三种冷冻保护剂对新生小鼠睾丸组织冷冻效果的比较

目的探讨三种不同冷冻保护剂对新生C57BL／6J小鼠睾丸冷冻的效果。方法5日龄C57BL／6J雄鼠睾丸组织,分别用3种不同冷冻保护剂（二甲基亚砜〈dimethyl sulfoxide,DMSO〉、丙二醇〈propylene Glycol,PROH〉、EFS20／40）进行玻璃化冷冻。复苏冷冻后的睾丸组织,每组一部分进行组织学分析,另一部分进行组织移植,移植后检测关键基因表达水平以及卵胞质内单精注射,并设相应对照组。结果3个实验组睾丸组织形态改变与对照组相比均具有显著性差异（P〈0．01）,其中EFS组分值最低即改变最小;原位移植8周后组织块生长发育,存活率分别为DMSO组16．7％、PROH组13．3％、EFS组23．3％,存在成熟精子;睾丸组织特异性基因pgk-2和TESK1正常表达;卵胞质内单精注射表明移植后睾丸内精子均具有受精能力,胚胎移植后可得到正常子代小鼠（DMSO：PROH：EFS=5：2：8）。结论3种冷冻保护剂均能良好保存睾丸组织结构和功能。其中,EFS方法保存的睾丸组织形态改变最小、功能完善更适于睾丸组织的冷冻。     

米非司酮固体脂质纳米粒冷冻干燥性能的研究

目的:考察几种冻干保护剂对载药SLN冻干过程中的保护作用,并筛选出最佳的配方.方法:通过测定载药SLN冻干前后的载药量和粒度变化,来评价保护效果和优选配方,并通过TEM和AFM进行了形态学观察.结果:浓度为20%的海藻糖可以对载药SLN起到较佳的保护效果,试样的包封率高达79%(冻干前88%),粒径247 nm(冻干前109 nm),并且多分散系数0.121大大低于冻干前的(0.246),为SLN载体系统的建立稳定剂型和长期保存开辟了前景.结论:选用合适浓度的冻干保护剂在合理的冻干条件下可以获得令人满意的载药冻干SLN试样.     

卵巢组织冷冻保存的研究进展

卵巢冷冻技术可保存卵巢中大量的生殖细胞,将冷冻的卵巢组织解冻移植后可恢复卵巢的内分泌功能和生育能力,是癌症患者保存生育能力的一个理想途径。在该技术中诸多因素,如冷冻保护剂(CPA)、冷冻载体、皮质块大小以及冷冻方法等均影响着卵巢冷冻保存的效果;且目前尚缺乏高效、统一和标准的技术程序,因而限制了该技术在临床和科研中的广泛使用。为优化该技术程序,改善冷冻后卵巢功能的恢复情况,本文对影响该技术使用效果的一些因素进行了论述。     

深低温冷冻保护剂研究进展

【摘要】作为再生医学的重要组成部分,组织工程在受损组织和器官的修复再生方面显示了良好的临床应用前景。组织工程相关细胞及组织的深低温保存是组织工程技术平台的重要部分。冷冻保护剂是深低温保存细胞和组织不可缺少的部分,具有很高的临床应用和研究价值。就传统低温冷冻保护剂的分类和组成、其他冷冻剂的研究进展、改善深低温冷冻保护剂效果的技术手段及冷冻保护剂在组织工程研究中的重要价值进行了综述;探讨最优的深低温保护剂,为组织工程产品的保存及临床应用提供理论依据,为冷冻保护剂的研究指出了新的发展方向;最后指出了冷冻保护剂在组织工程应用方面存在的相关问题。     

Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report

The aims of this investigation were to test a novel technology comprising cryoprotectant‐free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo‐injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland^® medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: CM + 1% BSA + 40% seminal plasma + 0.125 m sucrose. For cooling, 20‐μl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM + 1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR‐14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post‐warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM + 1% BSA + 40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant‐free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA + 40% seminal plasma.     

Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

AIM:To optimize a xeno-free cryopreservation protocol for primary human hepatocytes.METHODS:The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes.Despite several hepatocyte cryopreservation protocols being available,improvements are urgently needed.We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups.Using the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) medium.The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation.The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and CYP3A7.RESULTS:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P < 0.01).There was no significant difference in viability estimation between both the trypan blue(TB) and the Live-Dead Assay methods.There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols(r 2 = 0.69) using the TB method.However,due to high within-group variability in the activities of the major CYPs,any statistical between-group differences were precluded.Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability.Thus,it may be a better alternative to the standard DMSO-UW protocol.Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples.CONCLUSION:The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.     

The effects of cryoprotectants on the freeze-drying of ibuprofen-loaded solid lipid microparticles (SLM)

The effects of cryoprotectants on the diameter and the entrapment efficiency of ibuprofen-loaded solid lipid microparticles (SLM) during the freeze-drying process were investigated extensively. The SLM were prepared by the emulsion-congealing technique in which a glycerol behenate was used as the lipid matrix for the SLM and a soybean lecithin/bile salt used as the stabilizer. Also, trehalose, glucose, mannitol, and sucrose were chosen as the cryoprotectants. Trehalose and glucose proved to be the most effective in preventing particles aggregation and in inhibiting leakage from drug-loaded particles during the SLM freeze-drying process. The most suitable concentrations were proved to be 15% and 5% (wt), respectively.     

不同玻璃化冷冻方案对小鼠卵巢组织的影响

目的比较不同组合的冷冻保护剂对小鼠卵巢组织的影响,为人类卵巢组织玻璃化冷冻的保护剂选择提供实验依据。方法将20只小鼠的卵巢进行玻璃化冷冻,组织标本随机分为4组,新鲜的和冻融后的卵巢皮质分别进行光学显微镜观察、TUNEL实验和免疫组织化学分析。结果光镜观察发现各实验组的各级卵泡形态正常率均明显低于对照组,B组的始基卵泡形态正常率和初级卵泡形态正常率均高于A组和C组,差异有统计学意义（P〈0．05）。A组和C组冻融后卵泡凋亡率、卵母细胞凋亡率和颗粒细胞凋亡率均高于B组,有统计学差异（P〈0．05）。另外,B组卵泡Bax蛋白阳性表达率低于A组和C组,而B组Bcl-2蛋白阳性表达率高于A组和C组,差异具有统计学意义（P〈0．05）。结论实验结果提示：与其它组合相比,EG与PROH的组合更适合小鼠卵巢组织的玻璃化冷冻。     

胚胎发育期和冷冻保护剂剂量对Nakagata小鼠胚胎冷冻效果的影响

目的利用不同剂量的冷冻保护剂,对ICR和Km小鼠不同发育期的胚胎进行冷冻和解冻实验。探讨Nakagata小鼠胚胎玻璃化冷冻管方法,使用不同剂量的冷冻保护剂等因素对冷冻效果的影响。方法2细胞期胚胎在KSOM微滴培养液内分别培养到2细胞4、细胞和8细胞期胚胎,分别加入50μL,100μL,200μL的DAP213冷冻保护剂,利用Nakagata方法冷冻,解冻以及观测解冻后培养至囊胚期胚胎的冷冻效果。结果冷冻保护剂DAP213剂量和ICR,KM小鼠胚胎不同细胞发育期对冷冻效果具有显著的交互影响(P<0.01)。相对而言,冷冻保护剂剂量对结果的影响(P>0.05)低于胚胎细胞发育期对冷冻效果的影响(P<0.01)。结论冷冻保护剂的剂量和胚胎细胞发育期均会影响冷冻实验的结果。胚胎的细胞发育期对冷冻效果的影响更大。对2细胞期,4细胞期和8细胞期的胚胎进行冷冻。8细胞期胚胎的冷冻效果最好,4细胞期胚胎冷冻效果最差。利用Nakagata冷冻管方法建立小鼠胚胎冷冻库,100μL冷冻保护剂效果较好。不同小鼠的品系也可影响冷冻结果。     

雄性生殖细胞冷冻保存研究进展

随着单精子及精子细胞卵胞浆内显微受精技术的发展,雄性生殖细胞冷冻保存技术已成为辅助生殖技术的重要内容,为男性因素不育患者的临床助孕提供了物质保障基础。目前,雄性生殖细胞冷冻方法包括程序化冷冻和玻璃化冷冻;冷冻保护剂可有效降低细胞的冷冻损伤,根据其特性分为渗透性保护剂和非渗透性保护剂两大类;冷冻载体中冷冻环、冷冻叶片等应用较广泛。雄性生殖细胞冷冻的效果与冷冻方法、冷冻保护剂和冷冻载体等密切相关。