去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

人胚胎干细胞程序降温保存的实验研究

本文采用升降式程序降温仪对人胚胎于细胞进行了程序降温保存,并探讨和比较了降温速率、置核温度、保护剂和投入液氮前温度对冻存复苏后胚胎干细胞的存活率、活力及分化特性的影响。结果表明:采用Me_2SO 血清 DMEM(体积比为1∶3∶6)的保护剂,从0℃开始,以0.5℃/min的速率对细胞悬液降温;至-10℃时对其进行置核,并于-35℃时将其快速投入液氮中保存,复温后效果最佳。冻存复温后细胞存活率可达81.8%,复苏后的胚胎干细胞形态和集落生长方式都与冻前的生长形态相同,且胚胎干细胞标志之一碱性磷酸酶(AKP)反应阳性,同时染色体组型仍正常。     

恒河猴(Macaca mulatta)肾细胞冻存复苏培养及其生物学特性的研究

本文使用深低温(-196℃)冻结保存动物细胞技术,对胰酶分散的恒河猴肾细胞的冻存,复苏培养及其生物学特性进行了研究。结果表明、液氮冻存2—58周的恒河猴肾细胞,其平均存活率为63.3—74.4％。复苏培养细胞于6—7天形成緻密单层,对脊髓灰质炎病毒敏感;国产二甲基亚砜可以用作冻存细胞的保护剂;复苏培养的细胞核型正常;其生物学特性与未冻的原代细胞一致。     

菌种保藏方法对裂殖壶菌生长及发酵性能的影响

考察保护剂、保藏温度及预冷冻方法对Schizochytrium sp.HX-308菌种存活率及发酵性能保持的影响。结果显示:在-80℃低温保藏6个月后,渗透性保护剂的细胞存活率均比非渗透性保护剂高了5%,其中用60%(质量分数)海藻糖的保护剂最终的株细胞存活率达到80.02%,明显优于其他保护剂。采用液氮-196℃保藏菌种(两步预冷冻法、60%海藻糖保护剂),存储6个月后存活率高达90.70%,生物量、油产量和二十二碳六烯酸(DHA)产量分别达到了61.65、26.41和11.10 g/L,为最优的保藏方法,为裂殖壶菌的实验室研究及工业化生产提供了一种长期安全的保藏法。     

人胚胎干细胞程序降温保存的实验研究

本文采用升降式程序降温仪对人胚胎干细胞进行了程序降温保存,并探讨和比较了降温速率、置核温度、保护剂和投入液氮前温度对冻存复苏后胚胎干细胞的存活率、活力及分化特性的影响。结果表明：采用Me2SO 血清 DMEM(体积比为1：3：6)的保护剂,从0℃开始,以0．5℃／min的速率对细胞悬液降温;至-10℃时对其进行置核,并于-35℃时将其快速投入液氮中保存,复温后效果最佳。冻存复温后细胞存活率可达81．8％,复苏后的胚胎干细胞形态和集落生长方式都与冻前的生长形态相同,且胚胎干细胞标志之一碱性磷酸酶(AKP)反应阳性,同时染色体组型仍正常。     

基于电容测定的罗汉果细胞超低温保藏快速方法

建立了一种基于活细胞电容值定量测定的植物细胞超低温保藏的快速评价方法,优化了罗汉果细胞超低温保藏方法。通过采用活细胞传感仪测定冻存后细胞的存活率并结合细胞生活力(细胞线粒体活性/TTC)对罗汉果细胞的低温保藏过程进行优化,确定了罗汉果细胞较为适宜的冷冻保护剂组分为基本培养基中添加10%的蔗糖和10%的DMSO。预处理剂的考察实验表明,采用0.2 mol/L蔗糖的预处理剂处理细胞时冻存后细胞存活率和细胞活力较高;采用0.2 mol/L蔗糖预处理剂处理细胞时,随着预处理时间的增加,细胞存活率先增加后降低,预处理时间为9 h时,细胞存活率和细胞活力最高。保藏后的细胞复苏实验结果表明:细胞存活率与采用活细胞电容值得到的细胞存活率具有很好的一致性,同时经过冻存的细胞复苏培养后,仍保留了原始细胞的形态和合成甜苷V的特性,说明该冻存方法适用于罗汉果细胞的超低温保藏。因此基于活细胞传感仪测得的电容值进行细胞冻存过程细胞活性的快速评价方法具有较好的可行性和可靠性。     

BALB/C小鼠杂交瘤细胞冻存初探

自从Polge(1949)用甘油作为保护剂成功的冻存了Hela和L细胞以来,细胞冻存技术已日趋完善。本文对细胞株的冻存分别采用-70℃冰箱和常用的液氮深低温的方法。定期复苏,测定抗体效价,最后作染色体比较,至今已一年多,其结果令人满意。现将结果简要报告如下。方法1.细胞冻存:选择生长旺盛期,形态良好的细胞,按每ml细胞冻存液内含活细胞约1×106个,每支冻存管1ml。细胞冻存管置-20℃冰箱内15小时左右,分别放入液氮和-70℃冰箱内。经不同时期后取出作复苏及抗体效价比较。2.细胞复苏:取出冻存管,迅速浸入37℃水浴中,在1分钟内解冻后转种到已预制…     

螺旋藻的超低温保存法

【目的】建立螺旋藻藻种的超低温保存法,并探究该方法对不同种类螺旋藻藻种保存的适用性。【方法】采用碘量法筛选出耐低温螺旋藻藻株,通过单因素和正交试验设计对耐低温螺旋藻超低温保存法进行条件优化,并以优化后的超低温保存法对8株不同种类的螺旋藻进行保藏实验。【结果】FACHB-351为筛选出的耐低温螺旋藻藻株;优化后的超低温保存方案为:以10%蔗糖溶液做冷冻保护剂,将藻丝体密度为1.0×107 CFU/m L的藻悬液于4°C驯化72 h,再将藻液和保护剂分别在0°C预冷30 min后混匀,混匀后于0°C停留3 h,然后投入液氮保存。保藏实验结果表明,保藏6个月时除了耐低温性较差的FACHB-350、FACHB-1070、FACHB-902螺旋藻存活率为0,不能恢复生长繁殖,其它5种耐低温性较好的螺旋藻均能在一定时间内恢复正常的生长繁殖,其中FACHB-351的存活率最高,为39.33%。【结论】建立的超低温冷冻保存法可用于耐低温性较好的螺旋藻藻种的长期保存。     

渗透与非渗透性抗冻剂联用技术对铜绿微囊藻的超低温保藏研究

铜绿微囊藻(Microcystis aeruginosa Kütz)是一种在全世界分部很广的淡水水华蓝藻。在实验室长期的研究工作中, 为了使铜绿微囊藻能维持稳定的生理学特征, 通常使用超低温保藏技术长期冻存藻细胞。研究发现, 同时使用渗透性和非渗透性的抗冻剂比只使用传统的渗透性保护剂能显著提高Microcystis aeruginosa超低温保藏的存活率。以三株铜绿微囊藻为材料进行二步法超低温保藏, 对4 种抗冻剂(甲醇、二甲亚砜、丙三醇、聚乙烯吡咯烷酮), 两种降温速率(-1 /℃min、-0.5℃/min), 第一步温度设置(-30℃、-40℃、-80℃)进行筛选; 用流式细胞仪和细胞计数检测存活率, 并监测冻后相关生理参数、PSII、细胞色素、生长曲线等以确保该方法可保持藻株的活性和生理状态。结果表明, 5%的二甲亚砜和30%的聚乙烯吡咯烷酮(PVP)同时使用时能达到最好效果, 并能保持生理活性与冻前一致。       

人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能

研究人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能。应用包含10%二甲基亚砜、90%小牛血清的冻存液冻存6个T细胞系(5个CD4 T细胞系,1个CD8 T细胞系)细胞,液氮中冻存32~54个月后复苏,台盼蓝染色法检测复苏后T细胞系细胞的存活率,用酶联免疫斑点法(enzyme-linked immunospot assay,ELISPOT)检测复苏后T细胞系细胞的功能。结果显示,6个T细胞系细胞液氮冻存解冻后细胞的存活率为24.7%~93.5%,过夜培养后细胞的存活率为2.5%~72.2%。CD8 T细胞系细胞的存活率高于CD4 T细胞系细胞。6个复苏后的T细胞系细胞在PHA诱导后均能分泌IFN-γ。人乳头瘤病毒特异性T细胞系细胞冻存复苏后能够保持较好的存活率和功能。     

Cryopreservation of Dunaliella salina and Platymonas helgolandica var. tsingtaoensis

Dunaliella salina (Dunal) Teod. and Platymonas helgolandica Kylin var. tsingtaoensis were cryopreserved in liquid nitrogen with a two-step cryopreservation method. With 5% and 20% dimethylsulphoxyde (DMSO) concentration respectively through 30 min equilibrium time, and under -40℃, 60 min and -30℃, 30 min respectively as prefreezing temperature and sustained time, and by using slow dilution method to remove DMSO from the sample at 0℃ and room temperature respectively after sample thawing, D. salina and P. helgolandica var. tsingtaoensis gained the highest survival rates that were 68.5% and 78.3% respectively.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

脱水速率和降温速率对葡萄柚种子超低温保存的影响

以新鲜葡萄柚(Citrus paradise)种子为材料,使用3种脱水速率处理至约20%含水量,然后用饱和盐溶液平衡至10种不同的含水量,再进行简化的二步法超低温保存;同时,以经过15 ℃、50%相对湿度(RH)条件脱水至含水量约12%的葡萄柚种子为材料,使用程序降温仪进行5个降温速率和3个预冷温度的传统二步法超低温保存。结果表明,葡萄柚种子对脱水敏感,种子含水量越低,成苗率越低。而且,脱水速率越快,对种子的损伤越大,成苗率呈现75% RH>50% RH>15% RH;冷冻处理对种子有进一步的伤害,含水量6%～8%的种子超低温保存效果最佳,但脱水速率对超低温保存的种子成苗率的影响不明显。用传统二步法超低温保存葡萄柚种子,与预冷对照相比,经液氮冷冻的种子成苗率都有明显下降。在预冷对照组中,-2.5 ℃·min^-1降温速率的种子成苗率最高,在冷冻保存中-0.5 ℃·min^-1和-0.25 ℃·min^-1的降温速率最有利于超低温保存。预冷温度从-40 ℃降至-60 ℃,未超低温处理种子的发芽率降低,但超低温处理种子的发芽率提高。     

Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.     

Preservation of Pseudomonas putida bacteria at low temperatures]

We have found that the mode of cooling, composition of cryopreservation medium, original concentration of cells and storage temperature affect viability of Pseudomonas putida bacteria during low-temperature preservation. We have elaborated the conditions of preservation, providing for a high survival of bacteria, namely: one-stage cooling with the rates of 30, 40 degrees C/min or immersion into liquid nitrogen in the culturing medium with addition of sucrose, glycerol or dimexide in the concentration of 0.5 M; storage temperature is -80 degrees C divided by -196 degrees C.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Long-term viability of preserved eukaryotic algae

Levels of viability of Chlorella emersonii after storage of dried material for one year were 0.1% on rehydration, all other dried organisms examined in this study failed to recover after prolonged storage. In addition, no detectable recovery was observed in any of the algae tested after storage of freeze-dried cultures. Methods have also been developed to cryopreserve a range of microalgae, but no single protocol has been found to be universally satisfactory. Some strains are apparently not able to withstand cryopreservation using known methods, whilst others may be frozen successfully in the absence of cryoprotectant by plunging directly into liquid nitrogen. A two-step protocol (cooling to an intermediate subzero temperature prior to plunging into liquid nitrogen) has been used to cryopreserve the majority of strains. Where this has proven successful, post-thaw viability levels of over 95% have been attained for some algae. This paper demonstrates that, where applicable, cryopreservation allows the long-term preservation of frozen algae with no significant reduction in viability up to 22 years storage. (Previous location of Culture Collection of Algae and Protozoa) This revised version was published online in September 2006 with corrections to the Cover Date.