Objective To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.Methods In the 4thgeneration of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342(Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguishthem. The seeding cells were collected to make them to be the cell suspension of suitable concentration(6.0×105 cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and didnot cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to noncryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreservedcell to non-cryopreserved cell and the cell survival rates. We compared the cll survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. Results The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r=0.970，Plt;0.05), image analysis study also showed the correlation was significant (r=0.982，Plt;0.05).The cell survival rate decreased by use of flow cytometry twohours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (Pgt;0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly.Conclusion Flow cytometry and fluorescence image afterPI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservationof tissue engineered tendons.
ObjectiveTo investigate the feasibility of immoribund skin fibroblast cell line derived from Leber′s hereditary optic neuropathy (LHON) patients as a cell model. MethodsA basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD+), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD+, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. ResultsCompared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD+/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. ConclusionImmortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.