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 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 effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
【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 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.
Objective To observe the long-term effectiveness of tendon allograft to repair tendon defect. Methods Between October 1996 and September 1999, 24 patients with tendon defect were treated with tendon allograft which was cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature. There were 19 males and 5 females, aged from 12 to 46 years with an average of 25.9 years. These patients included 7 cases of total extensor tendon defect of 2nd-5th fingers, 7 cases of index finger extensor tendon defect, 3 cases of deep flexor tendon defect of 2nd- 5th fingers, 1 case of ring finger deep flexor tendon defect, 3 cases of long extensor tendon defect of 2nd-5th toes, 2 cases of long extensor hallucis tendon defect, and 1 case of shoulder adduction missing. The sizes of tendon defect ranged from 5 to 15 cm. The mean time from injury to operation was 1.3 months (range, 2 hours to 3 months). Results Incisions healed by first intention. No deep infection, infectious diseases, and obvious immune rejection occurred. All patients were followed up from 10 to 12 years with an average of 10.8 years. When compared with contralateral sides, at 10 years of follow-up, 1 patient lost 6-10° flexion function; after 10.6 years, flexion tendon releasing was performed; allografted tendon had normal color and elasticity with decreased diameter and with mild and moderate adherence; and after releasing, function was improved. According to Hand Surgery Association assessment standard, the results were excellent in 12 cases, good in 6, and poor in 6; the excellent and good rate was 75%. Conclusion Tendon allograft which is cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature is safe to use in cl inical, which has good long-term effectiveness in treating tendon defect.
Objective As one of the adult stem cells, adi pose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs invitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cool ing with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in l iquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkal ine phosphatase (ALP) staining and Al izarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% ± 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Al izarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P gt; 0.05). Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.
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 o study the feasibility of homologous vascularized nerve transplantation after ultra deep cryopreservation. Methods Vascularized sciatic nerve from 12 female dogs was transplanted after ultra deep cryopreservation. Fortyeight male dogs were divided into 4 groups: ultra deep cryopreservation homologous vascularized nerve (group A), ultra deep cryopreservation homologous nerve (group B), fresh homologous vascularized nerve (group C), and fresh autologous vascularized nerve (group D). The gross appearance, patency rate of arteryand morphological transplanted nerve were observed 1, 4 and 12 weeks after transplantation respectively. Immunological analysis was performed using IL 2 assay and T lymphocyte subpopulations assay after 4 weeks. Image pattern analysis andelectromyogram were observed after 12 weeks. Results In groups A and D, no toe ulcer occurred, the atrophy of later limb and the sense of pain from skin of calf were restore significantly in the postoperative 12th week. In groups B and C, toe ulcer occurred, the atrophy of later limb and the sense of pain from skin of calf were not restored significantly in the postoperative 12thweek. The vessel patency rate of groups A and D was 83.3%, which was significantly higher than that of group C (50%,Plt;0.05). The changes of IL2 and Th, Ts in group C were significantly higher than that in groups A,B,D(Plt;0.01). There were increased vessel and regenerated nerve in transplanted nerve under optical microscope and image pattern analysis in groups A and D. There were shorter latent period of motor evoked potential, greater amplitude of action potenlial and faster motor nerve conducting velocity in groups A and D after 12 weeks. Conclusion The antigenicity of the homologous never and vessel may be reduced significantly by being frozen, and cryopreserved vascularized nerve can transferred successfully without the use of immunosuppressive agents. Vascularized nerve may restore good significantly for the thick nerve.
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 ℃.