The application of SELDI-TOF mass spectrometry to mammalian cell culture

Surface Enhanced Laser Desorption/Ionisation Time-of-Fight Mass Spectrometry (SELDI-TOF MS) is a technique by which protein profiles can be rapidly produced from a wide variety of biological samples. By employing chromatographic surfaces combined with the specificity and reproducibility of mass spectrometry it has allowed for profiles from complex biological samples to be analysed. Profiling and biomarker identification have been employed widely throughout the biological sciences. To date, however, the benefits of SELDI-TOF MS have not been realised in the area of mammalian cell culture. The advantages in identifying markers for cell stresses, apoptosis and other culture parameters mean that these tools could help greatly to enhance monitoring and control of bioreaction process and improve the production of therapeutics. Better characterisation of culture systems through proteome analysis will allow for improved productivity and better yields.     

Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL⁻¹ 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These ‘nucleotide ratios’ changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.     

一种稳定和高产的肝贮脂细胞分离法

改良国外文献方法,用链霉蛋白酶和胶原酶先后原位灌注大鼠肝脏,以11％Metriza-mide密度梯度分离肝贮脂细胞获得成功。贮脂细胞得率为3—4×10~7个/肝脏,存活率在98％以上,纯度达90—95％,传代培养后纯度在98％以上。     

人早孕胎盘绒毛膜滋养层细胞体外培养模型的建立

目的通过对早孕胎盘绒毛膜滋养层细胞的分离,纯化和培养,寻找一种稳定、简便可获得较高纯度滋养层细胞的培养方法。方法通过胰酶/DNA酶联合消化法对妊娠6-10周绒毛组织进行消化,获得单细胞悬液,比较Per-coll密度梯度离心和淋巴细胞分离液对滋养层细胞的分离纯化效果。含10?S的DMEM/F12培养基培养,并比较是否应用鼠尾胶原对细胞贴壁和生长的影响。通过免疫荧光方法对滋养层细胞进行鉴定。结果经简化Percoll密度梯度离心分离纯化的滋养层细胞纯度高,明显优于淋巴细胞分离液的分离效果(P<0.001);细胞生长表面预先经鼠尾胶原处理后,细胞贴壁良好,分裂生长旺盛。结论利用简化Percoll密度梯度离心法分离细胞,并在应用鼠尾胶原的条件下进行培养,可以获得满意的人绒毛膜滋养层细胞的体外培养模型。     

Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed in Chinese hamster ovary cells

The presence of aggregated forms of proteins can be problematic for therapeutics due to the potential for immunogenic and pharmacokinetic issues. Although downstream processing can remove the aggregated forms, inhibiting aggregate formation upstream during the cell culture stage could reduce the burden on downstream processing and potentially improve process yields. This study first examined the effects of environmental factors (temperature, pH, and dissolved oxygen) and medium components (bivalent copper ion, cysteine, and cystine) on the aggregation of two different recombinant fusion proteins expressed by Chinese hamster ovary (CHO) cells. Any strategy to reduce protein aggregation upstream during cell culture must also consider potential effects on critical upstream parameters such as cell growth, harvest titer, and protein sialylation levels. Manipulating the culture temperature shift and cystine concentration in the medium were both identified as effective and practical strategies for reducing protein aggregation in both CHO-cell expression systems. Furthermore, a combination of both strategies was more effective in reducing protein aggregation levels compared to either approach individually; and without any negative effects on harvest titer and protein sialylation. This study demonstrates a practical methodology for decreasing protein aggregation during upstream processing and emphasizes the importance of process understanding to ensure production of recombinant glycoprotein therapeutics with consistent product quality.     

提高哺乳动物工程细胞抗凋亡能力的基因策略

利用哺乳动物细胞发酵生产重组蛋白药物具有细菌、酵母等表达系统所不具备的显著优势,因此在生物制药工程中的重要性越来越突出。哺乳动物细胞对工业生产环境下的各种应激环境耐受能力差,易发生细胞凋亡,严重阻碍了大规模生产,降低了生产效率。细胞凋亡是细胞必经的生物学过程,随着对凋亡机制的深入了解,发展出各种抗凋亡策略,并有望应用于重构更适合工业生产的工程细胞。常用的抗凋亡策略包括:下调凋亡蛋白、上调抗凋亡蛋白、增强生长因子自表达、减少有毒代谢产物生成等。以上策略虽然能在一定程度上提高细胞的抗凋亡能力,但距离满足生产的工程细胞重构还有距离,围绕提高工程细胞的抗凋亡能力,已发展出\"凋亡工程\"这一重要的技术领域。     

高通量微型生物反应器的研究进展

近年来,哺乳动物细胞培养技术发展迅猛,基于此技术的生物制药行业更是异军突起。在激烈的生物药市场竞争中,缩短研发时间和降低研发成本是制胜的关键。与传统的生物反应器相比,高通量微型生物反应器具有操作简单、运行通量高、实验重复性好等优点,可大大缩短研发周期,降低人力、物力成本,因此成为了生物制药行业最新的研究热点之一。目前,已成功应用于生物药物研发的微型生物反应器有Simcell ^TM、Ambr 15 ^TM、Ambr 250 ^TM等,分别适用于工艺开发中的不同阶段。以上述三种微型生物反应器为例,介绍高通量微型反应器在哺乳动物细胞培养工艺开发中的研究现状及发展前景。     

正常人肝脏星形细胞的分离,培养及鉴定

参考Friedman等分离人肝脏星形细胞(HSC)的方法,采用校正密度的淋巴细胞分离液进行一步梯度离心,成功地分离得到了正常人HSC。HSC的收获量约为1×10~7个/10克肝脏组织,存活率在95％以上,纯度达85％以上。传代后纯度接近100％。对人HSC的标志物进行检测发现,结蛋白不宜作为鉴定初分离的及原代培养初期人HSC纯度的指标,而α-平滑肌肌动蛋白可作为鉴定激活的人HSC的可靠指标。     

Genome-wide analysis of histone modifications by ChIP-on-chip

Post-translational modifications to histone proteins regulate the packaging of genomic DNA into chromatin, gene activity and other functions of the genome. They are understood to play key roles in embryonic development and disease pathogenesis. Recent advances in technology have made it possible to analyze chromatin structure genome-wide in mammalian cells. Global patterns of histone modifications can be observed using a technique called ChIP-on-chip, which combines the specificity of chromatin immunoprecipitation with the unbiased, high-throughput capabilities of microarrays. The resulting maps provide insight into the functions of, and relationships between, different modifications. Here, we provide validated ChIP-on-chip methods for analyzing histone modification patterns at genome-scale in mammalian cells.     

A method to maintain mammalian cells for days alive at 4 °C

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 °C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.     

外周血树突状细胞的体外培养

本文用塑料贴壁的外周血单个核细胞(PBMNCs)在含自体血浆、rhGM-CSF、rhIL-4和TNFα的RPMI1640培养基,37℃,5%CO2湿化空气培养树突状细胞(DCs).经10天培养,可获得大量具DCs形态学特征的细胞,其有很强的刺激同种淋巴细胞增殖功能,约30%的细胞表达HLA-DR和CD1a.健康志愿者每1×107PBMNCs可收获细胞约5×105.本文培养方法产率高,培养体系简单,用自体血浆代替小牛血清,培养过程简便,这些均适应临床治疗要求.     

Scale-up of suspension and anchorage-dependent animal cells

Alternative culture processes for laboratory scale-up (to 20 L) are described for both suspension and anchorage-dependent cells. Systems range from simple multiple culture units such as the roller bottle, through stirred suspension and microcarrier unit bioreactors, to highly sophisticated perfusion culture capable of maintaining cells at densities of about 10⁸/mL. Critical parameters in scale-up are discussed, and the advantages and disadvantages of each culture system are critically evaluated.     

培养原始生殖细胞的新方法

为探讨原始生殖细胞(primordial germ cells,PGCs)在体外长期增殖、生长并长期保持分化潜能的新方法,我们将PGCs分别与睾丸支持细胞(Sertoli cells,SCs)和同源生殖嵴成纤维细胞共培养。结果与SCs共培养的PGCs集落明显多于同源生殖嵴成纤维细胞共培养PGCs集落,传代次数也显著多于同源生殖嵴成纤维细胞．目前与SCs共培养的PGCs已成功传代培养至了第51代。因此我们认为PGCs与SCs共培养,可有效提高原始生殖细胞在体外的增殖能力并可长期维持干细胞的特性。     

培养原始生殖细胞的新方法

为探讨原始生殖细胞（primordial germ cells,PGCs）在体外长期增殖、生长并长期保持分化潜能的新方法,我们将PGCs分别与睾丸支持细胞（Sertoli cells,SCs）和同源生殖嵴成纤维细胞共培养。结果与SCs共培养的PGCs集落明显多于同源生殖嵴成纤维细胞共培养PGCs集落,传代次数也显著多于同源生殖嵴成纤维细胞,目前与SCs共培养的PGCs已成功传代培养至了第51代。因此我们认为PGCs与SCs共培养．可有效提高原始生殖细胞在体外的增殖能力并可长期维持干细胞的特性。     

单克隆抗体制备的细胞工程学研究进展

单克隆抗体是近年来发展最快、最成功的大分子药物之一,哺乳动物细胞作为单抗大规模制备最适宜的宿主,在工业生产中仍然存在成本高、产率低等缺点。近年来,抗细胞凋亡、控制细胞周期、优化代谢过程等细胞工程学方面的研究极大地推动了抗体表达及翻译后修饰技术的发展。以下对近年来单克隆抗体制备在细胞工程学方面取得的进展作一综述,并探讨该领域未来可能的研究方向。