Objective To investigate an effect of differenttemperature cryopreservation of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat. Methods Eighty femaleSD rats were randomly divided into 8 groups of 10 rats each. One was the control group and 7 were the experimental groups. Two 2-cm-long sciatic nerve segments were respectively taken from both legs of each rat. In the control group, the sciatic nerve segments did not undergo the treatment of cryopreservation; however, in the 7 experimental groups, the sciatic nerve segments respectively underwent the different temperature cryopreservation of the twostep freezing method at -20℃, -30℃, -40℃, -50℃, -60℃, -70℃ and -80℃. The sciatic nerve segments were cryopreserved for 2 hours，and then placed into the liquid nigrtrogen at -196℃. After 48 hours of storage，the nerve segments werethawed quickly in the 37℃ water bath box for 1 minute. Then, the sciatic nerve segments each group were harvested. The cells of the sciatic nerve were incubated with Calcein-AM for 15 minutes. The average fluorescence intensity of the cells was measured by the flow cytometry. The nerve fibers were also incubated with Calcein-AM for 15 minutes. The fluorescence intensity of the cells was analyzed by the confocal fluorescence microscope. The Schwann cell biological activity intensity was measured. Results The fluorescence intensity in the -40℃ group was the best and the Schwann cell biological activity in this group was thebest among all the groups（P＜0.01）. The fluorescence intensity in the 8 groups measured by the flow cytometry was as follows：242.522 0±9.568 4 in the control group，168.677 0±10.207 0 in the -20 ℃ group，214.992 0±8.329 1 in the -30 ℃ group，235.526 0±9.280 5 in the -40 ℃ group，222.434 0±8.515 5 in the -50 ℃ group，217.409 0±9.515 7 inthe -60 ℃ group，132.376 0±13.459 7 in the -70 ℃ group， and 108.132 0±16.033 1 in the -80 ℃ group. The fluorescence intensity detected by the confocal fluorescence microscope was as follows：143.700 0±5.567 8 in the control group，119.700 0±5.161 5 in the -20 ℃ group，121.300 0±4.347 4 in the -30 ℃ group，700 0±5.012 2 in the -40 ℃ group，121.000 0±4.546 1 in the -50 ℃ group，118.400 0±4.9261 in the -60 ℃ group，81.200 0±5.116 4in the -70 ℃ group，and 79. 000 0±5.716 4 in the -80 ℃ group. Conclusion The Schwann cell biological activity treated by the two-step freezing methodcan be preserved and the activity is cryopreserved best at -40 ℃.
ObjectiveTo investigate the effectiveness and technical key points of limb salvage surgery by allotransplantation of cryopreservated vascularized bone in children and adolescents with osteosarcoma. MethodsA retrospective analysis was made on the clinical data of 21 children and adolescents with osteosarcoma receiving limb salvage surgery by allotransplantation of cryopreservated vascularized bone from their relatives between February 2004 and April 2012. There were 13 males and 8 females, aged from 7 to 16 years (mean, 12.6 years). According to Enneking stage system, 15 cases were rated as stage ⅡA and 6 cases as stage ⅡB. The tumors located at the distal femur in 10 cases, at the proximal femur in 1 case, at the proximal tibia in 8 cases, at the proximal humerus in 1 case, and at the distal radius in 1 case. Imaging examination showed that epiphyseal extension of malignant bone tumors in 7 cases. The iliac bone allograft with deep iliac vessels was obtained from their lineal consanguinity. After preservation by a twostep freezing schedule, the iliac bone allograft with deep iliac vessels was implanted into the bone defect area after tumor resection. The size of iliac bone flap was 8.0 cm×3.0 cm×2.0 cm-14.0 cm×5.0 cm×2.5 cm. Reserved joint surgery was performed on 16 cases and joint fusion surgery on 5 cases, and external fixation was used in all cases. The chemotherapy was given according to sequential high-dose methotraxate, adriamycin, and cisplatine before and after operation. ResultsAll 21 cases were followed up from 5 months to 11 years (mean, 6.4 years). At 2 weeks after operation, the erythrocyte rosette forming cells accounted for 56.7%±3.9%, showing no significant difference when compared with that of normal control (58.3%±4.3%) (t=1.56, P=0.13), which suggested no acute rejection. At 4 weeks after operation, single photon emission computerized tomography bone scan indicated that the blood supply of bone graft was rich, and the metabolism was active. At 12 weeks after operation, the digital subtraction angiography showed the artery of iliac bone flap kept patency. X-ray films showed that malunion and non-union occurred at 5 and 6 months after operation in 1 case, respectively. The bone graft healed in the other patients, and the healing time was 3.2-6.0 months (mean, 4.4 months). At last follow-up, American Musculoskeletal Tumor Society (MSTS) score was significantly improved to 26.80±2.14 from preoperative value (17.15±1.86) (t=-4.15, P=0.00). The survival rate was 85.7% (18/21) and the recurrence rate was 9.5% (2/21). ConclusionAllotransplantation of cryopreservated vascularized bone from the relatives provides a new method for the treatment of osteosarcoma in children and adolescents. A combination of allotransplantation and chemotherapy can achieve the ideal treatment effect. The correct cutting, preservation, and transplantation of the donor bone, and indication are the key to improve the effectiveness.
【Abstract】 Objective To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs),to investigate a better cryopreservation protocol of HAFMSCs and to observe the biocharacteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and cl inical appl ications. Methods HAFMSCswere isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method.HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO atcryoprotectant) in l iquid nitrogen for 12 weeks. The biocharacteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adi pogenic and osteogenic differentiation abil ities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservations. Results At 12 weeks after cryopreservation, different protocols had different effects on the cell viabil ity; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed “S” shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adi pogenic differentiation, the l ipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralizations were verified by von Kossa staining. There was no significant difference (P gt; 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservations. Conclusion HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabil ities and no changes of the biocharacteristics and differentiation potential ities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
Objective To investigate the effect of tissue engineered bone with cryopreservation on healing of bone defects and to explore feasibility of cryopreservation for tissue engineered bone. Methods Tissue engineeredbones were constructed with osteoblasts being seeded onto bio-derived materials made from freshhuman bones,and they were preserved at 4℃ and －196℃ for 3 months and 6 monthsrespectively.They were applied to repair segmental bone defects of rabbit’s radius while the tissue engineered bone without cryopreservation and bio-derived materials were brought into control groups.The experiment was divided into groups A3,A6,B3,B6,C and D(group A3:tissue engineered bones were preserved at 4℃ for 3 months; group A6:tissue engineered bones were preserved at 4℃ for 6 months;group B3:tissue engineered bones were preserved at －196℃ for 3 months; group B6:tissue engineered bones were preserved at －196℃ for 6 months; group C: tissueengineered bones without cryopreservation; group D: bio-derived materials). Macroscopical and histologial examination were done at the 2nd,4th,6th,12th weeks, X-ray examination was done at the 6th,12th weeks and biomechanics were determined at 12th weeks after operation respectively. Results Macroscopical observation showed no significant differences among group A3, A6, B3, B6 and C, but less new bone formation and more obvious boundary in group D were observed. Histological observation showed more collagen and new bone around the edge of implant of group A3, A6, B3, B6 and C than group D, and histological evaluation showed significant differences between group D and other groups(P＜0.05). Radiographic observation showed no absorbability of the implant cortex and less new bone formation in group D, but the unity between implant and host bone, medullary cavity reopened, disappearance of fracture line and fine bone modelling were observed in other groups at 12 weeks after operation. Biomechanics between group D and other groups showed significant differences(P＜0.05). Conclusion Cryopreservation (4℃ and -196℃) were capable of preserving tissue engineered bone for long time, and tissue engineered bone withcryopreservation has significant effect on healing of bone defects. The methods f it clinical application.
OBJECTIVE To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P lt; 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P lt; 0.01). CONCLUSION If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
Abstract: Objective To investigate the influence of cryopreservation on cellular viability of latepregnancy fetal valved allografts in human. Methods The fetal valved allografts with gestational ages ranged from 24 to 40 weeks were sterilely procured within 6 hours after brain death. Each sample was bisected into control group and experiment group. The cellular viability of control group was directly tested and that of experiment group was examined after being storaged in liquid nitrogen for a week through a programmed frozen procedure. The light microscopy, tissue culture and Methylthiazol tetrazolium assay (MTT assay) were used to determine the cellular viability. Results Twelve latepregnancy fetal valved aortic allografts were procured. Light microscopy showed the integrity of the basic structure of the thawed aorta, the normal structure of the collagen and elastic fibers, with part of vascular endothelium lost. There were lots of cells deriving from both groups,but the cellular growing rate of the experiment group was relatively slower. At 490 nm, MTT assay valve of control group was 0.442±0.046, and that of experiment group was 0.424±0.041. The difference between two groups failed to statistically significance(t=1.617, P=0.328). Conclusion There were viable cells in latepregnancy fetal valved allografts after cryopreservation.
Objective To investigate the effect of cryopreservation (CP) on the expression of connective tissue growth factor (CTGF) in the renal tubular epithel ial cells. Methods A total of 40 male Wistar rats (weighing 230-250 g) were used in this study. En bloc removal with in situ cooling both kidneys and hypertonic citrate adenine preservation solution were adopted. The rat kidney was be preserved 0, 12, 24, 36 and 48 hours at 0-4℃ (n=8), respectively. The expression of CTGF of renal tubularepithel ial cells was detected by using immunohistochemistry and in situ hybridization analysis. Results The expression of CTGF was less in CP 0 hour group and CP 12 hours group, the positive unit (PU) values of CTGF protein were 5.91 ± 2.30 and 5.57 ± 2.40 (P gt; 0.05), respectively, and the PU values of CTGF mRNA were 6.24 ± 2.79 and 6.51 ± 2.43 (P gt; 0.05), respectively. The PU values of CTGF protein increased at CP 24 hours group (10.25 ± 2.92), CP 36 hours group (14.31 ± 2.83) and CP 48 hours group (18.11 ± 3.94, P lt; 0.05), respectively, and the PU values of CTGF mRNA increased at CP 24 hours group (15.24 ± 3.95), CP 36 hours group (19.20 ± 4.73) and CP 48 hours group (23.09 ± 4.40, P lt; 0.05), respectively; showing significant differences when compared with CP 0 hour group and CP 12 hours group (P lt; 0.05). Conclusion CTGF expression may increase with severe cold ischemia injury, and might play an important role in regeneration and repair of renal tubular epithel ial cell injury.
Objective To observe the configuration and viability of full thickness human fetal retina after short-, mid- and long-term preservation. Methods Twenty-two full thickness human fetal retinae of gestational age of 12-24 weeks were coated by glutin and cut into 88 pieces, and then preserved in Ames' solution, DX solution, -80℃ refrigerator or under cryopreservation condition. The cell viability of retinal neuroepithelial layer was determined by trypan blue staining, retinal configuration was determined by light microscope and electromicroscope. Results The viability of neuroepithelial layer was (94.79plusmn;2.85) % in fresh fetal retina, gt;80% in Ames' solution within 4 hours, and gt;77% in DX solution within 2 days. There was no significant difference between those solution-preservations and the fresh fetal. In -80℃ refrigerator, the viability was (65.83plusmn;5.06)% after 7 days, and then dropped to (57.54plusmn;16.18)% at the end of the first month. Under the cryopreservation condition, the viability was (69.46plusmn;9.31)% at the end of first month. Light and transmission electron microscopy had not deteced any abnormals in the full thickness human fetal retina preserved in Ames' solution within 2 hours, but showed clear retinal layers with bigger intercellular space after preserved in DX solution for 2 days, in -80℃ refrigerator for 7 days and under cryopreservation condition for 1 month. Conclusion Ames' solution and DX solution can preserve good viability and configuration of full thickness human fetal retina in a certain time period.
Objective To summarize the experience of l iving donor l iver transplantation using cryopreserved il iac vein for middle hepatic vein reconstruction. Methods Between July 2006 and June 2009, right l iver transplantation without middle hepatic vein was performed in 37 cases of 85 patients undergoing l iving donor l iver transplantation; of 37 cases, 30 received middle hepatic vein reconstruction using cryopreserved il iac vein. There were 27 males and 3 females, aged from 10 to 57 years (median, 44 years). Thirty cases included 11 hepatocellular carcinoma, 10 hepatic cirrhosis, 2 Wilson’ sdisease, 1 cholangiocarcinoma, 1 hepatoblastoma, 1 congenital hepatic fibrosis, 1 chronic severe hepatitis, and 1 congenital bil iary atresia. Il iac veins harvested from donors were put into 0-4℃ mixed antibiotics sal ine and transported to the operating room. The il iac veins were trimmed, placed into sterile bags (containing RMPI 1640 + 20% DMSO + 10% calf protein solution) and frozen at —70 . In l iving donor l iver transplantation process, the veins were melt and used for middle hepatic vein reconstruction. After operation, the patency of veins was monitored by regular Doppler ultrasound examination or enhanced CT for 3 months. Results In 30 patients, 30 il iac veins were used. The average cryopreserve time was 14 days (range, 3-44 days). Anastomosis were all successful; after cryopreservation, the blood vessels texture and elasticity were fit for surgery. No easily tearing or severe suture bleeding was observed. In 30 patients, 6 had segment V veins reconstruction; 3 had segment VIII; and 21 had both segments V and VIII. The patency rate of reconstructed vessels was 93% at 1 week, 90% at 2 weeks, 90% at 1 month, and 67% at 3 months. No serious compl ication was observed in donors. The prognosis was good with no small-for-size syndrome. Conclusion Cryopreserved il iac vein is an ideal material for the right hepatic l iving donor l iver transplantation in the reconstruction of middle hepatic vein.