ABSTRACT
BACKGROUND:Umbilical cord-derived mesenchymal stem cel s are gaining more attention in clinical treatments. Cel viability prior to transplantation has a direct impact on clinical prognosis. Despite trypan blue staining is a widely performed procedure to assess the viability of umbilical cord-derived mesenchymal stem cel s, it cannot reflect the functional capacity of those cel s accurately because of some subjective factors. OBJECTIVE:To explore sensitive and accurate assay for the functions of umbilical cord-derived mesenchymal stem cel s. METHODS:Human umbilical cord-derived mesenchymal stem cel s were isolated and cultured in vitro. Cultured umbilical cord-derived mesenchymal stem cel s were preserved in 0.9%saline for 0, 2, 4 and 6 hours at 4 ℃. Various methods (trypan blue staining, AnnexinV-PI, terminal deoxynucleotidyl transferase dutp nick end labeling, cel counting kit-8, live-dead assay, cel adherent assay) were used to determine the viability of post-storage umbilical cord-derived mesenchymal stem cel s, and the results were compared with colony-forming efficiency, a measure of cel function. RESULTS AND CONCLUSION:Human umbilical cord-derived mesenchymal stem cel s cultured in vitro showed a spindle shape and attached growth, the third-generation umbilical cord-derived mesenchymal stem cel s were positive for CD29, CD44, CD105, and negative for CD 34 and CD 45. Umbilical cord-derived mesenchymal stem cel s incubated in the adipogenic and osteogenic medium were both positive. Cel viability measured with trypan blue correlated moderately with colony-forming efficiency, while the percentage of viable cel s measured with other methods correlated better with colony-forming efficiency, among which adherent assay was the most obvious. It is proved that cel adherent assay-measured viability is the most accurate indicator.
ABSTRACT
BACKGROUND:The viability of human umbilical cord-derived mesenchymal stem cel s is often declined with the commonly used transplantation storage solution in clinics, which may influence the therapeutic effects of cel ular transplantation. However, reasons for this are stil unknown. OBJECTIVE:To investigate the role of oxidative stress in the reduction of human umbilical cord-derived mesenchymal stem cel s viability in the storage process during clinical transplantation and to observe the effects of radical scavenger on the results. METHODS:Human umbilical cord-derived mesenchymal stem cel s were harvested and cultured in normal saline for 0, 2, 4 and 6 hours at room temperature. Intracel ular reactive oxygen levels were detected at those time points. Antioxidant enzyme activities and levels of malondialdehyde were measured to determine the intracel ular oxidative stress levels after storage. Cel adhesion rate changes were retested after adding N-acetyl cysteine to the storage solution. RESULTS AND CONCLUSION:The reactive oxygen levels in human umbilical cord-derived mesenchymal stem cel s were increased significantly after normal saline storage and levels of malondialdehyde were increased in a time-dependent manner. Activities of superoxide dismutase, catalase and glutathione peroxidase were al reduced. Addition of N-acetyl cysteine into the storage medium decreased the reactive oxygen levels and improved the human umbilical cord-derived mesenchymal stem cel s viabilities. Experimental findings indicate that, increased reactive oxygen species in human umbilical cord-derived mesenchymal stem cel s is one of the reasons for reduced cel viability. Adding the radical scavenger N-acetyl cysteine can improve the storage effects of human umbilical cord-derived mesenchymal stem cel s.