ABSTRACT
The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.
ABSTRACT
The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol (EG) and dimethylsulphoxide (DMSO) were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group (1.28 vs. 1.70, P<0.05). The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group (31% vs. 63%, P<0.05). It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.
Subject(s)
Animals , Female , Mice , Cryopreservation , Methods , Cryoprotective Agents , Pharmacology , Dimethyl Sulfoxide , Pharmacology , Ethylene Glycol , Pharmacology , Fluorescent Dyes , Metabolism , Membrane Potential, Mitochondrial , Physiology , Microscopy, Fluorescence , Mitochondria , Metabolism , Oocytes , Physiology , Temperature , VitrificationABSTRACT
Background The multipotent differentiation features of induced pluripotent stem cells (iPSCs) offer a new option for cell replacement therapy of many clinical diseases.In ophthalmology,iPSCs are a good model in studying the pathogenic mechanism of degenerative ocular diseases.A better identification method for iPSCs is critical for analyzing the in vivo biological characteristics of iPSCs.Objective This study was to investigate the feasibility and stability of labeling iPSCs with quantum dots.Methods Human umbilical mesenchymal stromal cells-iPSC lines were cultured and amplified on matrigel,and the characteristics of iPSCs were evaluated by immunofluorescence.Different concentrations (5.0,7.5 and 10.0 nmol/L) of quantum dots with a CdSe/ZnS nuclear shell structure were used to label iPSCs after passaging and proliferation.The labeling outcome was observed with a three-dimensional deconvolution real-time live cells imaging system.The labeled iPSCs were subsequently cultivated,and then changes in fluorescence intensity were examined 7 days after the first and the second passaging of iPSCs.Results iPSCs were observed to grow in a clonal manner under the inverted microscope.The iPSC markers,OCT4 and Nanog,were detected by immunofluorescence.With increasing concentrations of quantum dots,the fluorescence intensities representing the levels of OCT4 and Nanog in iPSCs were gradually elevated,with optimal levels of fluorescence observed at a concentration of 10 nmol/L of quantum dots.The fluorescent labeling of OCT4 and Nanog in iPSCs remained and weakened gradually till day 7 even after the second passage.Conclusions Quantum dots labeling could be used to track iPSCs in a dose-independent manner.The fluorescent signal from the quantum dots labeling the iPSCs lasts 2 weeks at least.
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Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.
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Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.