

The final drug product was manufactured as an investigational medicinal product to be tested in various clinical trials (Table 1). It was formulated as a ready-to-use suspension, with total cell count and packaging size depending on the intended application. The required quantity of cryopreserved drug substance aliquots, originating from one and the donor skin tissue, were thawed, pooled, washed, and suspended in HRG solution (Ringer’s lactate solution containing 2.5% human serum albumin and 0.4% glucose) at a target concentration of 1 × 10⁷ cells/ml. The cell suspension was filled in one or multiple 1-ml polycarbonate or 10-ml polypropylene syringes (BD Plastipak™, Becton Dickinson, Heidelberg, Germany), as required, which were sealed with polyethylene closing cones (Combi-Stopper, B. Braun, Melsungen, Germany).