Objective To investigate the effects of ovarian tissue cryopreservation by needle immersed vitrification (NIV) method and subsequently orthotopic transplantation on ovarian function reconstruction in chemotherapy-induced ovary damage rat model. Methods A total of 52 matured virginal female Wistar rats at age of 8-9 weeks housed in specific-pathogen-free facilities, weighing 250-300 g. Vaginal smears were obtained daily, 50 rats having at least 2 consecutive normal estrous cycles were included in the experiment. Ten rats were selected as donors randomly, and NIV method was used for cryopreserving ovarian tissues. The remaining 40 rats were divided into 3 groups according to different treatments: cyclophosphamide group (C group, n=14), cyclophosphamide/transplantation group (C/T group, n=12), and control group (NS group, n=14). In C group and C/T group, the rats received peritoneal injection of cyclophosphamide every day for 21 days to establish the chemotherapy-induced ovary damage models; and then the frozen-thawed ovarian tissues orthotopically transplanted into the left ovarian bursae in C/ T group. The rats received peritoneal injections of 0.9% saline solution every day for 21 days in NS group. Estrous cycle recovery time, ovary weight, morphology change of ovarian tissues, and follicle count were compared among 3 groups. Results One rat died at 2 days after transplantation in C/T group; the other rats survived to the completion of the experiment. At 4 weeks after the end of injection, no significant difference in body weight was found among 3 groups (P gt; 0.05). The rats of NS group had regular estrous cycle, but cyclic changes in vaginal smears were observed in C group and C/T group during cyclophosphamide treatment. The median estrous cycle recovery was 9 days (95%CI: 7.9-10.1 days) in C group, and was 6 days (95%CI: 4.9-7.1 days) in C/ T group, showing significant difference (χ2=6.571, P=0.010). The ovarian weight showed an obvious downtrend in C group at 4 weeks after cyclophosphamide treatment, and an upward trend was observed in C/T group. The ovarian grafts survived and grew well in C/T group. Primordium follicles and primary follicles in C/T group and NS group were significantly more than those in C group (P lt; 0.05), but no significant difference was found between NS group and C/T group (P gt; 0.05). There was no significant difference in secondary follicles and antral follicles among the 3 groups (P gt; 0.05). Conclusion The method of ovarian tissue cryopreservation by NIV and subsequently orthotopic transplantation can significantly shorten the estrous cycle recovery time in chemotherapy-induced ovary damage rat model. Ovarian grafts grow well, follicle count is similar to normal level. So it has the potential ability of ovarian endocrine and fertility reconstruction after chemotherapy.
Objective To investigate the effect of tetramethylpyrazine (TMP) with a certain concentration added to vitrification solution on peripheral nerve allografts regeneration. Methods Forty-eight healthy clean SD male rats were selected as donors, and 96 healthy clean Wistar male rats as recipients, all rats being 3 months old and weighing 200-250 g. The sciatic nerves segments of 15 mm were removed from the donors, then randomly divided into 4 groups according to vitrificationsolution containing TMP. No TMP was used in group A as the control group; 100 mg/L, 200 mg/L and 400 mg/L TMP were used in group B, group C and group D, respectively. Then them were cryo-preserved at — 196 ℃ for 3 weeks. Nerve defect of 10 mm in length was made in the sciatic nerves of recipients. After rewarming, the allografts were transplanted to the corresponding rats. The gross appearance, the morphological and electrophysiological changes, the image analysis of axons and motor end-plate were detected at 4, 8, 12 and 16 weeks. Results All rates survived to the end of the experiment. The adhesion and edema of allografts in group A and group B were obvious 4 weeks after operation; then adhesion and edema was obvious in group A and were improved in the other groups 8 weeks after operation. Adhesion was observed in groups A and B; no adhesion was observed in groups C and D at 12 weeks. The number of regeneration nerve, the latent, the ampl itude, the nerve conduction velocity, the medullary sheath/μm2, the medullary sheath density/μm2 and the image analysis of axons and motor end-plate in groups A and B were significantly lower than those in groups C and D (P lt; 0.01); and there were no significant differences between groups C and D (P gt; 0.05). The observation of transmission electron microscope showed that medullated nerve fibers and myel in sheath of groups C and D were thicker than groups A and B, layers of groups C and D were clear. Conclusion The vitrification solution with 200 mg/L tetramethylpyrazine has protective effect on regeneration of peripheral nerve allografts．
Objective To evaluate the feasibility of preservation of arteriesby vitrification and the effectiveness of vitrified arteries as allografts. Methods Sixty rabbits were used in the research. Forty-eight femoral arteries wereharvested from 24 rabbits as the transplanted materials，and 24 femoral arteries were preserved by vitrification, 24 by freezing for 14 days，respectively. Theother 36 rabbits were used as the transplanted subjects，and were divided into three groups， 12 rabbits including 24 femoral arteries per group: Group A(fresharterial autografts), Group B(vitrified arterial allografts) and Group C(frozen arterial allografts). The morphologic changes of arterial grafts were observed macroscopically and histologically. The patent rate of arterial grafts were measured by angiography, and the rabbits were sacrificed on the 14th day, the 30th day, the 60th day and the 120th day after transplantation respectively. Arterial grafts were harvested to observe the morphological changes，and the immunological rejection was evaluated by measuring the ratio of tunica intima and tunica media. The results were compared between these groups. Results Before transplantation，theintegrated rate of Group B was 91.67%，which was significantly better than that of Group C（54.17%, Plt;0.01）. After transplantation, the accumulative patent rate of Group B was 87.50%，which was significantly better than that of Group C（66.67%, Plt;0.05）. There was statistically significant difference in the ratioof tunica intima and tunica media between Group B and Groups A, C（Plt;0.05）.Conclusion The above results show that vitrification does less damage to cells and tissues because of ice-free in the process of cryopreservation. So vitrification can be used to preserve arteries， and the arterial allografts preserved by vitrification are better than those preserved by freezing.
ObjectiveTo compare the effect of cryoprotective vitrification agent with different concentrations on protecting human ovarian tissue. MethodsHuman ovarian biopsy tissues were obtained from nine patients between August 2013 and May 2014. The cortical tissue pieces obtained from each patient were cryopreserved using direct cover vitrification (DCV) with two different concentrations. The vitrification solutions were divided into two groups: high concentration [15% (V/V) dimethyl sulphoxide (DMSO)+15% (V/V) ethylene glycol (EG)+0.5 mol/L sucrose] and low concentration group (12% DMSO+12% EG+0.5 mol/L sucrose). The preservation effects in the two different concentration groups were compared by histologic evaluation using light microscope and apoptosis assessed by terminal dexoynucleotidyl transferase-mediated nick end labeling staining. ResultsThere was no significant difference in the proportion of morphologically normal primordial follicles between high concentration group and low concentration group (P ＞ 0.05) . The proportion of apoptotic primordial follicles in the low concentration group was 29.7% (58/195) , and was 42.1% (69/164) in the high concentration group, with a significant difference between the two groups (P ＜ 0.05) . The proportion of apoptotic stromal cells in the low and high concentration group was 30.2% (162/537) and 41.9% (206/492) respectively with a significant difference (P ＜ 0.05) . ConclusionsVitrification solutions with lower concentration can improve the preservation of the primordial follicles and stromal cells in human ovarian tissue. It is a method worth trying in order to improve the protective effect of vitrification by decreasing the toxicity of vitrification solutions.
ObjectiveTo determine if the anti-apoptosis agent can improve the protective effect of human ovarian tissue with novel vitrification method. MethodsHuman ovarian cortical tissues were collected from ten patients treated between October 2012 and August 2013. The samples obtained from each patient were divided into two groups:control (the novel immersed vitrification) and experimental group (the novel immersed vitrification supplemented with vitamin C). The preservation effects in the two groups were compared by ultrastructure using electronic microscopy, and apoptosis was assessed by terminal deoxynucleotidyl transferase-medicated dUTP nick-end labeling (TUNEL) staining. ResultsThe proportion of normal ultrastructure of oocytes and granulosa cells in the experimental group was higher than that in the control group (P<0.05). The proportion of TUNEL-positive primordial follicles in the experimental group was 21.6% (49/227) and the proportion of TUNEL-positive primordial follicles in the control group was 38.6% (91/236), with a significant difference between the two groups (P<0.001). The proportion of TUNEL-positive stromal cells in the experimental group was (21.33±3.41)% and the proportion of TUNEL-positive stromal cells in the control group was (33.46±3.09)%, also with a significant difference between the two groups (P<0.001). ConclusionAnti-apoptosis agents can improve the preservation of primordial follicles and stromal cells by inhibiting of apoptosis. It may be a method worthy of trying in order to improve the protective effect of human ovarian tissue vitrification.
Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×103 °C/min. The volume range of 1–8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400–1 200 W laser power, and the rewarming rate was up to the order of 106 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400–1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.