Theraflex methylene blue technology

PBMCsec was produced in compliance with good manufacturing practice by the Austrian Red Cross, Blood Transfusion Service for Upper Austria (Linz, Austria) as previously described [30 (link),34 (link)]. Briefly, the PBMCs were enriched using Ficoll-Paque PLUS (GE Healthcare, Chicago IL, USA) density gradient centrifugation. Cell suspensions were adjusted to 2.5 × 10⁷ cells/mL and exposed to 60 Gy γ-irradiation (IBL 437, Isotopen Diagnostik CIS GmbH, Dreieich, Germany). Subsequently, cells were cultured in phenol red-free CellGenix GMP DC medium (CellGenix, Freiburg, Germany) for 24 h. Cells, as well as cellular debris, were removed by centrifugation. The conditioned supernatants containing the secretome were filtered through 0.22 µm filters followed by viral clearance using Theraflex methylene blue technology (MacoPharma, Mouvaux, France). The secretomes were lyophilized and sterilized by high-dose γ-irradiation (25,000 Gy, Gammatron 1500, Mediscan, Seibersdorf, Austria). CellGenix GMP DC medium without cells was used as vehicle control. The GMP batches A000918399131, A00918399136, and A000918399132 were used in this study. The stock concentration of one vial lyophilized secretome equals to 25 units/mL.

Secretomes of PBMCs were produced in compliance with good manufacturing practice (GMP) by the Austrian Red Cross, Blood Transfusion Service for Upper Austria (Linz, Austria) as described.²⁴ (link)^{,26 ,}33 (link), 34 , 35 (link) In brief, PBMCs were enriched by Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA)-assisted density gradient centrifugation. Cell suspensions were adjusted to a concentration of 2.5 × 10⁷ cells / mL and exposed to 60 Gy γ-irradiation (IBL 437C, Isotopen Diagnostik CIS GmbH, Dreieich, Germany). Cells were cultured in phenol red-free CellGenix GMP DC medium (CellGenix, Freiburg, Germany) for 24 ± 2 hours. Cells and cellular debris were removed by centrifugation and conditioned supernatants containing the secretome were passed through 0.22 µm filters. Viral clearance was performed using Theraflex methylene blue technology (MacoPharma, Mouvaux, France). Secretomes were lyophilized and terminally sterilized by high-dose γ-irradiation (25,000 Gy, Gammatron 1500, Mediscan, Seibersdorf, Austria) as described previously.²² (link)^,34 CellGenix GMP DC medium used for PBMC culture but without cells served as vehicle control, unless indicated otherwise. The GMP batches A000917399046, A000918399100, and A000918399102 were used in this study.