Abstract In order to determine the fasibility of reestablishment of circulation with cryopreserved microvenous allografts (1.0～1.4mm in diameter), 40 rabbits were divided into 2 groups. In the control group, the fresh autografts were used. In the experimental group, 20 rabbitsfemoral vein segments were treated by a two-step freezing procedure. After stored in liquid nitrogen for 48 hours, the segments were implanted into the femoral veins as allografts. The histological as well as the pathological studies were performed with light and electron microscope, and its patency was determined by angiography. The results showed that the preservation of vein was generally good. The rejective response was weak. The patency rates of 1 week and 12 weeks were 90% and 85% respectively, and there was no significant difference with that of the allogenic fresh autografts (Pgt;0.05). It was suggested that clinical use of cryoperserved allogenic microvein grafts instead of fresh autografts was possible.
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.
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 investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature. METHODS: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later. RESULTS: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked. CONCLUSION: The cryopreservation agent in group A can protect TET in deep low temperature.
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 observe the effects of cryopreservation and resuscitation on the biological characteristics of mesenchymal stem cells (MSCs) derived f rom human umbilical cord blood. Methods MSCs were isolated and cultured f rom human umbilical cord blood in vitro. The cells were passaged , and the third generation of MSCs were cryopreserved in－196 ℃ liquid nitrogen for 4 weeks with cryopreservation medium , which contained 10 % dimethyl sulfoxide (DMSO) and 90 % fetal calf serum ( FCS) . The morphology , proliferation and differentiation of MSCs were investigated and compared with those of MSCs before cryopreservation. Results There was no significant difference of morphology between pre-cryopreserved MSCs and the ones af ter resuscitation. It was observed that all MSCs were spindle-shaped and showed adherence growth characteristic before and af ter cryopreservation. The cell growth curves of MSCs were also similar before and af ter cryopreservation. Even though the curve of resuscitated MSCs descended a little as compared with that of pre-cryopreserved MSCs , there was no significant difference ( Pgt; 0. 05) . After 2-week adipocytic differentiation induction , fat drops could be found in the kytoplasm of MSCs and they were red when stained with oil-red O staining , which suggested that MSCs could be induced and differentiated into adipocytes. Af ter 4-week osteoblastic differentiation induction , MSCs could be induced and differentiated into osteoblasts , and calcium node showed black when stained with Von Kossa staining. There were no significant changes of the differentiating ability of MSCs into adipocyte and osteoblast before and after cryopreservation. Conclusion MSCs derived from human umbilical cord blood maintains their biological characteristics af ter cryopreservation and resuscitation.
【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.
Abstract In order to study the possibility of repairing bone defect by cryopreserved vascularized bone allograft, 8 dogs were divided into 2 groups. In the experimental group, 15% dimethylsulfoxide (DMSO) was used as a cryoprotective agent, the posterior segments of dog s rib, pedicled with intercostal vesseles, were cryopreserved by a two-step freezing procedure,stored in liquid nitrogen for 96 hours, and then transplanted as allografts to theiliac bone defects of recipients by vascular anastomosis. In the control group, the autografts were transplanted in the same procedure. Immunosuppersive agents were administrated postoperatively for 3 weeks. The specimens were analyzed by immune response monitoring (IL-2, T cell subsets), SPECT scanning, angiography and pathologic examination. The results showed that the allografts had good blood supply and active osteocyte metabolism, bone healing of the allografts was perfect at 3 months and no evidence of immunologic rejection. The process of bone healing of allografts should be further investigated.
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.