人羊膜间充质干细胞体外大量扩增的方法

背景：一个胎盘羊膜的面积约600 cm2,羊膜来源间充质干细胞即取材于胎盘上的羊膜。 目的：建立一种简单的体外分离培养人羊膜来源间充质干细胞的方法,并分析其生物学特性。 方法：采用胰酶和直接贴壁相结合的方法分离获取人羊膜来源间充质干细胞并进行体外培养,观察细胞形态,分析传代第5代细胞生长曲线,检测第5 代细胞表面标志表达和检测细胞周期。取传代第4 代细胞行体外成骨细胞诱导和成脂诱导,冻存第4代细胞6个月后复苏,计数复苏后细胞存活率并绘制复苏细胞生长曲线。 结果与结论：原代接种后第9天有少许细胞爬出,15 d左右细胞达80%-90%融合,细胞以梭形为主。细胞传代后,细胞形态均一,螺旋状排列。传代细胞潜伏期48 h,对数增殖期4 d左右,对数增殖期后进入平台期。间充质干细胞表面CD34、CD14、CD19、CD45、HLA-DR 呈阴性表达,CD73、CD105、CD90 呈阳性表达。茜素红染色及油红O染色阳性,证实具有向脂肪细胞、成骨细胞分化的能力。流式细胞术细胞周期检测,S期占28%。冻存复苏后细胞存活率达 90% 以上,且与未冻存传代细胞具有相同的生长特性。结果证实实验成功地建立了一种简化的人羊膜来源间充质干细胞大量扩增的方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

大鼠骨髓间充质干细胞的纯化、冻存及成瘤性分析

背景：骨髓间充质干细胞在骨髓中的含量极低,体外的长期培养过程中易丧失干细胞潜能以及体内移植后安全性等问题的存在,限制了骨髓间充质干细胞在临床上的广泛应用。 目的：探索体外分离纯化、冻存大鼠骨髓间充质干细胞的最适方法,并观察以此方法培养的骨髓间充质干细胞移植体内后是否具有成瘤性。 方法：分别采用差速贴壁结合24 h首次换液、24 h首次换液、48 h首次换液的方法纯化培养骨髓间充质干细胞,筛选最适纯化方法进行后续实验。配制含体积分数为10%,20%,30%,40%,50%胎牛血清的细胞冻存液冻存细胞,复苏后计算细胞存活率,测定复苏后细胞的生长曲线及成脂诱导能力。将第3,15代骨髓间充质干细胞进行裸鼠肌肉、肝脏局部注射体内移植,45 d后取注射部位行病理组织标本检查。 结果与结论：差速贴壁结合24 h首次换液法所获得骨髓间充质干细胞纯度最高,而细胞增殖能力与其他两组无明显差别,因此选用该方法纯化骨髓间充质干细胞,然后进行传代培养;含体积分数为30%血清冻存液既可保证细胞活性与增殖能力,又可保证干细胞特性及多向分化潜能。骨髓间充质干细胞传至15代仍保持间充质干细胞特性,骨髓间充质干细胞移植入裸鼠肝脏45 d后仍可在肝脏局部存活,生长状态与体外培养相似,无异型性及向周围浸润生长,提示体外长时间培养15代以内的骨髓间充质干细胞可在裸鼠体内存活且无成瘤性。     

雌激素影响冻存肾脂肪囊来源脂肪间充质干细胞的成脂分化

背景：雌激素对脂肪干细胞向脂肪细胞诱导分化会有负向调节作用,但对长期深低温保存的肾脂肪囊来源脂肪间充质干细胞向脂肪细胞分化的效果尚未见报道。 目的：探讨雌激素对长期深低温保存的及新鲜提取的肾脂肪囊来源脂肪间充质干细胞成脂分化能力的影响。 方法：将长期深低温保存后复苏的及新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞分为4组进行成脂诱导分化,新鲜+雌激素组和冻存+雌激素组诱导时添加10-7 mol/L雌激素,新鲜组和冻存组不添加。成脂诱导分化14 d进行油红O染色及成脂量定量检测。 结果与结论：长期深低温保存后复苏的第3代肾脂肪囊来源脂肪间充质干细胞与新鲜提取的细胞形态和排列无差异;长期深低温保存后复苏的与新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞表面抗原分子CD29、CD44均呈阳性,CD31均呈阴性表达。成脂诱导后可观察到细胞内有脂滴形成,油红O 染色呈阳性。在成脂诱导分化14 d对各组进行成脂量比较,新鲜组与新鲜+雌激素组,冻存组与冻存+雌激素组的吸光度之间比较,差异有显著性意义,但新鲜组与冻存组之间差异无显著性意义。结果表明：小剂量雌激素可抑制长期深度低温保存后复苏的肾脂肪囊来源脂肪间充质干细胞的成脂分化;长期深低温保存后复苏的与新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞在成脂诱导方面无显著性差异。     

不同直径神经干细胞球慢速低温冻存的研究

为提高神经干细胞冻存复苏后的效果及存活率,对不同尺寸与状态的神经干细胞球进行了在不同浓度DMSO(二甲基亚砜)下的慢速冻存比较.首先用CCK-8试剂盒测定神经干细胞的生长曲线,然后对处于对数生长期的3类神经干细胞(单细胞悬液,直径30～50 μm球,直径80～100 μm球)分别进行了7个不同浓度(3%,5%,7%,8%,10%,15%,20%)DMSO的冻存实验比较,并对冻后复苏的细胞进行活性检测和诱导分化.结果表明, 直径80～100 μm的神经干细胞球,DMSO浓度8%,复苏后细胞活率82.9%,且仍具有多项分化潜能.神经干细胞间的亲密联系和刻痕信号与直径尺寸共同作用,影响着神经干细胞的冻存复苏效果.     

p53抑制剂对扩增晚期人骨髓间充质干细胞活力的影响

背景：p53抑制剂能否直接干预骨髓间充质干细胞活力及其可能的机制尚不完全清楚。 目的：探讨p53抑制剂PFT-α对体外扩增培养晚期骨髓间充质干细胞衰老进程的影响,寻找延缓人骨髓间充质干细胞复制性衰老的关键靶点。 方法：qPCR检测扩增早、晚期人骨髓间充质干细胞p53、p21和p15 mRNA的表达。20 µmol/L p53抑制剂 PFT-α或等量二甲基亚砜分别作用晚期人骨髓间充质干细胞2周,β-半乳糖苷酶(SA-β-Gal)染色观察衰老细胞阳性率,TUNEL染色法检测细胞凋亡情况。300 µmol/L H2O2作用于人骨髓间充质干细胞30 min,CCK-8法检测细胞抗氧化应激能力。 结果与结论：qPCR显示晚期人骨髓间充质干细胞p15,p21,p53 mRNA表达水平分别较早期人骨髓间充质干细胞增高(1.45±0.23)倍,(1.51±0.14)倍,(1.78±0.14)倍(P < 0.05)。PFT-α组衰老细胞阳性率(41±5)%低于二甲基亚砜组(63±7)%(P < 0.05),但PFT-α组与二甲基亚砜组细胞凋亡率差异无显著性意义(P > 0.05)。经H2O2处理后,CCK-8结果显示PFT-α组吸光度值为二甲基亚砜组(1.27±0.13)倍(P < 0.001),以上结果表明p53信号通路激活可能是导致人骨髓间充质干细胞衰老的重要因素,应用p53抑制剂PFT-α能够增强晚期人骨髓间充质干细胞抗氧化应激损伤能力。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

深低温冻存羟基磷灰石/骨髓间充质干细胞修复兔桡骨缺损

背景：以往研究表明羟基磷灰石复合骨髓间充质干细胞具有良好的骨缺损修复效果,但这种复合材料在冻存后是否具有骨缺损修复效果还不清楚。 目的：观察深低温冻存羟基磷灰石/骨髓间充质干细胞复合修复骨缺损的效果。 方法：制备27只日本大耳白兔桡骨10 mm缺损模型,随机分为冻存复合材料组、新鲜复合材料组与羟基磷灰石组,3组分别于骨缺损处植入-80 ℃保存3个月的羟基磷灰石/同种异体骨髓间充质干细胞复合物、新鲜制备的羟基磷灰石/同种异体骨髓间充质干细胞复合物及单纯羟基磷灰石,植入后8,12周行大体观察、X射线观察及苏木精-伊红染色等组织学观察,并于12周行生物力学检测。 结果与结论：术后12周,冻存复合材料组、新鲜复合材料组骨缺损大部分愈合,有成熟骨小梁通过,有的可见髓腔通畅,塑形较好;羟基磷灰石组骨痂生成少,骨缺损部分愈合,塑形欠佳,新骨生成少于冻存复合材料组、新鲜复合材料组(P < 0.05)。冻存复合材料组、新鲜复合材料组最大载荷明显大于羟基磷灰石组(P < 0.05)。表明低温冻存羟基磷灰石/骨髓间充质干细胞复合植骨材料的骨缺损修复能力与新鲜制作的复合材料几乎一致,未因冻存受到影响。     

不同月龄人脐带间充质干细胞移植梗死心肌的血运重建

背景：干细胞移植有利于心肌梗死后的心肌血运重建及改善心功能,HLA-G分子在免疫耐受状态的形成及维持中具有重要作用。 目的：观察不同月龄有HLA-G表达差异的人脐带间充质干细胞移植后对急性心肌梗死兔血运重建的影响。 方法：健康新西兰大白兔30只,随机数字表法分为小月龄细胞移植组、足月龄细胞移植组及对照组。建立兔心肌梗死模型后2周,将小月龄人脐带间充质干细胞和足月人脐带间充质干细胞分别标记BrdU,多点注射心肌梗死的交界区和中心区,对照组注射无血清培养基。 结果与结论：移植后4周,小月龄和足月龄细胞移植组在心肌梗死区均发现有BrdU示踪细胞,且两组梗死区心肌纤维化程度、心肌梗死面积均少于对照组(P < 0.01),两移植组间差异也有显著性意义(P < 0.05)。Ⅷ因子染色见小月龄细胞移植组毛细血管密度高于足月龄细胞移植组(P < 0.01),且两移植组与对照组比较差异也有显著性意义(P < 0.05)。提示HLA-G表达量较高的小月龄脐带间充质干细胞能更好促进梗死区血管新生,改善血运重建,有潜力成为心肌细胞移植的更理想来源。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

不同冷冻方案保存人类卵巢组织的活性比较

背景：卵巢组织冷冻保存被认为是保存女性生殖内分泌功能安全、有效的方法,但目前尚无统一确定的方案。目的：探讨3种冷冻方案对人类卵巢组织保存冷冻效果及卵泡活性的影响。方法：采用丙二醇慢速程序冷冻法、二甲基亚砜玻璃化冷冻法、液氮直投法对20例人类卵巢组织进行冷冻保存,复苏后采用细胞存活/死亡荧光分析法计数有活性的细胞,体外培养测定培养液雌二醇浓度及各级卵泡计数,判断3种不同冷冻方案对人类卵巢组织活性的影响。结果与结论：不同冷冻方案冻融后卵巢组织块的活性卵泡率均低于新鲜卵巢组(P < 0.05),二甲基亚砜组最低,液氮直投法组与慢速程序冷冻法组差异无显著性意义。体外培养各冷冻组分泌的雌二醇水平比较,第4天时二甲基亚砜组低于液氮直投法组和慢速程序冷冻法组(P < 0.05),至第8天时各冷冻组雌二醇水平与新鲜卵巢组一致。培养14 d组织学观察,各组卵巢组织内生长期卵泡比例增多,始基卵泡仍然占最主要的。新鲜卵巢组织正常卵泡的总数高于冷冻组(P < 0.05),二甲基亚砜组的正常卵泡数低于液氮直投法组和慢速程序冷冻法组(P < 0.05)。提示冷冻保存对卵巢组织卵泡有一定的损伤,但仍能保存大部分始基卵泡的活性,经体外培养后可进一步发育并具有分泌功能,在3种冷冻方案中,慢速程序冷冻法和液氮直投法的冷冻效果优于二甲基亚砜玻璃化冷冻法,液氮直投法操作简便,冷冻效果稳定。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

骨髓间充质干细胞对肾病综合征大鼠CD4+CD25+调节性T细胞的影响

背景：CD4⁺CD25⁺调节性T细胞功能降低、数量下降已被公认为是肾病综合征患儿免疫失调的重要表现。骨髓间充质干细胞具有免疫调节功能,可上调CD4⁺CD25⁺调节性T细胞,抑制淋巴细胞增殖,并已在许多免疫性疾病方面成功应用。 目的：观察骨髓间充质干细胞移植对原发性肾病综合征大鼠外周血CD4⁺CD25⁺调节性T细胞的影响。 方法：自SD大鼠分离骨髓间充质干细胞,经体外传代培养及鉴定后制备细胞悬液。30只SD大鼠随机均分为3组,生理盐水组和干细胞移植组尾静脉注射阿霉素建立阿霉素肾病大鼠模型,干细胞移植组注射阿霉素当日尾静脉射干细胞1×10⁷;正常组不做处理。 结果与结论：①与正常组比较,生理盐水组大鼠均出现肾病综合征表现,以腹水、大量蛋白尿、低蛋白血症、高胆固醇血症为特征;干细胞移植组较生理盐水移植组则有明显改善(P < 0.05)。②造模第28天,干细胞移植组和生理盐水组大鼠造模后外周血CD4⁺CD25⁺Treg/ CD4⁺ Treg均明显高于正常组(P < 0.05);干细胞移植组与生理盐水组比较差异无显著性意义(P > 0.05)。③造模第28天,干细胞移植组大鼠外周血单个核细胞FoxP3mRNA的表达显著高于生理盐水组和正常组(P < 0.05)。提示骨髓间充质干细胞移植对阿霉素肾病综合征模型有一定的治疗作用,其机制可能与上调FoxP3在组织局部的表达而诱导CD4⁺CD25⁺Treg产生有关。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

低频电磁场在基因修饰表皮干细胞移植修复创面中的作用

背景：烧伤、慢性创面等大面积皮肤缺损的创面修复是临床上亟待解决的难题。表皮干细胞和角质化细胞生长因子可为创面的治疗提供新的策略。低频电磁场作为一种非侵入性物理刺激在创面修复中已被认为较佳的方法。 目的：探讨低频电磁场在角质化细胞生长因子基因修饰的表皮干细胞移植促进小鼠皮肤全层缺损创面修复中的作用。 方法：体外分离培养SD乳鼠表皮干细胞,用5-溴脱氧尿核苷进行标记,成功转染角质化细胞生长因子基因腺病毒表达载体后,移植于昆明小鼠全层缺损创面。建立全层缺损创面小鼠模型,分别进行表皮干细胞移植,角质化细胞生长因子修饰的表皮干细胞移植,角质化细胞生长因子修饰的表皮干细胞移植外加低频电磁场干预。 结果与结论：移植后第9,16天,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组创面收缩率优于其他各组(P < 0.05)。移植后第9天,免疫荧光染色法检测显示,各组组创面中均有5-溴脱氧尿核苷阳性细胞分布。移植后第16天,Western blot检测显示,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组和角质化细胞生长因子基因修饰的表皮干细胞移植组创面组织中角质化细胞生长因子蛋白表达高于表皮干细胞移植组(P < 0.05);移植后第16,30天,苏木精-伊红染色结果显示,低频电磁场干预的角质化细胞生长因子基因修饰的表皮干细胞移植组的创面组织上皮化现象较明显。结果证实,低频电磁场有促进角质化细胞生长因子基因修饰的表皮干细胞移植修复小鼠皮肤全层缺损创面的作用。     

人牙周膜干细胞与牙周膜细胞生物学特性的比较

背景：牙周膜干细胞生物作用是目前牙周病治疗研究的热点,牙周膜成纤维细胞是其分化的终末功能细胞之一,也是其主要的支持细胞,两者生物学特性的差异研究鲜有报道。 目的：比较牙周膜干细胞与牙周膜细胞生物学特性的差异。 方法：用组织块法体外对牙周膜细胞以及单细胞克隆分离纯化后的人牙周膜干细胞两种细胞分别进行显微镜下形态观察,CCK8法检测并绘制2种细胞的生长曲线。流式细胞分析比较2种细胞的细胞周期以及细胞表面标记物的表达、实时PCR对2种细胞碱性磷酸酶、增殖细胞核抗原和Scleraxis基因进行检测。 结果与结论：牙周膜干细胞与牙周膜成纤维细胞外观差别明显,人牙周膜干细胞的生长曲线培养前5 d要低于牙周膜细胞,但在5 d后明显高于牙周膜细胞。人牙周膜干细胞与牙周膜细胞的细胞周期分别为41.1%和23.9%。表面标记物检测结果显示2种细胞虽有相似的表达,但在表达率差异有有显著性意义。实时荧光定量PCR结果显示,人牙周膜干细胞在碱性磷酸酶、增殖细胞核抗原以及Scleraxis基因的表达检测均高于牙周膜细胞。表明牙周膜干细胞在成骨增殖等生物学功能上比牙周膜细胞具有更强的潜能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Comparison of different cryopreservation protocols for human umbilical cord tissue as source of mesenchymal stem cells

The main purpose of this study is to establish an effective cryopreservation protocol for the umbilical cord tissue as a source of mesenchymal stem cells (MSCs). In this context, it was aimed to use a cryoprotectant that could be an alternative to dimethyl sulfoxide (DMSO) which is commonly used despite the toxic side effects. Therefore, two different cryopreservation solutions were prepared using 10% DMSO and 10% 1,2 propanediol (PrOH). The fresh tissue group that was not performed cryopreservation was used as the control group. Following the cryopreservation step, MSCs were isolated from all groups and compared with each other to assess the efficiency of the cryopreservation solutions. The comparison was performed in terms of followings: morphology, immunophenotypes, growth kinetics, differentiation, and ultrastructural features. Based on the results, there were no significant morphological and immunophenotypic differences between the MSCs isolated from cryopreserved tissue groups and the MSCs isolated from the fresh tissue group. According to the growth kinetic analysis, the cells isolated from the PrOH group had a lower proliferation rate than the cells isolated from the fresh tissue. However, there was no significant difference between the cryopreserved groups in this respect. Osteogenic and adipogenic differentiation was observed in all groups. Upon comparison of the cryopreserved groups, PrOH group was discovered to hold a minor superiority in terms of these modes of differentiation. These results suggest that PrOH, which is considered as a cryoprotectant with low toxicity, could be used as a preferred cryoprotectant instead of DMSO concerning the process of cryopreservation of the umbilical cord.     

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.     

牙周膜干细胞成骨分化中细胞因子的作用

背景：牙周膜干细胞是牙周组织中的成体干细胞,具有高度增殖、自我更新能力和多分化潜能。促进牙周膜干细胞向成骨细胞分化有助于牙周疾病的治疗。 目的：观察胰岛素样生长因子1和成纤维细胞生长因子2对牙周膜干细胞向成骨细胞分化的影响。 方法：采用胶原酶消化人牙周膜组织,获得牙周膜干细胞,经体外鉴定、扩增后,通过倒置显微镜、苏木精-伊红染色、流式细胞仪对牙周膜干细胞进行生物学检测。分别在成骨细胞诱导培养液中加入成骨诱导液(对照组)及胰岛素样生长因子1和成纤维细胞生长因子2持续诱导7,14 d后,进行碱性磷酸酶染色、碱性磷酸酶活性检测,以及茜素红染色,并用实时定量PCR法检测向成骨细胞分化的标志性基因的表达情况。 结果与结论：胰岛素样生长因子1刺激组的碱性磷酸酶活性以及钙化结节明显高于对照组,Runx2、Alp、col-1的mRNA呈高表达;成纤维细胞生长因子2刺激组的碱性磷酸酶活性以及钙化结节也高于对照组,Runx2、Alp、col-1的mRNA表达量也高于对照组。提示胰岛素样生长因子1和成纤维细胞生长因子2在不同程度上促进体外培养的牙周膜干细胞向成骨细胞方向分化。     

Determination of cryoprotectant for magnetic cryopreservation of dental pulp tissue

Dental pulp, covered with dental hard tissue, is a promising source of mesenchymal stem cells and osteoprogenitor cells for regenerative medicine. Our previous studies showed that 73% of dental pulp cells isolated from magnetically cryopreserved teeth where their viability, morphology, and expression of stem cell surface markers were similar to the cells isolated from fresh teeth, suggesting that magnetic cryopreservation is an applicable method for intact tooth as well as dental pulp tissue banking. However, the cryoprotectant, concentration, contact surface, and equilibration time for magnetic cryopreservation of dental pulp require optimization. In addition, the integrity and viability of post-thawed dental pulp with and without dental hard tissue covering after magnetic cryopreservation were investigated. Lower concentration of the cryoprotectant (5% dimethyl sulfoxide [DMSO]) and shorter preequilibration time are required for magnetic cryopreservation compared with the conventional cryopreservation method. The structure of at least 33% of post-thawed pulp with dental hard tissue from the open end remained intact where >80% of cells were viable. The addition of the cryoprotectant additive trehalose did not replace or improve DMSO's efficacy for magnetic cryopreservation of dental pulp or intact tooth. Tooth banking for transplantation provides an alternative treatment to replace missing teeth. The optimized cryoprotectant conditions for dental pulp tissue during magnetic cryopreservation should lead to more satisfactory outcomes in clinical applications such as autotransplantation and the isolation and expansion of dental pulp stem cells for tissue repair.     

多发性骨髓瘤患者骨髓间充质干细胞冻存前后的生物学特性

背景：多发性骨髓瘤患者的骨髓间充质干细胞具有多向分化、免疫调节和支持造血作用,但是这些功能是否受冻存的影响目前尚不清楚。 目的：探讨冻存对多发性骨髓瘤患者骨髓间充质干细胞生物学特性的影响。 方法：采用细胞贴壁法获取多发性骨髓瘤患者骨髓间充质干细胞,将传代后的细胞用IMDM细胞冻存液(含10%的二甲基亚砜和体积分数40%的胎牛血清)保存在-196 ℃液氮中。检测短期(1个月)和长期(12个月)冻存复苏后间充质干细胞的活性和增殖能力;将冻存后多发性骨髓瘤患者骨髓间充质干细胞作为滋养层,应用甲基纤维素半固体培养,检测其支持造血的能力;混合淋巴细胞反应检测冻存后多发性骨髓瘤患者骨髓间充质干细胞调控免疫能力。 结果与结论：经过短、长期冻存后多发性骨髓瘤患者骨髓间充质干细胞的细胞活性分别为(92.9±7.5)%和(86.7±9.2)%;短、长期冻存后细胞的增殖能力与冻存前间充质干细胞相似;冻存后多发性骨髓瘤患者骨髓间充质干细胞仍具有支持造血祖细胞生长的作用和抑制T淋巴细胞增殖的能力,与冻存前相比,没有明显差别。说明冻存可以降低多发性骨髓瘤患者骨髓间充质干细胞的细胞活性,但是并不影响间充质干细胞的增殖、支持造血和免疫调节的能力。     

Identification and reduction of cryoinjury in endothelial cells: a first step toward establishing a cell bank for vascular tissue engineering

We analyzed a cryopreservation protocol which improves long-term storage of endothelial cells (EC) for tissue engineering purposes. Human umbilical vein EC were frozen in a high-potassium solution containing 10% dimethyl sulfoxide using 3 different cooling rates. After a storage time in liquid nitrogen of 1, 4, or 12 months, samples were thawed and compared to fresh cells in terms of growth rates, anti-inflammatory, and anticoagulant functions. Independent of cooling rate and storage time, the retrieval after cryopreservation ranged between 60% and 80%. However, viability of the cells cryopreserved at 10 degrees C/min decreased significantly from 78 +/- 5% to 64 +/-3% with storage. Storage time of 4 months resulted in a decreased cell multiplication factor over 4 and 12 days in culture. The lag phases returned to normal in the next passage. Thawed cells showed increased metabolic activity, reduced expression of thrombomodulin, and unchanged basal expression of adhesion molecules. However, the tumor necrosis factor-induced expression of adhesion molecules was significantly increased after long-term storage. This effect was partially compensated after expansion of the cells, whereas the prostacyclin release increased. Expansion of cryopreserved/thawed EC resulted in highly proliferative cells with antithrombotic properties and a capacity for inflammatory reactions, which makes them suitable for vascular tissue engineering.     

SD大鼠骨髓间充质干细胞在-80℃低温条件下的冻存

BACKGROUND: We attempt to explore a low-cost, simple and effective way to cryopreserve bone marrow mesenchymal stem cells at -80 ℃. OBJECTIVE: To screen the optimal cryopreservation fluid for bone marrow mesenchymal stem cells and to verify the biological features of bone marrow mesenchymal stem cells after long-term cryopreservation. METHODS: Bone marrow mesenchymal stem cells were cultured using adherent method and the biological features and purity of cells were detected using immunofluorescence method. Bone marrow mesenchymal stem cells were cryopreserved in the cryoprotectant medium containing low-sugar DMEM, fetal bovine serum and dimethyl sulfoxide at different proportions at -80 ℃ for a short term. Then, the optimal cryoprotectant was selected to storage the bone marrow mesenchymal stem cells. After 1, 3, 6 months of cryopreservation, the cells were resuscitated, cultured and passaged. Passage cells were identified immunofluorescence method to determine the biological features of bone marrow mesenchymal stem cells cryopreserved at -80 ℃. RESULTS AND CONCLUSION: Cryoprotectant medium of 80% DMEM+10% fetal bovine serum+10% dimethyl sulfoxide was suitable for cryopreserving MSCs at -80 ℃, and resuscitated cells were able to proliferate in vitro, and passage normally, indicating the cryopreserved bone marrow mesenchymal stem cells still maintain the original biological activity.      

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.