拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6 mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p < 0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p < 0.001 for 2 mm and 4 mm plugs, and at p < 0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.     

Vitrification of rabbit tissues with propylene glycol and trehalose

A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues—carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing. This process was arrested when a concentration had been reached that should permit vitrification if the tissues were cooled rapidly to −175 °C. They were stored at that temperature; warmed rapidly by conduction; the cryoprotectants removed by stepwise dilution; and appropriate active functions measured. These were contraction and relaxation for arteries and endothelial integrity and ability to control stromal swelling for the corneas. In control experiments the exposure and functional assays were carried out without vitrification. It was shown that the tissue concentration of propanediol was 33%w/w in artery and 30% in cornea. These permitted cooling to −175 °C without freezing but devitrification occurred during the warming of the arteries, though not of the corneas, despite the lower tissue concentration reached in the cornea. The function of the vitrified arteries was severely reduced but the endothelium of the corneas was substantially intact although we were unable to demonstrate any ability to control stromal swelling during normothermic perfusion. It appears that concentrations of cryoprotectants sufficient to prevent freezing in these tissues during cooling were well tolerated so long as appropriate stepwise means of addition and removal were used. Devitrification during warming remained a major problem with arteries, but not with corneas. We suggest that the composition of the aqueous phase in the tissue with respect to components other than the vitrifying agents may be crucial here and that the search for agents that will suppress devitrification is an important avenue for further study.     

Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.     

Vitrification of non-human primate immature testicular tissue allows maintenance of proliferating spermatogonial cells after xenografting to recipient mice

This study demonstrates preservation of tissue integrity, maintenance of proliferating spermatogonia and Leydig cell functionality after vitrification and transplantation of non-human primate immature testicular tissue. The objective was to assess the potential of vitrification of non-human primate immature testicular tissue (ITT) in an in vivo xenotransplantation model. Testicular tissue was obtained from one immature rhesus monkey (Macaca mulatta) aged 4 years. Collection and vitrification of testicular tissue, followed by short-term xenografting (3 wks) to nude mice were performed to evaluate and compare vitrified/warmed and fresh tissue. Fresh ungrafted tissue was used for control purposes. Cell density and seminiferous tubule (ST) integrity were assessed by light microscopy. Presence of spermatogonia (SG) (MAGE-A4), proliferation (Ki-67) and Leydig cell (LC) functionality (3β-hydroxysteroid dehydrogenase; 3β-HSD) were evaluated by immunohistochemistry (IHC). Qualitative analysis revealed preservation of the histologic characteristics of SG and Sertoli cells (SCs), as well as cell-cell cohesion and cell adhesion to the basement membrane, in both vitrified and fresh grafted tissues. Survival of SG able to proliferate and functional LCs was confirmed by IHC in fresh and vitrified grafts. In conclusion, vitrification appears to be a promising approach, representing an alternative strategy to slow-freezing in the emerging field of ITT cryopreservation and cryobanking.     

两种手术方式治疗真菌性角膜炎的临床观察

目的 观察深板层角膜移植术（deep lamellar keratoplasty,DLKP）和穿透性角膜移植术（penetrating keratoplasty,PKP）治疗真菌性角膜炎的临床疗效.方法 回顾44例（44眼）临床确诊为真菌性角膜炎的患者,根据共焦激光显微镜检查是否累及角膜内皮分别行DLKP 28例（28眼）或PKP 16例（16眼）.术后随访6～24个月,观察术后真菌复发情况以及拆线后两周视力和角膜地形图散光.结果 术后角膜病理切片检查均检出真菌.术后DLKP组有1例、PKP组有3例出现排斥反应,经抗排斥治疗后均好转.拆线后两周最佳矫正视力DLKP组为0.59×0.04,PKP组为0.41 ×0.05,两组间相比较差异具有统计学意义（t =2.577,P =0.01）;角膜地形图散光DLKP组为（2.0 ×0.17） D;PKP组为（2.9×0.30）D,两组间相比较差异具有统计学意义（t =0.088,P=0.016）.结论 激光共焦显微镜检查对于手术方式的选择提供了良好的客观依据.两种手术方式治疗真菌性角膜炎均有良好的疗效,DLKP术后视觉疗效优于PKP.     

Thermal expansion of blood vessels in low cryogenic temperatures, Part II: Vitrification with VS55, DP6, and 7.05 M DMSO

As part of the ongoing effort to study the mechanical behavior of biological materials in cryopreservation processes, the current study focuses on thermal expansion during vitrification (vitreous in Latin means glassy). A new device is utilized in this study, which has been described in detail in the companion paper (Part I). The current study (Part II) focuses on measurements of vitrified blood vessels permeated with the cryoprotectants VS55, DP6, and DMSO. Data analysis in this study includes polynomial approximation of experimental results in the lower part of the cryogenic temperature range, where the material behaves as solid over a practical time scale. The study further includes a unified thermal expansion analysis throughout the entire cryogenic temperature range by compiling the current results with previously reported data. Finally, analysis of the glass transition temperature, based on thermal strain data is presented.     

小鼠原核胚玻璃化冷冻保存技术的研究

目的　原核胚是转基因等生物技术所必需的主要材料 ,其冷冻保存使操作不受时间和空间的限制。另外 ,冷冻保存还可以避免体外培养过程中的细胞发育阻断期。方法　在室温 (2 0℃或 2 5℃ )条件下 ,以乙二醇、DMSO为主体抗冻保护剂配制成的玻璃化溶液 (EFS、EDT、EDFS) ,不借助冷冻仪 ,对小鼠原核胚进行一步法和二步法玻璃化冷冻保存。结果　 2 0℃室温条件下 ,用EDFS4 0平衡 1min一步法冷冻解冻后的原核胚 ,经培养后囊胚发育率最高仅为 4 7% ,与新鲜原核体外培养的对照组 (75 % )的差异极显著 (P <0 0 1) ;当原核在 10 %E +10 %D溶液中预处理 5min ,移入EDFS30中平衡 30s二步法冷冻保存 ,解冻后的囊胚发育率达 6 8%。而室温升至 2 5℃ ,二步法冷冻保存后原核胚的囊胚发育率高达 77% ,与对照组差异无显著性 (P >0 0 5 )。用最佳冷冻组的原核胚或解冻后培养到囊胚移植给受体母鼠均获得产仔。结论　本研究对小鼠原核胚实施玻璃化冷冻保存 ,经体外培养和移植结果与对照组无显著性差异 ,证明了本方法的可行性     

Vitrification of turbot embryos: preliminary assays

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.     

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Cryogenic protection of oocytes with antifreeze proteins

Proteins belonging to a family of compounds known as “antifreeze proteins” interact with occytes and protect the oolemma from damage at cryogenic temperatures. Experiments were performed with pig oocytes rapidly cooled to cryogenic temperatures in vitrifying solutions with and without antifreeze proteins. Four different types of antifreeze polypeptides and glycoproteins were tested. The integrity of the oolemma was examined with Fluoroscein Diacetate (FDA) staining and morphological examinations. Results show that the pig oocyte oolemma is a primary site of injury during exposure to low temperatures and that all the different proteins have a similar ability to interact with and protect the oolemma. Our results may be important in developing solutions for long-term preservation of oocytes at cryogenic temperatures (cryopreservation). © 1993 Wiley-Liss, Inc.     

Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method

The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me(2)SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me(2)SO required at low temperatures are reached only at those low temperatures. In this paper, we describe some new experiments with discs of ovine articular cartilage similar to those used in our previous studies and we show that continuous stirring throughout the process resulted in a significant increase in the rate of (35)S sulphate incorporation into glycosoaminoglycans (GAGs), now reaching 87% of the corresponding fresh control values. We confirmed that the method is also effective for human knee joint cartilage, which gave 70% of fresh control ability to synthesise GAGs; continuous stirring was also used in this experiment. We then extended the method to ovine knee joint osteochondral dowels and showed that, again with continuous stirring, the method produced tissue concentrations of Me(2)SO that were sufficient to prevent freezing in dowels too, and to permit cell function at 60% of control. The most important mechanical property (instantaneous compressive modulus) was unaffected by the process. Finally, we experimented with some technical variations to facilitate clinical use-a more rapid process for warming and removal of Me(2)SO was developed and a method of short-term storage before or after cryopreservation was developed. Finally, pilot experiments were carried out to provide proof of principle for a closed, continuous flow method in which both temperature and Me(2)SO concentration were computer-controlled.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Evaluation of the Cryotech Vitrification Kit for bovine embryos

The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit® and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production.     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.     

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (?183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.     

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.