利用气液界面无血清培养原代兔气管上皮细胞的研究

用低温酶消化法分离兔气管上皮细胞,具有细胞损伤小,活力及纯度高的优点,成纤维细胞污染极低。人胎盘胶原提高了气管上皮细胞贴壁性。无血清培养基能促进细胞增殖,分化和成熟。气液界面培养方式更好地模拟了气管上皮细胞的天然生长环境,在膜上呈复层生长,有利于细胞的分化成熟及功能表达。光镜下细胞形态及免疫组化细胞角蛋白染色阳性证实培养细胞为气管上皮细胞。本文所建立的兔气管上皮细胞体外气液界面无血清培养方法为研究气 管上皮细胞的生理和病理提供了一个十分有用的模型。     

不同冻存条件对甘蔗原生质体活力和再生能力的影响

为了获得活力高和再生能力强的甘蔗原生质体,该文对甘蔗原生质体的冻存液浓度、冻存温度和冻存部位进行了研究。结果表明:(1)不同的冻存液、不同的冻存温度和不同的取材部位原生质体冻存后复苏对甘蔗原生质体的活力影响有显著差异性,三个冻存液组合比较,在组合2(70%培养基+20%血清+10%DMSO),冻存30 d后复苏活力最强,高达72%;冻存90 d内复苏,-196℃液氮和-80℃冰箱冻存,甘蔗原生质体的活力差异不显著,活力均在75%以上,但90 d冻存后复苏,-196℃液氮冻存后复苏比-80℃冰箱冻存冻存后复苏原生质活力强;不同取材部位比较,幼叶冻存30 d后复苏所得原生质体活力较高(达79. 2%),茎尖冻存30 d后复苏所得原生质体活力仅为42.7%。(2)不同的冻存液和不同的冻存温度,细胞第一次启动分裂和形成细胞团的时间差异不显著,一般培养5～6 d,细胞壁基本形成完整,培养6 d后,细胞启动分裂,培养15 d后形成细胞团。不同的材料部位相比较,茎尖酶解所得原生质体再生能力最强,较幼叶酶解原生质体,形成细胞壁的时间早3 d,第一次分裂时间早2 d。     

一种无血清冻存液在猪胎儿成纤维细胞上的应用研究

在猪胎儿成纤维细胞(porcine fetal fibroblasts, PFF)冻存过程中,血清品质常常制约着细胞的冻存效果。为了解决这个问题,本研究旨在开发一种无血清冻存液应用于猪胎儿成纤维细胞冻存。用3种不同冻存液冻存猪胎儿成纤维细胞,每种冻存10管。冻存30 d后复苏细胞,测定冻存细胞存活率,细胞增殖活力以及电转后细胞活性。结果显示:自制无血清细胞冻存液,冻存猪胎儿成纤维细胞后存活率达95.33%;细胞增殖活力以及电转后细胞活性均显著高于标准胎牛血清冻存液(p<0.05),与特级胎牛血清冻存液效果相当(p>0.05)。因此,自制冻存液冻存猪胎儿成纤维细胞效果稳定,能够替代含血清冻存液,有良好的推广应用前景。     

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。     

软骨细胞上清液诱导冻存复苏后人骨髓间充质干细胞的研究

目的:探讨人软骨细胞培养上清诱导冻存人骨髓充质干细胞向软骨细胞分化的可行性.方法:取进行全髋关节置换术老年患者的骨髓和软骨组织,利用密度梯度离心法、全骨髓培养法分别培养骨髓间充质干细胞,冻存备用.培养软骨细胞,观察细胞生长,收集软骨细胞培养上清.复苏冻存的人骨髓间充质干细胞,观察复苏后细胞生长状态.利用收集的软骨细胞培养上清对复苏间充质干细胞进行定向诱导,诱导培养2周,观察细胞外观表型变化,Ⅱ型胶原免疫组化检测诱导后人骨髓间充质干细胞Ⅱ型胶原的表达.结果:密度梯度离心法与全骨髓培养法均可分离获得人骨髓间充质干细胞,原代生长前者优于后者.复苏细胞仍进行可传代,与正常生长骨髓间充质细胞无明显差异,均可传至第8代.软骨细胞培养上清诱导2周后,细胞形状向圆形,多角形转变,冻存骨髓间充质干细胞Ⅱ型胶原免疫组化检测Ⅱ型胶原表达阳性.结论:老年人骨髓间充质干细胞仍具有向软骨细胞转化的能力,冻存不影响其转化能力.     

大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

人正常食管鳞状上皮不同原代培养方法的比较

该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实...     

胶原酶Ⅰ消化法体外分离培养奶牛乳腺上皮细胞

为获得纯净的奶牛乳腺上皮细胞,该研究采用胶原酶I消化法,在不添加外源激素及生长因子的条件下培养奶牛乳腺上皮细胞。采用差时消化与差速贴壁方法,对奶牛乳腺上皮细胞进行纯化。采用Western blot方法检测细胞中β-酪蛋白(β-casein,CNS2)的水平。通过免疫荧光技术对细胞进行鉴定。结果显示,纯化后的细胞呈现典型的"铺路石"或"鹅卵石"样。细胞角蛋白-18(cytokeratin-18,CK-18)反应呈阳性,波形蛋白反应呈阴性。细胞传至15代冻存并复苏后生长状态依然良好。这提示,胶原酶I消化法在不添加外源激素及生长因子的条件下可以获得奶牛乳腺上皮细胞,以用于后续实验。     

深低温冻存组织工程化软骨在喉狭窄功能重建中的应用

目的:探讨低温保存组织工程化软骨在喉狭窄功能重建中的应用价值。方法:取3周龄新西兰兔关节软骨细胞,体外培养,取第2代对数生长期培养细胞,制成细胞悬液,调整软骨细胞悬液浓度约为5×107个/ml左右,接种于PGA三维支架材料上,复合物体外培养2周后冻存,冻存6个月后解冻复苏,再行体外培养观察,2周后接种于已建立的喉甲状软骨缺损模型的软骨缺损处,并设对照组。术后12周取材,行大体及组织学观察。结果:经低温冻存的组织工程化软骨生长良好,组织学观察有软骨形成,与周围软骨组织结合紧密,与非冻存组相比差异无统计学意义。结论:深低温冻存对组织工程化软骨的生物活性无明显的影响,低温冻存的组织工程化软骨可用于喉软骨缺损的修复,重建喉功能。     

深低温冻存组织工程化软骨在喉狭窄功能重建中的应用

目的：探讨低温保存组织工程化软骨在喉狭窄功能重建中的应用价值。方法：取3周龄新西兰兔关节软骨细胞,体外培养,取第2代对数生长期培养细胞,制成细胞悬液,调整软骨细胞悬液浓度约为5×10^7个／ml左右,接种于PGA三维支架材料上,复合物体外培养2周后冻存,冻存6个月后解冻复苏,再行体外培养观察,2周后接种于已建立的喉甲状软骨缺损模型的软骨缺损处,并设对照组。术后12周取材,行大体及组织学观察。结果：经低温冻存的组织工程化软骨生长良好,组织学观察有软骨形成,与周围软骨组织结合紧密,与非冻存组相比差异无统计学意义。结论：深低温冻存对组织工程化软骨的生物活性无明显的影响,低温冻存的组织工程化软骨可用于喉软骨缺损的修复,重建喉功能。     

The influence of culture media on embryonic renal collecting duct cell differentiation

Summary During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove’s modified Dulbecco’s medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams’ medium E, McCoys 5A medium, and Dulbecco’s modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P_CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.     

培养和冷冻对新生大鼠胰岛活性的影响

新生大鼠胰碎片在MEM、DMEM、M-199和RPMI-1640中培养,比较培养不同天数冷冻保存,不同条件处理及液氮贮存不同时间的实验结果。表明新生大鼠胰碎片在RPMI-1640中培养胰岛素分泌量最高,随着培养时间的延长,胰岛素分泌量逐渐升高。培养6d进行冷冻保存为宜。新生大鼠胰碎片在液氮中贮存12个月分泌功能无明显变化。     

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.     

Large-scale, high-density freezing of hybridomas and its application to high-density culture

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x 10(8)/mL. The number of cells stored in a bag (3.0 x 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.     

Cultured gill epithelia from freshwater tilapia (Oreochromis niloticus): effect of cortisol and homologous serum supplements from stressed and unstressed fish

Procedures for the preparation and culture of branchial epithelia from dispersed gill cells of freshwater tilapia (Oreochromis niloticus) are described. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on both the apical and basolateral side with isotonic media containing 6% fetal bovine serum (FBS). When the apical medium was replaced with freshwater (pseudo in vivo asymmetrical culture conditions), transepithelial resistance (TER) increased markedly, transepithelial potential became negative, and paracellular permeability decreased. The physiological effects of cortisol and 10% homologous (tilapia) serum were investigated. Tilapia serum (TS) was prepared from unstressed and stressed fish and therefore allowed comparison between the effects of homologous serum derived from fish in differing physiological states. Under both symmetrical and asymmetrical culture conditions, cortisol significantly elevated TER across cultured tilapia gill epithelia, indicative of a significant increase in epithelial "tightness." Cortisol reduced transepithelial Na + and Cl? movement and paracellular permeability. The glucocorticoid agonist dexamethasone elicited a similar response, which was inhibited by the glucocorticoid antagonist (receptor blocker) RU486. Cortisol did not stimulate active ion transport across epithelia under either symmetrical or asymmetrical culture conditions. In epithelia supplemented with TS from stressed fish, physiological changes in cultured preparations were consistent with those observed in FBS + cortisol-supplemented epithelia. Differences between the physiological status of epithelia supplemented with TS from unstressed and stressed fish could be abolished with RU486. Using TS as a medium supplement did not stimulate active ion transport under asymmetrical culture conditions, although Na +-K +-ATPase activity increased in TS-supplemented epithelia relative to FBS-supplemented preparations.     

Organ and species specificity of epithelial growth

Summary Previous work in this laboratory demonstrated that rat respiratory airway epithelial cells grown from tissue explants undergo concurrent division and differentiation in culture. In the present studies, this model system has been used to optimize conditions for epithelial growth, with the goal of facilitating the culture of epithelium from other organs and species. Fibroblasts appeared to limit the expansion of epithelial outgrowths; their growth was controlled by using delipidated serum, rather than serum, in the culture media, with addition of adrenergic antagonists. In nonsupplemented media, rat tracheal fibroblasts doubled in number in about 3 d. The doubling time was slowed to more than 9 d by the serum substitution in conjunction with the α-antagonist phenoxybenzamine. Rat tracheal epithelial cultures maintained under identical conditions doubled in less than 4 d, so that a growth advantage of threefold was achieved. The combination of conditions also seemed to inhibit growth of fibroblasts from other species. However, even when the problem of fibroblast overgrowth was obviated, the respiratory airway epithelia of the mouse, hamster, and marmoset failed to show self-sustaining growth in culture. The rat epithelium continued to grow for over 6 wk. A number of other organs from the rat, notably esophagus, skin, and salivary gland, showed self-sustaining growth after removal of explants. Although epithelial outgrowths were formed by explants from certain organs of the hamster, only those from the kidney and thyroid continued to grow after removal of the tissue explants. Therefore, growth failure in cultured epithelia, quantitatively defined, is common and frequently unrelated to the problem of fibroblast overgrowth. The rat seems to be the species of choice for studies on the isolated epithelia from most organ sites. The research was sponsored jointly by Grant PCM 8110597 from the National Science Foundation, Washington, D.C., the Environmental Protection Agency under Interagency Agreement 81-D-X 0533, the National Institute of Health Requisition 375014 GPCL, and the Office of Energy Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. A preliminary report of this work appeared in the J. Cell Biol. 91: 32a, 1981.     

A simple freezing medium for serum-free cultured cells

Methylcellulose was found to protect serum-free cultured cells from the deleterious effects of freezing and thawing. We have formulated a simple medium suitable for freezing serum-free cultured cells that consists of 0.1% methylcellulose, 10% dimethylsulfoxide, and MEM or any other serum-free culture medium.     

Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: Challenges for fertility preservation

Cryopreservation of ovarian tissue and in vitro follicle maturation are two emerging techniques for fertility preservation, especially in cancer patients. These treatment regimes are opening up more options and allow for more suitable choices to preserve fertility according to the patient's specific circumstances. If these technologies are to become widely accepted, they need to be safe, easy to perform and must obtain favorable results. The generation of healthy eggs with the normal genetic complement and the ability to develop into viable and healthy embryos requires tight regulation of oocyte development and maturation. Novel freezing techniques such as vitrification, along with whole ovary cryopreservation and three-dimensional follicle cultures, have shown favorable outcomes. The scope of this article is to take a comprehensively look at the challenges still faced in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.