RESUMO
Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.
RESUMO
Objective To develop simple sequence repeat (SSR) molecular markers based on Stephania kwangsiensis transcriptome and to evaluate genetic diversity of S. kwangsiensis in natural populations. Methods The unigenes from S. kwangsiensis transcriptome were used to explore SSR loci. SSR primers were designed by oligo 7.0. Polymorphic primers were selected by electrophoresis and were applied to the investigation of the genetic diversity of S. kwangsiensis. Results A total of 6 833 SSR loci (33.11%) were obtained from 20 637 unigenes in the S. kwangsiensis transcriptome. Of the 50 SSR markers tested, ten pairs of highly polymorphic primers were selected to analyze the genetic polymorphisms of 63 individuals from five populations. Ten primers produced 83 band loci, all (100%) of which were polymorphic, and the polymorphism information content (PIC) was 0.688 9. At the species level and population level, the Nei’ s gene diversity (H) was 0.725 2 and 0.613 4, respectively; the Shannon’s Information index (I) was 1.576 6 and 1.220 3, respectively; The observed heterozygosity (Ho) was 0.584 5 and 0.558 4, respectively; The expected heterozygosity (He) was 0.731 2 and 0.643 5, respectively. The genetic differentiation coefficient (Fst) was 0.146 5 and the gene flow (Nm) was 1.456 9. Based on UPGMA cluster analysis, the five populations of S. kwangsiensis were divided into three clusters. Conclusion The developed SSR markers were applicable to the genetic diversity evaluation of S. kwangsiensis. The genetic diversity of this plant was relatively high at both species level and population level. The genetic differentiation mainly existed within populations.