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1.
马皮肤成纤维细胞的体外培养与冷冻保存 总被引:1,自引:0,他引:1
本研究利用小组织块直接培养法得到了成年马皮肤成纤维细胞的原代培养物,再用酶消化法处理,能够纯化马皮肤成纤维细胞。成纤维细胞的冷冻保存,首先采用手工冷冻法,选用含有不同浓度保护剂如:二甲基亚砜(DMSO)、乙二醇(EG)、甘油(GC))和新生牛血清(NCS)的12种冷冻液对马皮肤成纤维细胞进行冷冻保存;其次用2种冷冻方法对马皮肤成纤维细胞进行冷冻保存,以24 h贴壁率评价冻存效果。结果表明,10% DMSO和20% NCS的DMEM冻存液,对马皮肤成纤维细胞的冻存效果好(24 h贴壁率84.98%)。从冷冻方法来看,程序冷冻法(86.32%)优于手工冷冻法(79.98%)(P<0.05)。 相似文献
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通过比较不同冷冻保存方法和冷冻保护剂对小鼠耳皮肤成纤维细胞冷冻-解冻复苏后细胞存活率、48h贴壁率和细胞生长曲线的影响,筛选适宜的小鼠耳皮肤成纤维细胞冷冻保存方法和冷冻保护剂。结果表明,以100mL/L二甲基亚砜(DMSO)作为冷冻保护剂进行小鼠耳皮肤成纤维细胞冷冻保存时,方法3处理组解冻复苏后细胞存活率和培养48h细胞贴壁率均高于方法2处理组(P>0.05),并分别极显著高于方法1和方法4。采用方法3进行冷冻保存时,以100mL/L DMSO作为冷冻剂的细胞贴壁率显著高于100mL/L甘油(GL)组(P<0.05),且冷冻解冻后的细胞呈现正常的分裂增殖生长模式。因此,宜选择100mL/L胎牛血清(FBS)+100mL/L DMSO+DMEM作为冷冻保护液,采用方法3进行小鼠耳皮肤成纤维细胞的冷冻保存。 相似文献
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成年小鼠雄性生殖细胞的冷冻保存 总被引:1,自引:0,他引:1
在含10%小牛血清(NBS)的DMEM培养基中,分别各添加5%,10%,15%,20%和25%的二甲基亚砜(DMSO),丙二醇(PG),乙二醇(EG)和甘油(G),对成年小鼠睾丸生殖细胞冷冻保存;复苏后台盼蓝染色测定细胞复苏率。结果显示,5%-25%DMSO冻存液组的细胞复苏率分别为88.5%,88.0%,65.6%及51.3%;5%-25%PG冻存液组的细胞复苏率分别为87.2%,86.4%,79.0%,73.4%及40.1%.;5%-25%EG冻存液组的细胞复苏率分别为6.6%,80.9%,60.8%,51.3%及30.0%;5%-25%G冻存液组的细胞复苏率分别为86.5%,86.3%,65.3%,36.0%及31.4%。其中各抗冻剂5%和10%组的细胞复苏率最高,与15%组相比均存在显著或极显著差异。4种抗冻剂的最高细胞复苏率之间无显著差异。DMSO,PG,EG和G分别冷冻保存成年小鼠睾丸生殖细胞对的最小损失率分别为4.8%,6.1%,6.7%,6.8%。结果表明,采用慢速冷冻时,DMSO,PG,EG及G均适宜用作成年小鼠睾丸生殖细胞的抗冷冻剂,最佳使用含量均为5%-10%。成年小鼠睾丸生殖细胞分别在含5%-10%的DMSO,PG,EG和G的DMEM(含10%NBS)冻存液中,2步慢速降温,液氮储存,37℃水浴复苏,是一种具有较高复苏率的冷冻保存方法。 相似文献
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《畜牧兽医学报》2016,(5)
为了改进奶牛乳腺上皮细胞原代培养技术和探究乳腺组织冻存方法,选用24月龄无泌乳史荷斯坦奶牛,采用组织块种植法,通过侧置和倒置两种方式,分别从新鲜和冻存(8个月)的乳腺组织中分离培养奶牛乳腺上皮细胞。结果表明,侧置处理能使乳腺细胞爬出时间缩短1~2d,并改善组织块贴壁速度和效果;回收于前次培养后的乳腺组织块再分离培养,贴壁第2天即有乳腺上皮细胞爬出;一步冷冻(-80℃)液氮保存较大奶牛乳腺组织块,冻存液A(FBS∶DMSO∶DMEM/F12=7∶2∶1)冻存的组织块存活率为50%,冻存液B(FBS∶DMEM/F12∶DMSO∶HEPES/丙酮酸钠贮存液=10∶7∶2∶1)冻存的组织块存活率为56%。可见,作者建立的侧置处理法和组织块回收法可以用于奶牛乳腺上皮细胞的分离培养,可以缩短组织块种植法实验周期,冻存液中加入缓冲液HEPES/丙酮酸钠后有利于组织块生物活性保持。 相似文献
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贵德黑裘皮羊耳成纤维细胞系的建立和生物学特性的研究 总被引:4,自引:1,他引:4
以贵德黑裘皮羊耳缘组织为材料,采用组织块贴壁培养法和细胞冷冻技术成功构建了成纤维细胞系,并对培养细胞进行了形态学、生长动力学观察、细胞活力测定、核型分析和微生物检测。结果表明:细胞群体倍增时间(PDT)约为36h,冻存前细胞活力为96.2%,以DMEM/F-12+20%FBS中添加10%DMSO冻存成纤维细胞,解冻后细胞活力为93.6%,24h后的贴壁率为85%。在传代10次之前,细胞染色体中二倍体(2n=54)占主体,约为82%~90%,细菌、真菌、病毒、支原体检测为阴性。该细胞系的建立,使贵德黑裘皮羊这一国家重要种质资源在细胞水平上保存下来,也为体细胞克隆等研究提供了理想的生物材料。 相似文献
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7日龄小鼠生精上皮单细胞冷冻保存 总被引:12,自引:3,他引:12
在DMEM中添加10%小牛血清(NBS),并分别添加不同浓度的抗冻剂二甲基亚砚(DMSO)、丙二醇(PG)、乙二醇(EG)及甘油(G),对7日龄小鼠生精上皮单细胞进行冷冻保存,复苏后台齿蓝染色测定细胞复苏率。结果:当冻存液中DMSO分别为5%、10%、15%、20%时,细胞复苏率分别为77.1%、88.2%、86.5%、73.5%、65.5%,其中10%和15%DMSO组细胞复苏率最高,与其作各组间均差异极显著(P<0.01)。当冻存液中PG分别为5%、10%、15%、20%、25%时,细胞复苏率分别为66.2%、84.3%、72.1%、69.9%、47.5%,其中10%PG组细胞复苏率最高,与其余各组间均差异极显著(P<0.01)。当冻存液中EG分别为5%、10%、15%、20%时,细胞复苏率分别为64.9%、81.6%、60.9%、44.7%,其中10%EG组细胞复苏率最高,与其余各组间均差异极显著(P<0.01)。当冻存液中G分别为5%、10%、20%、25%时,细胞腹苏率均低于10%。10%DMSO组细胞复苏率与10%PG组和10%EG组之间均差异显著(P<0.05)或极显著(P<0.01)。结果表明,10%DMSO、10%PG及10%EG均适宜冷冻保存小鼠精原细胞,以及10%DMSO的冻存效果最好,而则不适宜冷冻保存。在含10%DMSO的冻存液中,二步慢速冷冻,液氮储存,37C水浴复苏小鼠精原细胞,是一种具有较高细胞复苏率的冷冻保存方法。 相似文献
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冷冻保护剂和胎牛血清对牛成纤维细胞冷冻效果的影响 总被引:1,自引:0,他引:1
以甘油与DMSO为冷冻保护剂,添加5%-20%胎牛血清的DMEM为基础冻存液,对鲁西黄牛皮肤成纤维细胞进行冷冻保存,结果表明:①同浓度DMSO为冷冻保护剂效果优于同浓度的甘油(P<0.05),但添加50%血清时并不明显(P>0.05),可能与较高血清浓度有关,血清对冻存细胞的影响高于冷冻保护剂的作用。②血清浓度一定时,DMSO和甘油浓度在10%-15%时,冻存效果较好。③当甘油与DMSO浓度一定时,血清浓度越高冷冻保存效果越好。④甘油与DMSO浓度一定时(DMSO浓度5%除外),添加10%和20%血清效果无差异(P>0.05)。因此,在实际应用中,考虑成本等因素,血清浓度达到10%时就已经能达到一般试验要求。 相似文献
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试验旨在探究奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)最佳的冻存液以改善乳腺上皮细胞的冻存质量.BMECs传至第5代后分别加入以下10种不同配方的冻存液.A组:85% DMEM+10%胎牛血清+5% DMSO;B组:80% DMEM+ 10%胎牛血清+10% DMSO;C组:75% DMEM+10%胎牛血清+15% DMSO;D组:70% DMEM+10%胎牛血清+20% DMSO;E组:85% DMEM+5%胎牛血清+10% DMSO;F组:75% DMEM+15%胎牛血清+10% DMSO;G组:70%DMEM+20%胎牛血清+10%DMSO;H组:80%DMEM+10%胎牛血清+10%甘油;I组:70% DMEM+10%胎牛血清+20%甘油;J组:60% DMEM+10%胎牛血清+30%甘油,冻存前统一调整细胞密度到1×106个/mL冻存.分别对复苏后的细胞进行台盼蓝染色计算存活率和PI/Hoechst 33258双染计算凋亡率.结果表明,BMECs经不同冻存剂冻存复苏后,细胞活力、形态学及凋亡率表现有所不同,其中B组和G组的活力和24h贴壁率较其他组高,二者的凋亡率较低,二者之间差异无显著性(P>0.05);传代后B组细胞的生长状况最好. 相似文献
11.
为了提高野牦牛成纤维细胞分离培养效果,研究了组织保存方法、原代培养方法、培养液类型、添加细胞生长因子和冷冻保存液配方对野牦牛体细胞分离培养与传代的作用。结果表明,PBS、D/F12和TCM199适合野牦牛皮肤组织的保存,D/F12和TCM199培养液适合组织块培养,组织块成活率达到(86.29±4.62)%,组织块法适合野牦牛成纤维细胞的原代培养;D/F12培养液培养野牦牛成纤维细胞效果较好,群体倍增时间和平台期密度分别为38.47 h和2.075×106/mL,正常二倍体核型百分率为84.33%;表皮生长因子(EGF)和碱性成纤维细胞因子(bFGF)产生了较明显的促增殖作用,平台期细胞密度明显增加;2种冷冻保存液(D/F12添加胎牛血清和二甲亚砜及胎牛血清添加二甲亚砜)均能用于野牦牛细胞冷冻,细胞存活率分别是87.33%和89.00%。 相似文献
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鸡胚精原干细胞体外保存能力的研究 总被引:4,自引:1,他引:4
采用二酶3步法获取孵化第19 d的鸡胚精原干细胞(SSCs),比较在快速冷冻条件下3种冷冻保护剂(DMSO、乙二醇、甘油)在3个浓度5%、10%、15%条件下对鸡胚精原干细胞的冷冻保存效果进行研究。结果显示:(1)当DMSO的浓度为5%、10%及15%时,复苏后细胞存活率分别为73.1%、88.6%和74.8%,三者差异显著(P〈0.05);而10%DMSO保存精原干细胞的存活率、复苏后培养细胞生长和集落形成均显著高于其它2个浓度;当乙二醇浓度为5%、10%和15%时,复苏后细胞存活率分别为69.4%、83.1%和65.2%,三者差异显著(P〈0.05);复苏后无论饲养层存在与否,SSCs均能增殖,但未有AKP阳性集落生成;当甘油浓度为5%、10%、15%时,复苏后细胞存活率均小于15%,三者差异不显著(P〉0.05);复苏后细胞存活时间极短,仅存活12 h左右;(2)当在10%DMSO浓度条件,复苏后SSCs在有饲养细胞层的条件下,培养5 d形成集落,表明10%DMSO是鸡胚精原干细胞适宜的冷冻保护剂。 相似文献
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H. Kuil 《Veterinary immunology and immunopathology》1984,7(1):89-98
Cryopreservation of bovine peripheral lymphocytes and its effect on the response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to ?30°C at 1.3°C/minute and further to ?80°C at 6°C/minute and then rapidly to ?196°C by dropping in liquid nitrogen. The cells were recovered by rapid thawing in water at 30–35°C and washed twice before use in the stimulation test. Ten percent DMSO had a much better protective effect than 5%; addition of 25% fetal bovine serum to the freezing had no favourable effect. In most of the 16 animals used in the experiments the frozen lymphocytes gave the same or a higher response to Con A than those kept in the DMSO containing medium at 4°C for two hours.The responses of the frozen cells were comparable to those of fresh lymphocytes (kept at 4°C for two hours in medium without DMSO). 相似文献
14.
采用两步酶解和差异贴壁法分离、纯化鸡精原干细胞(SSCs),比较了6种冷冻保护液对其冷冻保存效果,结果表明:FBS浓度为10%时,10%DMSO与10%甘油解冻后细胞存活率分别为54.05%和37.49%,差异显著(P<0.05);以10%DMSO为冷冻保护剂时,FBS浓度从10%增加到20%,解冻后细胞存活率分别为54.05%和69.06%,差异显著(P<0.05);在冷冻液Ⅰ基础上,添加3种不同浓度的蔗糖溶液,解冻后细胞存活率分别为52.49%、47.65%和51.94%,三者之间差异不显著(P>0.05),且与冷冻液Ⅰ组差异也不显著(P>0.05);将解冻后细胞接种饲养层上培养,除甘油组外,SSCs均能增殖并形成AKP阳性集落,但10%DMSO加上20%FBS组细胞增殖速度和集落数优于其他组合,表明10%DMSO加上20%FBS为鸡SSCs最佳冷冻保护剂。 相似文献
15.
V Myhrvold 《Acta veterinaria Scandinavica》1979,20(4):525-530
The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 109 cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G). 相似文献
16.
O Abas Mazni Y Takahashi C A Valdez M Hishinuma H Kanagawa 《The Japanese journal of veterinary research》1989,37(2):29-39
The effects of concentrations of glycerol, ethylene glycol or dimethylsulphoxide (DMSO) in the presence of either 0.25 M lactose or sucrose on the post-thaw survival of mouse quickly-frozen compacted morulae were studied. In this method, the embryos were directly frozen in liquid nitrogen (LN2) vapor at approximately -170 degrees C for 2 min before being plunged into LN2. High survival rates of frozen-thawed embryos were obtained when the freezing medium contained 3 M ethylene glycol with either 0.25 M lactose or sucrose (76.5 and 70.2%, respectively). When the embryos were frozen in glycerol, significantly high survival was obtained with 3 M glycerol + 0.25 M sucrose (73.5%, P less than 0.001). However, a freezing medium containing DMSO with either sugar gave lower survival rates. At a higher concentration of 4 M, ethylene glycol with 0.25 M lactose gave significantly higher survival rate than glycerol or DMSO (P less than 0.05). Significantly higher rates were obtained at 2 M with all 3 cryoprotectants when the freezing medium contained lactose rather than sucrose (P less than 0.05). This study showed that glycerol and ethylene glycol were effective cryoprotectants in the quick freezing of mouse embryos, while DMSO was less effective. In addition, the protective effects of these cryoprotectants are affected by their concentrations and the type of sugar used. 相似文献
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OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml. 相似文献