无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

阿糖胞苷对大鼠海马神经元原代培养的影响

目的：探讨在大鼠海马神经元原代培养过程中,阿糖胞苷对培养神经元的影响。方法：将新生24 h大鼠,分离出海马组织,进行原代海马神经元培养,再将细胞分为阿糖胞苷组和对照组,阿糖胞苷组加入1μmol/L阿糖胞苷,通过检测神经元特异性标志物微管相关蛋白-2（Map-2）计算培养神经元的数量,通过台盼蓝染色法观察细胞的存活率。结果：培养第7天,阿糖胞苷组神经元数量为（11±3）个,对照组为（10±4）个,两组无明显差异;阿糖胞苷组神经元细胞在培养第14天时存活率为74%,培养第21天时存活率为49%,而对照组神经元14天时存活率为96%,21天存活率为88%,两组神经元存活率差异明显。结论：原代培养海马神经元时,阿糖胞苷对神经元产量及形态影响不明显,但是由于阿糖胞苷的毒性作用,明显缩短神经元的存活时间,影响长期培养神经元的存活率。     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

新生大鼠脊髓神经干细胞的分离培养及鉴定

目的　从新生大鼠的脊髓中分离培养神经干细胞并观察其增殖和分化能力。方法　采用细胞培养技术结合间接免疫荧光细胞化学法。结果　分离的细胞生长旺盛 ,单克隆化生成的细胞团 ,BrdU掺入呈强阳性。分离培养获得的细胞团呈Nestin强阳性 ,至今已在体外连续传代 8个月。培养的细胞团经 1%小牛血清诱导可分化为神经元和星形胶质细胞。结论　成功分离培养了新生大鼠脊髓神经干细胞     

成人宫颈上皮细胞的原代培养及鉴定

目的:探索采用无血清培养基原代培养成人宫颈上皮细胞的方法。方法:以成人的宫颈上皮组织为研究对象,采用胰蛋白酶-EDTA消化法获得宫颈上皮细胞悬液,于上皮细胞专用无血清培养基中培养,采用免疫细胞化学法测定细胞中角蛋白及波形蛋白的表达,对细胞纯度进行鉴定。结果:原代培养10-15天细胞融合达60%,传代至4-6代,细胞出现生长衰退。早期细胞生长状态良好,细胞纯度在90%以上。结论:采用酶消化法及K-SFM无血清培养基培养可获得纯度高的成人宫颈上皮细胞。     

马桑内酯对在体和离体小胶质细胞 CD11b/c表达的影响

目的研究致痫剂马桑内酯(CL)对在体和离体小胶质细胞CD11b/c表达的影响。方法①正常SD大鼠行马桑内酯侧脑室注射,观察大鼠的行为改变;利用免疫荧光染色的方法观察大鼠大脑皮质、海马内CD11b/c表达的变化。②纯化培养的小胶质细胞无血清培养,给予马桑内酯(5×10-5mol/L)刺激,利用免疫荧光染色结合流式细胞仪检测CD11b/c的表达。结果①马桑内酯侧脑室注射30min后均出现强烈的癫痫样发作,持续约4h;②马桑内酯侧脑室注射后大脑皮质及海马各区CD11b/c阳性细胞表达均出现明显增强,4-6h为表达高峰,至24h大脑皮质恢复至正常水平,但海马各区仍保持较高水平。③纯化培养的小胶质细胞在马桑内酯作用1h出现CD11b/c表达增强,2h达到高峰,至24h恢复正常。结论马桑内酯对小胶质细胞具有直接的活化作用;小胶质细胞的活化参与了马桑内酯的致痫过程。     

大鼠耳蜗雪旺细胞的体外培养和纯化

目的:探讨利用免疫磁珠从新生SD大鼠耳蜗螺旋神经节分离培养获得大量、高纯度雪旺细胞的方法。方法:选用1-3d SD大鼠,无菌条件下暴露双侧听泡,在高倍镜下仔细剥离蜗壳,开放耳蜗,完整取出耳蜗组织,分离并且除去膜蜗管外侧壁的血管纹和基底膜组织,然后剪碎。用0.25%的胰蛋白酶消化,用胎牛血清中止消化,离心以后加入DMEM/F12培养液培养。3-5天后对细胞应用免疫磁珠阳性分选方法进行纯化,培养2天后进行传代接种,培养过程中对提纯后的大鼠耳蜗雪旺细胞进行形态学观察、并绘制其生长曲线,采用细胞免疫荧光染色对细胞进行S-100免疫荧光鉴定并且计算细胞纯度。结果:分离培养后所得的细胞即为雪旺细胞;利用免疫磁珠阳性分选法对培养所得的细胞进行纯化,纯化后的大鼠耳蜗雪旺细胞纯度为97%±1.2%。结论:免疫磁珠法是一种有效的分离纯化新生大鼠仔鼠耳蜗螺旋神经节雪旺细胞的方法。所得耳蜗雪旺细胞活力强、纯度高,可以用于耳蜗雪旺细胞与螺旋神经节轴突的生长和再生等相关研究。     

小鼠Ⅱ型肺泡上皮细胞类器官三维培养体系的建立

本研究旨在建立小鼠Ⅱ型肺泡上皮细胞(type 2 alveolar epithelial cells, AT2)类器官三维(three-dimensional, 3D)培养体系的方法。采用酶消化与磁珠分选分离和纯化ICR小鼠肺AT2细胞,proSPC免疫荧光染色鉴定AT2细胞纯度;2维(two-dimensional, 2D)培养8 d,5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine, EdU)掺入和荧光染色法观察AT2细胞的增殖与分化;将AT2细胞与小鼠肺成纤维细胞(mouse lung fibroblasts, Mlg) 3D共培养,光学显微镜下观察类器官生长情况,收集生长13d的类器官,2%多聚甲醛固定后行HE染色,荧光染色观察类器官内部形态结构。结果显示,提取AT2细胞纯度超过95%;在体外培养1～8 d期间,AT2细胞EdU荧光染色阴性,未见增殖;AT2细胞形状逐渐趋向扁平鳞状、细胞表面积显著增大;在体外培养3 d后,有部分AT2细胞特异性标志物proSPC阳性细胞开始出现I型肺泡上皮细胞(type 1 alveolar epithelia...     

嗅神经鞘细胞的培养纯化及体外生长特性

采用原代培养的方法,从2,5月成年大鼠的嗅球分离培养嗅神经鞘细胞（OECs),培养6天后,用阿糖胞苷（Ara-C)抑制,差速贴壁,Forskolin和BPE营养物质处理,根据P75蛋白免疫细胞化学染色和形态学特征分析了所得细胞的纯度,同时对不同培养时期的OECs 的形态进行观察和纯化后的活力测定。实验结果显示：（1）这种纯化方法简单,经济,快捷,所得的OECS纯度可达95％以上,并且随培养时间延长,细胞仍保持较高的纯度。（2）在培养早期2天到5天主要以巨噬细胞状,多极状,不规则状为主,培养中期7天到20天主要以扁平的双极,三极为主。晚期20天以后呈现双极,三极形态,其起上有许多细小的棘突。93）其中以培养早中期细胞的活力较好,培养20天以后,细胞活力较差,本研究为以OECs 作为移植材料对促进神经再生的研究获得丰富的细胞来源奠定了基础。     

On-line purification of monoclonal antibodies using an integrated stirred-tank reactor/expanded-bed adsorption system

While expanded-bed adsorption (EBA) units have been used to recover proteins from whole cell cultures, the development of a more efficient, on-line process could streamline the traditional multistep process. This study implements a bench-scale on-line purification system in which whole cell cultures are loaded directly into a chromatography column to capture a monoclonal antibody (mAb) in a single step. The on-line purification system used here integrates a stirred-tank reactor (STR) and an EBA unit into a new hybrid (STR-EBA) system. To conduct this work, first, column and buffer conditions were optimized to capture immunoglobulin G from a hybridoma cell culture. A high cell removal (>95%) was achieved in part by removing the top flow distributor and mesh. Then, the 95% extent of removal was sustained for four successive cycles, each using PBS. With 20 mM phosphate buffer, however, the removal decreased from 95% to 75% stepwise. Next, the operational constraints of the EBA system were determined for the hybridoma cell culture, focusing on the effects of cell viability and density on cell removal. This study shows that the cell removal was not significantly different in the range of 80% to 0% viability. Cell density was also varied between 1 x 10(6) and 1 x 10(8) cells/mL. From 0.1 to 6 x 10(7) cells/mL, cell retention in the column was less than 5% and product recovery remained high, approximately 95%. After characterizing the working conditions of the EBA system, on-line purification was performed. With 1.1 L of culture containing 3 x 10(6) cells/mL and 100 mg/L of IgG, repeated-batch cultures were implemented. Half of the culture volume (550 mL) was directly sent to the EBA system every day, for 11 days, and the same amount of fresh medium was fed into the STR. During on-line purification, productivity was 58 mg of IgG/cycle (day) and purity was greater than 95%. Simple batch culture alone produced 17 mg of IgG/day. This result suggests that the on-line STR-EBA system can achieve higher and faster production compared with STR batch and off-line EBA purification. Overall, the STR-EBA system with repeated-batch mode was an effective and flexible system for bench-scale mAb production.     

Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression

Rationale

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.

Method and Result

We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30–70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7–8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95–98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5−10×10⁵ VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.

Conclusion

We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

Superfused pituitary cell cultures: effects of culture conditions on apparent responsiveness to LHRH stimulation administered as short duration pulses

We wished to study estrous cycle related differences in LH and FSH responsiveness to pulsatile LHRH. Such studies are very difficult to perform in vivo under controlled conditions; therefore, an in vitro superfused anterior pituitary cell culture system was evaluated for its capacity to support differences in estrous stage associated LHRH responsiveness. Three vital culture system parameters were evaluated; these parameters were (1) culture medium composition, (2) duration allowed for cell attachment to microcarrier beads and (3) superfusion flow rate utilized during pulsatile LHRH stimulation. It was found that a culture system which utilized 10% Nu Serum in DMEM (final protein concentration of 1.8 mg/ml; final serum concentration of 2.5%), an attachment time of 48 hrs and a flow rate of 0.125 ml/min most successfully maximized LH responsiveness at the lowest serum concentration. These studies indicated that although one may be able to observe LHRH responsiveness under a wide range of culture conditions, responsiveness may nonetheless be maximized by judicious adjustment of culture conditions.     

MC3T3-E1前成骨细胞培养时间对矿化结节表达的影响

目的:观察MC3T3-E1前成骨细胞不同培养时间点矿化结节的形态,探讨一个既节省实验时间与经费,又便于观察矿化结节形态差异的实验方法。方法:将MC3T3-E1前成骨细胞按培养时间分为四组(14、21、28、35天组),各组实验结束时行茜素红染色,光学显微镜下观察矿化结节的形态变化。结果:各组均见红色的矿化结节形成,随培养时间延长,染色面积增大,密度增高,14天时结节轮廓清晰,结节间距较大,21天时结节面积增大,28天时结节边界超出视野,35天时视野内大片深染,结节轮廓不清。结论:在本实验周期内,MC3T3-E1前成骨细胞培养14至21天通过茜素红染色可以较清晰地观察矿化结节,其中培养14天时即可观察到结节大小、数量及形态,考虑到实验时间及经费的因素,我们认为MC3T3-E1前成骨细胞培养14天后行茜素红染色是观察不同因素对其矿化产生影响的适宜时间点。     

新生BALB/c小鼠大脑皮质神经元细胞培养方法的建立

目的:经改良和优化,建立高纯度BALB/c小鼠大脑皮质神经元培养的方法.方法:采用L-多聚赖氨酸包被细胞培养板,取新生BALB/c小鼠(出生24 h内)大脑皮质组织,经0.25%胰酶消化后吹打成单个细胞,按1×106/孔接种于35 mm的六孔板中,用神经元细胞培养种植液培养6 h后换神经元细胞培养饲养液,培养40 h时...     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Optimal time for passaging neurospheres based on primary neural stem cell cultures

Cultured neural stem cells (NSCs) provide a powerful means for investigating central nervous system disease, neuron development, differentiation, and regeneration. To obtain sufficient neurospheres, subculturing is essential following establishment of the primary NSC culture. Passaging the primary neurospheres is a key issue that is often ignored. We evaluated the influence of different passaging schedules on primary cultured NSCs. Passaging was performed on day 5, 7 or 9. We observed more neurospheres with diameters of 200–250 μm on day 7 than on day 5 or 9. Prolonging the time of primary culture reduced the cell metabolic activity by the MTT assay and cell proliferation by colony-forming assay and the differentiation to neurons from cells at P2 and later decreased. Additionally, more cells were in G0/G1 phase, and higher expression of p16 ^INK4a and lower expression of cyclin D1 was found when the time of primary culture was prolonged to 9 days compared to 7-days cultures. Thus, in this study, we established that the optimal time for subculturing aggregated NSCs was on day 7 based on the primary culture.     

Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification.

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.