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Introduction: We previously developed 3% trehalose-added lactated Ringer's solution (LR-3T) and 3% trehalose- and 5% dextran-40-added lactated Ringer's solution (LR-3T-5D), which can be used to preserve adipose-derived mesenchymal stem cells (hADSCs) for 24 h at 5 and 25 °C. However, it is necessary to further extend the storage duration of cells to expand transportation zones and ensure time for quality control testing of final cell products. Therefore, we attempted to prolong the preservation duration of hADSCs by adding supplements to LR-3T-5D. We focused on ascorbic acid as an antioxidant because it is widely clinically as a nutrient. Methods: We added the antioxidant ascorbic acid to LR-3T-5D and evaluated the viability, colony formation rate, proliferative capacity, and surface markers of hADSCs before and after preservation at 5 °C. Results: Analysis of the concentration of ascorbic acid added to LR-3T-5D indicated that 1000 mg/L was the optimal concentration for maintaining the viability of hADSCs after 72 h of cold preservation. No changes were observed in the expression of specific cell surface markers or in the potential of hADSCs to differentiate into adipocytes, osteoblasts, or chondrocytes before and after cold preservation. Discussion: These results suggest that cold preservation of hADSCs in LR-3T-5D supplemented with ascorbic acid helps maintain the quality of cells for use in cell therapy.
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INTRODUCTION: Stem cells for therapy are often suspended in a preservation solution, such as normal saline or lactated Ringer's solution, for a short time before intravenous infusion. However, these solutions are not necessarily ideal for maintaining cell viability and preventing the sedimentation of cells during storage and infusion. In this study, we attempted to optimize the compositions of preservation solutions, which could affect the efficacy and safety of stem cell therapy. METHODS: We determined the characteristics of a preservation solution that would optimize cell viability and the percentage of cells in the supernatant using human adipose-derived mesenchymal stromal cells (hADSCs). We compared solutions that differed by electrolytes (e.g., normal saline and Ringer's solution) and the concentrations of dextran 40 and trehalose. The effects of the solutions on hADSCs were evaluated by assessing cell surface markers, colony-forming capacity, differentiation potential, and cell concentrations in the infusion line. RESULTS: Optimized preservation solutions consisted of lactated Ringer's solution with 3% trehalose without or with 5% dextran 40 (LR-3T and LR-3T-5D, respectively). The cell viabilities after 24 h of storage at 5 °C in LR-3T and LR-3T-5D were 94.9% ± 2.4% and 97.6% ± 2.4%, respectively. The percentage of cells in the supernatant after 1 h of storage at room temperature in LR-3T-5D was 83.5% ± 7.6%. These solutions preserved the percentage of cell surface marker-positive cells, the colony-forming capacity, and the adipogenic and osteogenic differentiation ability in hADSCs for at least 24 h after preservation at 5 °C and 25 °C. DISCUSSION: We determined the optimal composition of preservation solutions for hADSCs and confirmed the effects of these solutions on cell viability and the stability of cell characteristics in vitro. Our results suggest that LR-3T and LR-3T-5D can help maintain the quality of stem cells for therapy during preservation and infusion. However, further in vivo research is needed on the efficacy and safety of the solutions in different therapeutic cell lines before clinical use.
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PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, (1)H or (15)N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB-ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57-Ser65) and in ß4-loop3-ß5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5'-Cy3- and 3'-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB-ssDNA complex and on the positive cooperativity of PriB.
