FIG. 3.

Follow-up of the survival of hMSC (A) and foreskin fibroblast (FF) (B) loaded on FMB at different time points after sealing in CryoTubes at room temperature. The results are compared with trypsinized cells in suspension or cells on FMB that were left in optimal conditions in the CO₂ incubator 37°C. For each time point, the results are based on triplicate tubes. The range of similar experiments done with four different donors of hMSC (A) or from three FF lines (B) are within the shaded area within each graph. Initially, the cells were mixed with FMB or kept in suspension at 37°C for 24 h in slow rotation; then, the number of residual living cells in each arm was evaluated (A1, B1). The cells on FMB or in suspension were then sealed at room temperature, and MTS assay for the survival at different time points was done. The results were normalized to the initial cell number at day 1 (A2, B2). In parallel, hMSC nuclei stained by propidium iodide (PI) show cell density on minute FMB samples taken and fixed at the different time points (C). A stable density of cells that were kept at room temperature at different time points is evident (C1–C3). The elevated density of the cells that continued to proliferate in the optimal conditions at the incubator to reach confluence are also shown (C1, C5, C6). Eventually, at the end of the follow-up, at day 12, the FMB with cells were placed on a plastic surface, and downloading of functional living cells from both arms was evident (C4, C7). Samples of the medium before and after storage for 6 days in room temperature were taken in syringes and immediately tested for the partial pressure of O₂, CO₂, and pH (D). The role of the serum in the medium in enhancing survival of hMSC on FMB sealed at room temperature was also evaluated. A comparison of the effect of serum-free medium (Nutristem) relative to the regular FCS containing medium on the survival of cells on FMB sealed at room temperature was evaluated (E). MTS assay showed no significant difference in the survival at room temperature with the different media, though the proliferation rate in optimal conditions was slower with the serum-free Nutristem medium. Color images available online at www.liebertonline.com/tec