用天冬酰胺代替谷氨酰胺培养产尿激酶原CHO工程细胞

以产尿激酶原CHO工程细胞CL－11G的细胞生长和目的产物的表达为观察指标,对用天冬酰胺替代培养基中谷氨酰胺的可行性进行研究。结果表明,溶液中游离态的天冬酰胺的自发分解速度明显低于谷氨酰胺,用天冬酰胺替代培基中的谷氨酰胺培养11G细胞不影响细胞的生长和目的产物的表达,有助于克服因谷氨酰胺的自行分解而造成的培养基氨的过度积聚对细胞代谢的不利影响。     

Evaluating the toxicity of bDtBPP on CHO‐K1 cells for testing of single‐use bioprocessing systems considering media selection,cell culture volume,mixing, and exposure duration

Single‐use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale‐up. Bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4‐di‐tert‐butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single‐use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO‐K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO‐K1 cells. While CHO‐K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry‐standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO‐K1 cells. It was found that the sensitivity of cell culture to leachates from single‐use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318–1323, 2016     

Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-γ-producing CHO cell line

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.     

Apoptosis in CHO cell batch cultures: examination by flow cytometry

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G₁/G₀ and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1／Bcl-2或Bcl-2／CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1／Bcl-2和Bcl-2／CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

改进培养基配方对提高EPO产量的作用

研究了用中国仓鼠卵巢细胞(CHO)生产促红细胞生成素(EPO)的配方．改进配方前生产3批EPO,培养液样品的平均活性为4236．7IU／ml;改进配方后生产3批EPO,培养液样品的平均活性为6846．4IU／ml．后者比前者高出61．6％,即EPO产量提高达到61．6％．     

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO? and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

批次和流加培养中国仓鼠卵巢工程细胞的细胞周期相关基因转录谱分析

以表达人重组尿激酶原中国仓鼠卵巢 (CHO) 工程细胞系11G-S为研究对象,运用基因芯片技术比较了CHO工程细胞在批次及流加培养不同生长阶段基因表达水平的差异,在此基础上采用Genmapp软件,同时结合已知的细胞周期信号通路图,着重分析了批次及流加培养CHO工程细胞的细胞周期调控基因转录谱差异。在基因芯片涉及的19 191个目标基因中,批次和流加培养不同生长阶段CHO工程细胞的下调表达的基因数量多于上调表达基因数目;两种培养模式下的基因差异表达有着明显的不同,尤其是在细胞生长的衰退期,流加培养CHO工程细胞中下调表达的基因数量明显多于批次培养。有关调控细胞周期关键基因的转录谱分析表明,CHO工程细胞主要是通过下调表达CDKs、Cyclin及CKI家族中的Cdk6、Cdk2、Cdc2a、Ccne1、Ccne2基因及上调表达Smad4基因,来达到调控细胞增殖及维持自身活力的目的。     

Influence of protein and carbohydrate contents of soy protein hydrolysates on cell density and IgG production in animal cell cultures

The variety of compounds present in chemically defined media as well as media supplements makes it difficult to use a mechanistic approach to study the effect of supplement composition on culture functionality. Typical supplements, such as soy protein hydrolysates contain peptides, amino acids, carbohydrates, isoflavones, and saponins. To study the relative contribution of these compound classes, a set of hydrolysates were produced, containing 58‐83% proteinaceous material and 5‐21% carbohydrates. While the content of the different compounds classes varied, the composition (e.g., peptide profiles, carbohydrate composition) did not vary in hydrolysates. The hydrolysates were supplemented to a chemically defined medium in cell culture, based on equal weight and on equal protein levels. The latter showed that an increase in the carbohydrate concentration significantly (P value < 0.004) increased integral viable cell density (IVCD) (R = 0.7) and decreased total IgG (R = ?0.7) and specific IgG production (R = ?0.9). The extrapolation of effects of protein concentration showed that an increase in protein concentration increased total and specific IgG production and suppressed IVCD. In addition to proteins and carbohydrates, the functionality of soy protein hydrolysates may be modulated by the presence of other minor compounds. In the current study, the large differences in the balance between total proteins and total carbohydrates in the supplemented media seem to be a main factor influencing the balance between the viable cell density, total IgG, and specific IgG production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1396–1405, 2015     

改造中国仓鼠卵巢细胞

原核细胞、酵母细胞以及昆虫细胞相比,中国仓鼠卵巢细胞（CHO）作为宿主细胞表达的外源蛋白最接近其天然构象,因而CHO细胞表达系统是生物工程制药最为理想的表达系统。但这种系统也存在诸多缺点。如在大规模培养中CHO细胞会面临着对无血清培养基的适应性差、细胞无限度增殖以及细胞凋亡等很多难题。所以除了在培养基、培养条件和表达载体方面下功夫优化该系统外,对CHO细胞本身进行改造已成为优化CHO表达系统的另一热点。     

Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells

Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular -galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular -galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.     

Generating stable chinese hamster ovary cell clones to produce a truncated SARS‐CoV spike protein for vaccine development

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS‐CoV) is important for vaccine development. S_TR2 (an 88 kDa truncated SARS‐CoV TW1 S protein carrying the S fragments S‐74‐253, S‐294‐739, and S‐1129‐1255) is capable of expressing a major form of glycoprotein as endo H‐sensitive (～115 kDa) in CHO cells. To establish stable expressing cell clones, we transfected CHO/dhFr‐cells with the amplifiable vectors ISID (IRES‐driven dhfr) and ISIZ (SV40‐driven dhfr) to select stepwise MTX, and observed enhanced ～115 kDa glycoform generation through gene amplification. Following stepwise MTX selection, we compared gene amplification levels between two vectors in engineered CHO cell chromosomes. These results confirm that the IRES‐driven dhfr promoter generates greater gene amplification, which in turn enhances S_TR2 expression. Our results indicate that the ～115 kDa glycoform of S_TR2 protein was capable of increasing after gene amplification. The S_TR2 glycoform did not change between suspension and serum‐free cultures, suggesting that the stable and amplified cell clones analyzed in this study have potential for producing homologous S_TR2 on a large scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010