Ringer lactate

The effect of electrolyte solutions (5% glucoside solution, Grifols; Ringer lactate, Fresenius Kabi; and Viaflo Plasmalyte 148, Baxter) and albumins (recombinant albumins: AlbIX^® and Recombumin^® Alpha, from Albumedix; and HSA: Albutein, from Grifols) were used to evaluate their effect on the preservation of CQA in MSC, including cell recovery, viability, identity and potency, both pre- and post-thawing, to assess the stability of the final reconstituted cellular product (Table 1).
Cells were cryopreserved in a solution composed of Dulbecco’s Phosphate-Buffered Saline (DPBS; Gibco) supplemented with 10% (v/v) dimethyl sulfoxide (DMSO; OriGen Biomedical, Austin, TX, USA) and 2% (w/v) human serum albumin (HSA; Grifols, Barcelona, Spain), by applying a controlled freezing rate of 1 °C/min in a Mr. Frosty device (Nalgene, Rochester, NY, USA) kept in a − 80 °C freezer for 24 h before storage at − 196 °C in a liquid nitrogen tank until further use [17 (link)]. On the day of thawing, cells were rapidly thawed in a 37 °C water bath, then slowly diluted 1:10 using pre-cooled thawing solution consisting of 2% (w/v) albumin in Plasmalyte 148. DMSO was washout by centrifugation at 340g for 10 min. Finally, each experimental condition for assessing stability was created by resuspending in Plasmalyte 148 supplemented with 2% (w/v) of either one of the albumins and set up 10 in mL syringes.

A skeletal muscle biopsy to obtain autologous SMDC was taken as described previously [17 (link)]. Briefly, approximately 1 cm³ of skeletal muscle (Musculus pectoralis major) was removed and transported to a cGMP facility (Innovacell Biotechnology AG, Austria). Pure skeletal muscle tissue was enzymatically digested by incubation with collagenase (Serva/Nordmark, Germany). The SMDC obtained were maintained by standard cell culture methods. Cells were cultured in Ham’s F-10 basal medium supplemented with foetal calf serum (Life Technologies, UK) and bFGF (CellGenix, Freiburg, Germany) at 37 °C, 5% CO₂. The medium was changed every 3–4 days. SMDC were subcultivated three times corresponding to a mean ± SD cultivation time of 29.31 ± 5 days followed by harvest, characterization and cryo-preservation. A total of 79.4 ± 22.5 million cells per patient resuspended in Ringer lactate (Fresenius Kabi, Austria) supplemented with DMSO (OriGen, TX, USA) and human serum albumin (Baxter, IL, USA) were cryo-preserved in liquid nitrogen until implantation. Residual cells not used for implantation were subsequently analysed by microarray, flow cytometry and immunocytochemistry.

To assess the TF expression of MSCs in infusion solutions for up to 8 h of storage, either fresh or cryopreserved and thawed MSCs were suspended in NaCl solvent (STARVISION, VN) and Ringer lactate (RL, Fresenius Kabi, USA) at a concentration of 2.10⁶ cells/mL. The samples were stored at 4 °C for 0 h, 2 h, 4 h, and 8 h. At each time point, cells were stained with CD142-PE antibody (BD Biosciences, USA) and 7-AAD for flow cytometry analysis and tested for coagulation time.