PURPOSE:To establish methods for cryopreservation of human retinal pigment epithelial cells (RPEs)and cell culture from thawing of frozen cells. METHODS:Primary cultured RPEs or its first or second passages,added with 10 dimetbylsulfoxide,were kept in --20℃ for 1 to 2 hours,and then further froze to -40~C over night before being placed in liquid nitrogen. The frozen cells were thawed in 60℃ within 2 minutes. Trypan blue staining and immunocytochemical staining with anti-human keratin were performed for cell viability and differentiation. The growth curve was also determined by calculating the total number of cells/well/day. RESULTS:The viable rate from frozen RPEs was 90%. No differences were observed for growth activity between cultures from frozen cells and controls. The cells were positive with anti-human keratin staining. The logarithmic growth phase was during I to 4 days and the doubling time yeas 1.55 days. CONCLUSION: Cryopreservation of RPEs in liquid nitrogen can maintain biological activities of cells with normal growth and features after thaw- ing. This will provide cell lines for in vitro experiments and possibly for cell banks for RPE transplantation for some fundus diseases. （Chin J Ocul Fundus Dis,1997,13:157-159）
ObjectiveTo observe the changes of microstructure of rats'sciatic nerves with non-freezing cold injury after treated with dexamethasone. MethodsTwelve male Wistar rats were randomly divided into cooling group and treating group.Unilateral sciatic nerves of the rats in the cooling group received cooling treatment with 3-5℃ for 2 hours;while unilateral sciatic nerves of the rats in the treating group received cooling treatment with 3-5℃ for 2 hours and underwent the celiac injection with dexamethasone in addition.The other sciatic nerves were exposed,as the control.The bilateral sciatic nerves of rats in each group were harvested after 24 hours.The microstructure of nerves was examined under the light microscope and electron microscope. ResultsLight microscopic examination revealed extensive myelinated fibre degeneration in form of giant empty axons or shrunken dark axons on the first day after cooled.And the endoneurial capillary lumen was narrowed because of swollen endothelial cells.After the treatment,myelinated fibre degeneration was still similar to that before the treatment,but the endoneurial capillary lumen and endothelial cells were normal.By electron microscopy,sciatic nerves showed extensive myelinated fiber degeneration,and swollen endothelial cells.But unmyelinated fibers and tight junction were preserved on the first days after cooled.Aggregated red cells and platelet thrombus were not found.After the treatment,myelinated fibre degeneration was still similar to that before the treatment.Unmyelinated fibers and tight junction were preserved.The endoneurial capillary lumen and endothelial cells were normal. ConclusionAfter the treatment,the damage of endoneurial capillary had improved,but myelinated fiber degeneration was similar to that before the treatment.It suggested that dexamethasone may only improve the vascular system in non-freezing cold injury of sciatic nerve.
ObjectiveTo determine if the cryoprotectant solution supplementation with Vitamin C can improve the protective effect of human ovarian tissue cryopreservation by anti-apoptosis. MethodsHuman ovarian cortical tissues were collected from nine patients treated between March 2012 and April 2013. The cortical tissue pieces obtained from each patient were divided into two groups:control (conventional slow freezing) and trial group (slow freezing supplementation with Vitamin C). The preservation effects in the two groups were compared by histology using light microscope and apoptosis assessed by TUNEL assay. ResultsThe proportion of morphologically normal primordial follicles in the trial group was higher than that in the control group (P<0.05). The proportion of apoptotic primordial follicles and stromal cells in the trial group was lower than that in the control group (P<0.05). ConclusionCryoprotectant solutions supplementation with Vitamin C can improve the preservation of primordial follicles and stromal cells. It might be a method worth to try in order to improve the protective effect of human ovarian tissue cryopreservation by inhibiting of apoptosis.
ObjectiveTo discuss the clinical effects of intercostals nerve freezing technique for analgesia on post-operative chest. MethodsWe included 80 patients with thoracotomy in Zhongshan Hospital in Xiamen University between June 2013 and June 2014. The patients were divided into a trial group (30 males and 10 females at average age of 59.90± 10.62 years) and a control groups (28 males and 12 females at average age of 59.85± 10.52 years) by random digital table. The patients in the trial group were treated with frozen the intercostals nerve roots located in the incision and next intercostals before closing the chest by cryotherapy therapy apparatus (K520 type, Beijing Kulan Company). The patients in the control group were treated with self-control intravenous analgesia pump post-operation. We compared the postoperative incision pain and cough, sputum reflection score, and pulmonary complications between the two groups. ResultsThe pain degree in the trial group was significantly weaker than that in the control group on the first day, the second day and the seventh day after operation (t=-6.45, -4.95, -3.14, P < 0.05). Cough, sputum reflection score were significantly higher than those in the control group (P < 0.01). Pulmonary complications were significantly lower (t=3.023, P < 0.05). There were 4 patients with pneumonia and no pulmonary atelectasis in the trial group. While there were 8 patients with pneumonia in the control group and 1 patient with pulmonary atelectasis. ConclusionIntercostals nerve freezing technique can drastically reduce postoperative pain in the patients with open thoracic operation, effectively promote patient cough and expectoration, and reduce pulmonary complications rate.
In the present study, the performance of the liquid nitrogen frozen and thinned bovine pericardium was studied and compared with the porcine pericardium. The microstructure and mechanical properties of the bovine pericardium were observed and tested by hematoxylin-eosin (HE) staining and tensile test respectively. In all conditions, porcine pericardium was selected as a control group. The results showed that there was little difference in the performance of bovine pericardium after being frozen by liquid nitrogen. The secant modulus and ultimate strength of the thinned bovine pericardium were similar to those of porcine pericardium, however, the elastic modulus was a little higher than porcine pericardium. The study suggested that the performance of the thinned bovine pericardium was similar to those of porcine pericardium. It was easy for the thinned bovine pericardium to obtain a relatively ideal thickness and expected performance, therefore, the thinned bovine pericardium can be used as the materials of transcatheter aortic valve leaflets.
Cell freeze-drying can be divided into the freezing and drying processes. Mechanical damage caused by ice crystals and damage from solute during freezing shall not be ignored and lyoprotectants are commonly used to reduce those damages on cells. In order to study the mechanism of lyoprotectants to protect cells and determine an optimal lyoprotectant formula, the thermophysical properties and percentage of unfrozen water of different lyoprotectants in freezing were investigated with differential scanning calorimeter (DSC). The survival rate indicated by trypan blue exclusion test and cell-attachment rate after 24 h using different lyoprotectants to freeze hepatoma Hep-G2 cells were measured after cell cryopreservation. The results show that 40% (W/V) PVP + 10% (V/V) glycerol + 15% (V/V) fetal bovine serum + 20% (W/V) trehalose formula of lyoprotectant demonstrate the best effect in protecting cells during freezing, for cell-attachment rate after 24 h is 44.56% ± 2.73%. In conclusion, the formula of lyoprotectant mentioned above can effectively protect cells.