In recent years, Chinese hamster ovary (CHO) production vessel volume has reached more than 1 000 L in Chinese biopharms, and 10 000 L in foreign big biopharms, such as Lonza and Genetech. In general, there are some steps seed bioreactor for seed expansion, which decreases the efficiency of production process. In this work, a perfusion-based process was developed to drastically increase the split ratio during the scale-up of CHO cell cultures. Fed-batch cultures were inoculated with cells propagated in either batch or perfusion cultures that grown in disposable Cellbags using the WAVE Bioreactor system. The higher cell concentration of 2 x 10(7) cells/mL with 95% viability allowed to increase the split ratio to about 1:50-1:100 for inoculum propagated in perfusion culture. The method described here could reduce the number of required expansion steps and eliminate two or three bioreactors. Disposable perfusion bioreactor with only a few liters working volume have the potential to directly inoculate volumes of up to 1 000 liters. This would allow to shorten process time in these bioreactors, which often are the bottleneck in plant throughput.
Animals ; Bioreactors ; CHO Cells ; cytology ; Cell Culture Techniques ; instrumentation ; methods ; Cricetinae ; Cricetulus

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.
Animal ; Apoptosis/drug effects* ; CHO Cells/drug effects* ; CHO Cells/cytology ; Cell Cycle/drug effects* ; DNA Fragmentation/drug effects ; Flow Cytometry ; G2 Phase/drug effects ; Hamsters ; Mitosis/drug effects ; Nickel/pharmacology*

This study investigates whether kappa-opioid receptor and ORL1 receptor may interact to form a heterodimer. In immunofluorescence and co-immunoprecipitation experiments, differentially epitope-tagged receptors, colocalization and heterodimerization of kappa-opioid receptor and ORL1 receptor were used and examined in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. The results show that fluorescence of both kappa-opioid receptor and ORL1 receptor were overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified to have the ability to co-immunoprecipitate ORL1-receptors with kappa-opioid receptor and vice versa. In the current study, further evidence was provided for the direct interaction of two subtypes of opioid receptors, kappa-opioid receptor and ORL1-receptor, to form the heterodimerization. The finding represents the novel pharmacological mechanism for modulation of opioid receptor function as well as diversity of G protein-coupled receptors.
Animals ; CHO Cells ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Dimerization ; Female ; HEK293 Cells ; Hippocampus ; cytology ; metabolism ; Humans ; Immunoprecipitation ; Male ; Neurons ; cytology ; metabolism ; Rats ; Rats, Wistar ; Receptors, Opioid ; metabolism ; Receptors, Opioid, kappa ; metabolism

Microencapsulated recombinant cells technology is a novel approach to tumors therapy. It is necessary to prepare a plenty of the microcapsules with better cell viability and higher endostatin production in order to bring this technology into the clinic. The in vitro culture and cryopreservation are very important parameters in the preparation of microencapsulated cells. In this work, we studied the effect of the in vitro culture and cryopreservation on microencapsulated recombinant cells growth and endostatin production and the effect of the in vitro culture on the cryopreservation of microencapsulated recombinant cells. The results showed that the time of in vitro culture potently affected microencapsulated recombinant CHO cells growth in vivo, endostatin production and the microcapsule stability. The microcapsule kept intact after 36 days of implantation when the in vitro culture time was under 4 days. The thawed microencapsulated recombinant CHO cells had better cell growth and higher endostatin production after 40 days of cryopreservation when the in vitro culture time was 4 days and 8 days. Therefore, the best in vitro culture time was 4 days according to the results of the in vivo culture and cryopreservation and the cryopreservation did not affect microencapsulated recombinant CHO cells growth in vivo, endostatin production and the microcapsule stability.
Animals ; CHO Cells ; Capsules ; Cell Culture Techniques ; Cell Proliferation ; Cells, Immobilized ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Cryopreservation ; methods ; Endostatins ; biosynthesis ; Implants, Experimental ; Mice ; Technology, Pharmaceutical ; instrumentation ; methods ; Time Factors

To reseach GFP reporter gene under the control of chick ovalbumin gene regulatory elements express in the CHO cell and in the primary cell cultures of chicken oviduct. 1.5kb fragment and 2.9kb fragment were amplicated by PCR method, two fragments were subeloned to manmalian expression vector pGFP-N2 by recombinant DNA technology, the CMV promoter was cut off from pGFP-N2, so two expression vectors were constructed, one is the P2.9koval-GFP including promoter, first exon, first intron of chicken ovalbumin gene, the other is the P1.5koval-GFP including first intron of chicken ovalbumin gene. Restriction enzyme digestion and DNA sequence analysis revealed that 5'upstream regions of ovalbumin gene were not only identical to those of the published chicken ovalbumin gene, but also were contained in the recombinant vector. They were transfected into the CHO cell and the primary cell cultures of chicken oviduct by Lipofectin, they were used for fluorescence detection. GFP protein existed in GFP transfected the CHO cell and the primary cell cultures of chicken oviduct. It is demonstrated that GFP reporter gene under the direction of chick ovalbumin gene promoter could be expressed in the CHO cell and in the primary cell cultures of chicken oviduct.
Animals ; CHO Cells ; Cells, Cultured ; Chickens ; Cricetinae ; Cricetulus ; Epithelial Cells ; cytology ; Female ; Genes, Reporter ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Ovalbumin ; genetics ; Oviducts ; cytology ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Neural cell adhesion molecule (NCAM) is a glycoprotein expressing on the surface of neurons, glial cells, bone cells and natural killer cells. NCAM plays an important role in the process of cell - cell adhesion and cell migration, and is also a model protein to study polysialic acid. In this paper, NCAM gene from mouse mammary gland cells (NMuMG) was cloned into eukaryotic expression vectors pcDNA3.1(+) and transfected into mutant Chinese hamster ovary cells ldlD-14. The stable transfection over-expressing NCAM was obtained through the G418 selection and confirmed by Western blotting. Due to unique characters of ldlD-14 cells, carbohydrate chain of NCAM molecule can be easily manipulated with or without adding galactose in the serum free medium, and this modification can provide the basis for further studies on the effect of glycosylation on NCAM molecular function.
Animals ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Female ; Galactose ; Glycosylation ; Mammary Glands, Animal ; cytology ; Mice ; Neural Cell Adhesion Molecules ; biosynthesis ; Polysaccharides ; chemistry ; Sialic Acids ; chemistry ; Transfection

Nano-scale particles of silk fibroin peptide (SFP) were prepared from discarded materials of cocoon or filature by dissolving and enzymolysis. Polyvinyl Alcohol films inlaid with silk fibroin peptide nano-scale particles (SFP in PVA) were prepared by blending nano-SFP and PVA in water according to different blending ratios. The films' characteristics and their promoting cell growth functions were investigated. Silk fibroin fiber was dissolved in 60% NaSCN solution, and was decomposed with alpha-Chymotrypsin, Trypsin and Neutral, respectively. The uniformity of size of SFP nano-particles prepared by Neutral was better and appeared about 80-150 nm. (SFP in PVA) films were characterized by infrared spectroscopy (IR) measurement which demonstrated the combination of SFP and PVA. Scanning electron microscopy revealed the PVA films already inlaid with SFP micro-segment. The surface and form stability in water of the (SFP in PVA) films with blending ratios of 10/90, 20/80, 30/70 and 40/60 were observed. And the results showed that SFP/PVA film with the blending ratio of 30/70 has smoother surface and better stability in water. The Chinese hamster ovary (CHO) cells were cultured, and the promoting cell growth function of (SFP in PVA) films was assessed by MTT colorimetric assay. These findings indicate that SFP/PVA (30/70) film has excellent function of promoting cell growth.
Animals ; CHO Cells ; cytology ; Cell Proliferation ; drug effects ; Cricetinae ; Cricetulus ; Fibroins ; chemistry ; Membranes, Artificial ; Nanoparticles ; chemistry ; Peptides ; chemistry ; Polyvinyl Alcohol ; chemistry

Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
Animals ; Apoptosis ; CHO Cells ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; cytology ; virology ; Cricetinae ; Cricetulus ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Viral Structural Proteins ; genetics ; metabolism

The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.
Animals ; Bone Morphogenetic Protein 15 ; genetics ; CHO Cells ; Cell Differentiation ; Cricetinae ; Cricetulus ; Female ; Genes, Reporter ; Genetic Vectors ; Mice ; Microinjections ; Myoblasts ; cytology ; Oocytes ; metabolism ; Ovary ; metabolism ; Stem Cells ; cytology ; Swine

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.
ATP Citrate (pro-S)-Lyase/*genetics ; Animal ; Base Sequence ; CHO Cells ; Cells, Cultured ; Female ; *Gene Expression Regulation, Enzymologic ; Glucose/pharmacology ; Hamsters ; Liver/cytology/enzymology ; Molecular Sequence Data ; Promoter Regions (Genetics) ; Rats ; Support, Non-U.S. Gov't ; Transcription, Genetic