成纤维细胞饲养层饲养人角朊细胞的机制研究

目的 探讨人成纤维细胞(HFB)是否可作为饲养层支持人角朊细胞(KC)的生长.方法 分离培养人真皮FB,用丝裂霉素C处理制成饲养层,应用RT-PCR、免疫细胞化学染色方法检测Ⅰ型、Ⅲ型前胶原mRNA和胶原蛋白的表达;在FB饲养层上接种人KC,观察其促进KC贴壁、生长、移行及分化的作用;并设置NIH3T3细胞饲养层为对照组,在其上接种人KC,观察KC贴壁数和膜片完全融合时间.结果 FB饲养层Ⅰ型前胶原mRNA的表达有所增加(P＜0.05)、Ⅲ型前胶原mRNA的表达无明显变化,抗Ⅰ型、Ⅲ型胶原蛋白抗体阳性;以适宜密度接种的成纤维细胞饲养层,KC贴壁、生长和分化良好;二种饲养层KC贴壁数和膜片完全融合时间无差异.结论 人真皮FB饲养层能够分泌Ⅰ型和Ⅲ型胶原,促进人KC生长并形成复层细胞.     

以成纤维细胞作为饲养层供角朊细胞生长

体外角朊细胞培养已有多年的历史，随着人们对角朊细胞(Keratinocyte，KC)生长状况、结构功能及生物学特性的不断了解，培养技术日趋完善。比较成熟的是以NIH3T3细胞作为饲养层，有血清培养法和无血清培养法，前者可以培养出整张细胞膜片用于临床；后者用于对KC特性的实验研究，但3T3细胞作为异种细胞，存在伦理学问题及潜在的危害，因此本研究用成纤维细胞作为饲养层培养KC，旨在观察其促贴壁、生长和分化作用，为临床提供理论依据。     

人胚胎干细胞在血清和无血清培养体系中的特性比较

目的 探讨血清培养体系和无血清培养体系对人胚胎干细胞（hES cells）生长特性的影响。 方法 将人胚胎干细胞株BG02接种在丝裂霉素C处理灭活的小鼠胚胎成纤维细胞饲养层上,分别在含血清hES完全培养基或无血清培养基中连续培养25~30代。在不同培养体系下比较人胚胎干细胞形态、集落贴壁率;运用BrdU掺入法检测人胚胎干细胞增殖,用细胞计数法计算细胞倍增时间;采用免疫荧光染色检测人胚胎干细胞特异性分子标志的表达;用流式细胞仪检测人胚胎干细胞Oct-4,Nanog阳性的比例;RT-PCR检测成纤维细胞生长因子(FGFs)家族基因的表达。 结果 在两种培养体系的BG02细胞都具有人胚胎干细胞的形态特征。在含血清培养体系下,BG02细胞集落贴壁率和表达OCT-4,Nanog的阳性细胞明显高于无血清培养体系( P<0.05)。无血清培养体系中,BG02细胞生长速度明显高于含血清培养体系,细胞倍增时间分别为(33.8±4.3) h、(45.9±5.7) h,( P<0.05)。无血清培养体系的BG02细胞高表达 FGF2 、 FGFR2 、 FGFR4 。 结论 两种不同培养体系中人胚胎干细胞的体外培养特性存在一定的差异,可能与BG02细胞 FGFs 家族基因激活有关。     

小鼠胚胎干细胞饲养层培养体系的优化筛选

目的：建立小鼠胚胎干细胞饲养层培养体系。方法：用5种不同鼠胚成纤维细胞为饲养层，进行小鼠胚胎干细胞的分离培养，观察5种饲养层培养体系对小鼠胚泡发育，内细胞团增殖及胚胎干细胞分离培养的作用。结果：原代或冻存复苏后的原代培养鼠胚成纤维细胞用于制备饲养层，有利于胚泡的贴壁，孵化，内细胞团增殖形成巢式生长集落，离散后培养，可以观察到胚胎干细胞集落的出现，并可在短期内维持胚胎干细胞的正常形态，不发生分化，与其他三组有明显差异。结论：原代或冻存复苏后的原代培养鼠胚成纤维细胞饲养层是用于胚胎干细胞分离培养的有效的培养体系。     

用无血清培基培养杂交瘤细胞的初步报告

在杂交瘤细胞的研究中,培养基中的血清常会干扰单克隆抗体(McAb)的测定,由于血清所含成份复杂,动物个体差异较大,故使用前还须进行严格的筛选。血清还经常是污染的来源。为避免这些缺点,很多学者致力于研究用无血清培养基(serum free medium SFM)取代含血清的完全培养基(complete medium CM)进行细胞特别是杂交瘤细胞的培养。     

人气管上皮细胞气液界面无血清培养

用低温酶消化法分离人气管上皮细胞，具有细胞损伤小，活力及纯度高的优点，成纤维细胞污染低。人胎盘胶原提高了气管上皮细胞的贴壁性。无血清培养基能促进人气管上皮细胞增殖，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，细胞在膜上呈复层生长，有利于其分化成熟及功能表达。光镜下细胞形态及免疫组化角蛋白染色阳性证实培养细胞为气管上皮细胞。本文所建立的人气管上皮细胞体外气液界面无血清培养方法     

乳鼠角朊细胞分离表达共刺激分子CD80/CD86

以往研究认为角朊细胞不表达共刺激分子CD80／CD86，无法提供淋巴细胞增殖过程中关键的共刺激信号，但培养的角朊细胞膜片移植后，最终供者细胞被完全排斥，具体机理不明。本实验使用近交系小鼠，有和无血清两种体系培养角朊细胞1、4、7d，采用流式细胞技术、激光共聚焦技术，RT-PCR方法检测角朊细胞在培养过程中MHCⅠ类、Ⅱ类抗原，及CD80、CD86的表达。证实培养的细胞中不含郎格罕氏细胞，首次发现乳鼠角朊细胞在培养过程中表达CD80，但不表达CD86，可刺激异基因淋巴细胞增殖，执行抗原递呈细胞功能。本研究结果为角朊细胞膜片移植排斥提出另一种可能的解释：MHCⅠ／Ⅱ类分子和CD80的表达，使培养的异体角朊细胞兼具外来抗原和抗原递呈细胞的功能，刺激受体淋巴细胞增殖，参与排斥反应。     

梯度血清递减体外培养人脐带间充质干细胞

背景：人脐带间充质干细胞的扩增与培养条件密切相关,而含体积分数10%~20%胎牛血清的培养基可促进细胞生长。 目的：建立梯度血清递减扩增培养脐带间充质干细胞的培养技术。 方法：采用胶原酶Ⅱ消化分离获得脐带间充质干细胞悬液,并通过贴壁培养进行纯化,细胞贴壁后采用梯度血清递减方法,即第1代80%含血清培养基,20%无血清培养基;第2代50%含血清培养基,50%无血清培养基;第3代20%含血清培养基,80%无血清培养基;第4代100%无血清培养基。另一种传代中始终采用含体积分数10%胎牛血清的α-MEM完全培养液。用流式细胞仪检测细胞的表面标记,进行成骨诱导试验,同时与经典的含10%胎牛血清的α-MEM培养体系进行比较。 结果与结论：梯度血清递减培养法与经典α-MEM培养体系所获得的细胞在扩增能力、细胞形态、免疫表型等方面相似。细胞在两种培养体系中均能保持良好的分化潜能。但梯度血清浓度法培养间充质干细胞可使用更少量胎牛血清,提高临床应用安全性。     

视网膜祖细胞分离培养及鉴定

目的：探讨体外胚胎视网膜祖细胞的增殖与分化特性。方法：运用无血清培养技术，从培养第2～3代后的神经干细胞，按Lipofeefin?Reagent转染试剂盒的步骤，将CDK5-EGFP重组子和pEGFP-N1空载体分别转染进神经干细胞集落内，24h后加入5％胎牛血清，继续培养。于24h、48h、72h分别观察两组分化神经细胞的形态特征并计算细胞的突起长度。     

无血清培养促进脂肪干细胞向血管内皮细胞分化

背景：无血清培养对大鼠脂肪干细胞向血管内皮细胞诱导分化影响的报道甚少。 目的：观察无血清培养大鼠脂肪干细胞后向血管内皮细胞诱导分化的情况。 方法：采用酶消贴壁培养法获得雄性SD大鼠脂肪干细胞,传代培养至第3代。实验组细胞无血清培养24 h,对照组用含体积分数10%胎牛血清的L-DMEM完全培养基培养。然后应用血管内皮细胞诱导培养基培养3周。 结果与结论：大鼠脂肪干细胞经体外培养可呈多角形或梭形贴壁生长,并能稳定传代。传代后大鼠脂肪干细胞极低表达细胞表面标志CD31。大鼠脂肪干细胞定向血管内皮细胞诱导分化后细胞呈鹅卵石样,CD31阳性表达明显上升且实验组明显高于对照组。实验组诱导后大鼠脂肪干细胞能够吞噬Dil标记的乙酰化低密度脂蛋白及在基质胶上形成2D小管样结构,其能力明显强于对照组。结果证实无血清培养可促进体外大鼠脂肪干细胞向血管内皮细胞诱导分化。     

Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity

For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.     

A defined serum-free medium useful for monitoring anti-melanoma responses induced by dendritic cell immunotherapy

Animal sera provide a non-defined source of nutrients and growth factors for mammalian cell culture. Animal serum supplementation may also introduce experimental artefacts, including immune responses against foreign serum proteins. This artefact is particularly apparent in tumour immunotherapy experiments using dendritic cells (DC) and melanoma cells cultured in fetal calf serum (FCS)-replete media. FCS culture of both DC and melanoma cells significantly enhanced anti-tumour responses in mice immunized with DC that had not been pulsed with tumour antigen. Although serum-free media (SFM) may be used for short term culture of cells, most SFM do not support long term culture of tumour cell lines. In addition, in vivo propagation and re-isolation of tumour cells from rodents is expensive, time consuming and only low numbers of viable tumour cells can be recovered from solid tumours. We show that a defined SFM medium is ideal for routine culture of B16 for use in prophylactic DC immunizations, negating the need for in vivo propagation of tumours to avoid FCS effects in tumour implantation experiments.     

体外诱导人类B细胞分泌HLA抗体的实验研究

目的:探索体外诱导人类B细胞产生特异性HLA抗体的理想培养条件。方法:以EBV转化致敏肾移植患者B淋巴细胞系(BLCL)为模型,采用ELISA和ELISPOT方法,比较3种培养体系:(1)RPMI1640 抗OPTI单克隆抗体(mAb);(2)RPMI1640 0·1mmol/L非必需氨基酸液 1mmol/L丙酮酸钠 40mg/L转铁蛋白;(3)Hybridoma无血清培养液,以及3种培养条件:(1)对照组(仅培养液),(2)培养液 rIL-4 rIL-10 鼠抗人CD40(α-CD40),(3)培养液 rIL-4 rIL-10 重组人CD40配体(CD40L),人类B细胞产生免疫球蛋白(Ig)和特异性抗HLA抗体的能力。结果:BLCL细胞在Hybridoma SFM培养体系所产生的IgG和IgM浓度分别为25·2～28·1mg/L和7·14～7·95mg/L,高于其他两种培养条件(P<0·05)。BLCL细胞在添加了IL-4、IL-10和α-CD40或CD40L的杂交瘤无血清培养液中能产生特异性抗HLA-I抗体,所获得的斑点频数分别为3·06/每104个BLCL细胞和3·55/每104个BLCL细胞,高于对照组(1·77/每104个BLCL细胞)。结论:BLCL细胞在杂交瘤无血清培养液中和添加了IL-4、IL-10和α-CD40或CD40L的培养条件下,提高分泌IgG和IgM的能力和特异性抗HLA-I类分子的抗体。     

Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute

Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal differentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air-liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson's Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air-liquid interface showed lower frequencies of the CH(2) symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study.     

PI-3K在低氧大鼠肺动脉平滑肌细胞增殖中的表达

目的:研究低氧肺动脉平滑肌细胞(PASMC)增殖时磷脂酰肌醇3 激酶(PI-3K)的表达。方法:实验分为无血清培养基(SFM)组和低氧48 h(H48 h)组, 应用免疫组织化学法和流式细胞仪检测低氧PASMC。结果:SFM组和H48 h组PASMC细胞浆内均有PI-3K p110阳性表达, H48 h组(0.1891±0.0301)的表达明显高于SFM组(0.1025±0.0164, P＜0.05);PCNA在SFM组的少数PASMC细胞核内有阳性表达, H48h组(24.58±4.07)%的增殖指数(PI)明显高于SFM组(8.76±1.21)%(P＜0.05);流式细胞仪检测发现PASMC的S+G₂／M期细胞所占细胞总数的百分比在H48 h(27.15±5.43)%显著高于SFM组(10.64±1.52)%(P＜0.05)。结论:低氧可显著上调PI3K的表达, 提示PI-3K可能在低氧PASMC增殖中起重要作用。     

CHO细胞无血清培养研究

目的:用无血清培养基培养中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞。方法:应用无血清培养基采用小方瓶培养CHO细胞,观察细胞维持时间、培养过程的形态变化。结果:应用无血清培养基培养CHO细胞可维持细胞正常生长。结论:无血清培养基可以完全取代含血清培养基用于培养CHO细胞。     

Immunolocalization of protein C inhibitor in differentiation of human epidermal keratinocytes

Keratinocytes propagated in low calcium (0.05 mM) serum-free medium grow as monolayers and exhibit morphological and biosynthetic phenotypes similar to the keratinocytes of the basal layer in normal epidermis. When the calcium in the medium is increased to 1.5 mM, the keratinocytes start to stratify and differentiate. Such differentiation is important in the formation of an epidermal barrier. Proteolysis plays a crucial role in the process. The functions of most of the plasminogen activator cascade components in human skin have been studied, but little was known about the expression and role of protein C inhibitor in the differentiation of human epidermal keratinocytes. In the present study, we used immunohistochemistry and immunocytochemistry to examine the immunolocalization of protein C inhibitor in normal human skin and in cultured keratinocytes in serum-free medium with low and high calcium, respectively. The results indicated that protein C inhibitor is mainly localized in superficial and more differentiated keratinocytes in normal human epidermis. Keratinocytes positive for protein C inhibitor were detected in cultures containing both low and high calcium media, and the level of protein C inhibitor was increased in high calcium medium. This increase was accompanied by an altered intracellular distribution, from the perinuclear cytoplasm in undifferentiated keratinocytes to the whole cytoplasm in differentiated keratinocytes. Further study revealed that protein C inhibitor was incorporated into the cornified envelope in normal skin keratinocytes and cultured differentiated keratinocytes. Our results suggest that protein C inhibitor may be involved in the differentiation of keratinocytes.     

Efficient laboratory-scale production of monoclonal antibodies using membrane-based high-density cell culture technology

Monoclonal antibodies (MAbs) are important tools used in basic research as well as in the imaging and therapy of cancer. Many countries have limited the use of animals for large-scale production of MAbs, obliging laboratories to find efficient in vitro alternatives to ascites production. In this report we describe a protocol for laboratory-scale production of MAbs by culturing hybridoma cells in the two-chamber cell culture device CELLine 1000. This culture flask supports high cell densities (10⁷–10⁸ cells/ml) and generates high concentrations of MAbs (0.7–2.5 mg/ml). Two hybridomas producing MAbs directed against the gastrointestinal antigen GA733-2, GA733 MAb and CO17-1A MAb, were evaluated over culture periods of up to two months using several alternative conditions. Two different sets of conditions are reported; the first using serum-supplemented medium (20% v/v) and the second using serum-free medium (SFM). Average weekly yields of the purified MAbs in serum-supplemented medium were 24 mg and 33 mg, and in SFM were 21 mg and 17 mg for GA733 MAb and CO17-1A MAb, respectively. Experimental variables that can affect antibody production and economy include: nutrient medium and cell compartment medium compositions (cell line dependent), the proportion of the cell compartment medium harvested every 3 days (50% to 80% with 80% optimal) and the frequency of nutrient medium changes (3 to 9 days with 6 days as most cost effective). Protein-A Sepharose purification followed by antigen-specific affinity purification showed that MAbs obtained from serum-supplemented cultures contain less than 0.6% of bovine IgG contamination, while MAbs obtained from serum-free cultures contained no extraneous IgG. In addition, MAbs from both culture media were fully active (essentially 100%) as measured by their ability to bind to an antigen column. In contrast, the same MAbs purified from ascites using Protein-A-Sepharose typically contained a major portion of inactive IgG. This in vitro method for laboratory-scale production of MAbs (10 to 500 mg) proved to be simple, reproducible and cost effective. It represents a useful alternative to the in vivo production of MAbs in mice.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.