中华仓鼠卵巢(CHO)工程细胞无血清培养的研究

以DMEM：F12(1：1)为基础培养基,通过观察细胞生长状态和检测乙肝表面抗原的表达量作为评价指标,筛选适合于CHO工程细胞生长的生长因子,如：胰岛素、转铁蛋白、氢化可的松、硒酸钠,丁二胺等。并且建立了J5SFM培养基。该培养基与商品化的无血清培养基比较,能够使细胞生长维持较长的时间,表达产物分泌量也相对较高。     

alpha-Amanithin-resistant mutants of Chinese hamster ovary (CHO) cells

Spontaneous and EMS-induced alpha-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent RNA polymerase II in cell-free extracts from a mutant (ARM-1) was partially resistant to alpha-amanitin. Growing mutants for several generations in the presence or absence of alpha-amanitin did not change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without alpha-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Increased production of recombinant hIGFBP-1 in PEG induced autofusion of Chinese hamster ovary (CHO) cells

A recombinant Chinese hamster ovary (CHO) cell clone, S1, stably expressing human insulin-like growth factor binding protein-1 (hIGFBP-1), was treated with polyethylene glycol (PEG), resulting in cell fusion, in order to further enhance the protein expression by increasing the gene copy number and/or the amount of organelles important to the protein expression/-secretion. Both the fused cell line, Peg1, and its mother cell line, S1, were adapted to serum-free growth in suspension and were characterised with respect to growth and productivity. Peg1 was easier to adapt to the serum-free suspension conditions and had a higher viability during the adaptation period than S1. Furthermore, Peg1 showed a stable productivity of hIGFBP-1 that was twice as high as that for S1 under both adherent and suspension conditions. A considerable difference in the specific productivity (up to 3–4 times) was noticed during the growth phase. PEG fusion experiments have earlier been studied in our laboratory with CHO cells producing recombinant factor VIII and our results correlates very well with the results obtained with the factor VIII producing cells. Surprisingly, it was possible to obtain high producing recombinant cell lines, which were stable for more than 4 months. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells

The amount of acid or base consumed in yeast cultures has been recently assigned to the pathway of nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism (Castrillo et al., 1995). In this investigation, experiments under respirofermentative conditions have shown that production or consumption of ethanol does not contribute significantly to the specific rate of proton production (qH+), thus extending the previously obtained relationships for all aerobic conditions in which other major acid/base contributions are not involved. Tests in batch and chemostat culture confirm the validity of qH+ as a formal control parameter in aerobic fermentations.     

Freeze fracturing properties of native and heat-adapted Chinese hamster ovary (CHO) cells

Chinese hamster ovary cells were either maintained at 37 degrees C (native cells) or heat adapted by exposure to a temperature of 40 degrees C for 2 h. Thereafter, native and heat-adapted cells were incubated at different temperatures for various times, harvested, fixed with glutaraldehyde and glycerol, and studied by freeze fracture microscopy. We observed that the fracture plane either passed through the cell, exposing cytoplasm, or stayed within the plasma membrane, and that the location of the fracture plane was strongly determined by the previous thermal history of the cell. We believe that these differences reflect changes in membrane lipid packing arrangements.     

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.     

Morphological reverse transformation of Chinese hamster ovary (CHO) cells and surface fibronectin

Alterations in the distribution of surface fibronectin during reverse transformation of chinese hamster ovary (CHO) cells induced by dibutyryl adenosine cyclic monophosphate (dbcAMP) and theophylline was examined by indirect immunofluorescence. dbcAMP-induced reverse transformation was not followed by any significant increase in surface fibronectin up to 48 hrs after treatment. Reverse transformation induced by theophylliner by itself or in combination with dbcAMP is followed several hours later by a phenomenal increase in fibrillar surface fibronectin, which is largely persistent even in the presence of cytochalasin-B or colcemid but is sensitive to the presence of cycloheximide. It appears that reverse transformation consists at least of two steps: (a) morphological reversion to normal phenotype and (b) modulation of cell membrane properties or components favouring retention of fibronectin in the cell surface.     

SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either satin (GBI, GBII) at 1.4 x 10⁻³ M or soman (GD) at 1.1 and 2.2 x 10^t-3 M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 x 10⁻³ M and cyclophosphamide (CP) at 10⁻⁴ M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.     

Large-scale production of Erythroid Differentiation Factor (EDF) by gene-engineered Chinese hamster ovary (CHO) cells in suspension culture

From Chinese hamster ovary (CHO) cells which were producing Erythroid Differentiation Factor (EDF) in a culture medium, anchorage-independent cells, named as CHO-SPN, were produced by repeated cultivating in a suspension system. The growth time and maximum cell density of the CHO-SPN cells were 48 h and 7.8×10⁵ viable cells/ml. CHO-SPN cells accumulated 8,000 units/ml (corresponding to 4 mg/ml) of EDF in 4d. After 20 cycles of culture, CHO-SPN cells still possessed the same EDF productivity and the same growth kinetics. Furthermore, in an appropriate dissolved oxygen concentration and pH controlled culture system, the growth time and cell density became 24 h and 1×10⁶ viable cells/ml. The critical level of dissolved oxygen for cell growth was 0.015 atm. The maximum oxygen demand was 3.3×10⁻⁹ mole of O₂/ml/min.Fetal bovine serum (FBS) was indispensable for cell growth. However, a FBS-free medium (ASF201) was available for maintenance of the CHO-SPN cells, and EDF production occurred in the same medium.     

Mutants of Chinese hamster ovary (CHO) cells with altered colcemid-binding affinity.

Clones of CHO cells stably resistant to colcemid have been isolated in the presence of the nonionic detergent Tween 80 after mutagen treatment. Successive single-step selections for increasing resistance were performed resulting in lines after three selection steps about 10 fold more resistant to colcemid than the parental cells. Three observations indicate that these colcemid-resistant (CMR) mutants are different from the colchicine-resistant permeability mutants isolated previously. First, their relative resistance to colcemid was not diminished in the presence of detergent which promoted increased drug permeability. Second, the CMR clones displayed limited cross-resistances only to tubulin-binding compounds. Third, the binding affinity of labeled colcemid by cytoplasmic extracts from CMR clones was reduced, and the reduction was greater in the more resistant clones. No reduction in binding of labeled colcemid was observed in the membrane-altered colchicine-resistant mutants. All these observations are consistent with the CMR clones being tubulin-altered mutants. In further support of this conclusion, we observed that tubulin purified from a CMR mutant still possessed reduced colcemid-binding affinity compared with that from parental cells.     

中国仓鼠卵巢工程细胞无血清流加培养关键工艺参数的考察

主要考察流加培养基中不同营养成分、流加起始时间及初始接种密度对11G-S细胞无血清流加培养的影响。在研究中以悬浮适应的表达尿激酶原 (Pro-urokinase,Pro-UK) CHO工程细胞系11G-S为研究对象,在100 mL的摇瓶中无血清悬浮流加培养11G-S细胞,同时以活细胞密度、细胞活力及Pro-UK活性为评价依据。结果表明在培养基中氨基酸、无血清添加成分及无机盐对促进细胞生长、细胞活力维持及蛋白表达起着较为重要的作用;且流加起始时间为72 h及初始接种密度为3×105~4×105 cells/     

Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells

Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Lessons from peroxisome-deficient Chinese hamster ovary (CHO) cell mutants

Cells with a genetic defect affecting a biological activity and/or a cell phenotype are generally called "cell mutants" and are a highly useful tool in genetic, biochemical, as well as cell biological research. To investigate peroxisome biogenesis and human peroxisome biogenesis disorders, more than a dozen complementation groups of Chinese hamster ovary (CHO) cell mutants defective in peroxisome assembly have been successfully isolated and established as a model system. Moreover, successful PEX gene cloning studies by taking advantage of rapid functional complementation assay of CHO cell mutants invaluably contributed to the accomplishment of isolation of pathogenic genes responsible for peroxisome biogenesis diseases. Molecular mechanisms of peroxisome assembly are currently investigated by making use of such mammalian cell mutants.     

Localization of chromosome breakpoints induced by DNase I in Chinese hamster ovary (CHO) cells

DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed. Received: 15 January 1997; in revised form: 21 July 1997 / Accepted: 23 July 1997