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Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor‐β (TGF‐β) super family of proteins and is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post‐translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half‐life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in‐vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in‐vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO‐K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production. Significance statement: The present study demonstrates the suitability of primary goat mammary epithelial cells as a system for large scale production of therapeutic proteins in‐vitro. The cells of the mammary gland are highly secretory, and we reasoned that such cells may be useful for in‐vitro production of tough‐to‐produce therapeutics like human Bone Morphogenetic protein 2 (BMP2). We demonstrate that GMECs are a better alternative to the conventionally used CHO‐K1 cells for rhBMP‐2 production. Our results provide a strong case for developing and using GMECs as an alternative system for therapeutic protein expression. [ABSTRACT FROM AUTHOR]

In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus—the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy. [ABSTRACT FROM AUTHOR]

Site-specific integration is an important approach used to address the problem of unstable cell lines in industry. In this study, we observed a reduction in the gene copy number and antibody production in a CHOK1 cell line BA03 capable of high antibody expression. We identified a new integration site named locus 7 in the intron region of the parva gene through sequencing, FISH, and genome walking. We demonstrate that the integration of the exogenous gene at this locus does not affect the transcription of the parva and, therefore, has a minimal impact on cell growth. We designed sgRNA and donor vectors to integrate the etanercept-coding gene into locus 7 and obtained a cell line, SSI-4. We performed a passaged stability study on SSI-4 and proved the possibility of the stable, site-specific integration of exogenous genes at this locus in terms of integration site, copy number, expression level, and cell growth. In summary, our study has identified a new integration site suitable for site-specific integration, which lays the foundation for the subsequent development of site-specific integration cell lines. [ABSTRACT FROM AUTHOR]

Stabilized trimers of the HIV-1 envelope glycoprotein Env are capable of inducing a potent and sustained broadly neutralizing antibody response in laboratory animals and therefore are attractive targets for anti-HIV vaccine development. In this work, a stable producer of the trimer Env recombinant form CRF63_02A6 of HIV-1 was derived from the CHO-K1 cell line. Using immunochemical assays, the trimers synthesized in CHO-K1 cells were shown to be recognized by both monoclonal broadly neutralizing antibodies and sera from HIV-positive patients. The resulting trimers of the recombinant form CRF63_02A6 of HIV-1 can be used both for structural studies and as a candidate vaccine immunogen against HIV-1. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic threatened public health around the world at the same time as highlighting the urgency of vaccine development. Subunit vaccines are safe and effective vaccine types that utilize parts of viruses to trigger the body’s immune response. Previous research has shown that fusion of the spike protein with the foldon domain (fd) achieved the trimeric form to increase the protein stability of the recombinant subunit protein spike from SARS‐CoV and MERS‐CoV, thus exceeding the immune response in the body. The study aims to observe the expression of RBD‐fd and S1‐fd recombinant proteins from the spike protein of SARS‐CoV‐2 in CHO‐K1 mammalian cells and investigate the binding activity of those proteins with hACE2 receptor, expressed in HEK293T cells using immunofluorescence staining. The plasmids were transiently transfected into the cells, followed by antibiotic selection using G418 as an initial stage to select the positive stable transformants. Protein expression was confirmed by Western blotting and showed an estimated size for monomeric RBD‐fd of 35 kDa and S1‐fd of 55 kDa. However, the trimeric form of the proteins was not observed. In addition, immunofluorescence staining showed the binding activity between the RBD‐fd and S1‐fd proteins and hACE2 expressing cell line, revealing binding and an internalization process.

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1′ + S3′ specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C. [ABSTRACT FROM AUTHOR]

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine. [ABSTRACT FROM AUTHOR]

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy. [ABSTRACT FROM AUTHOR]

Background and Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine. Materials and Methods: The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants. Results: Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2. Conclusion: Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus—the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy.

Site-specific integration is an important approach used to address the problem of unstable cell lines in industry. In this study, we observed a reduction in the gene copy number and antibody production in a CHOK1 cell line BA03 capable of high antibody expression. We identified a new integration site named locus 7 in the intron region of the parva gene through sequencing, FISH, and genome walking. We demonstrate that the integration of the exogenous gene at this locus does not affect the transcription of the parva and, therefore, has a minimal impact on cell growth. We designed sgRNA and donor vectors to integrate the etanercept-coding gene into locus 7 and obtained a cell line, SSI-4. We performed a passaged stability study on SSI-4 and proved the possibility of the stable, site-specific integration of exogenous genes at this locus in terms of integration site, copy number, expression level, and cell growth. In summary, our study has identified a new integration site suitable for site-specific integration, which lays the foundation for the subsequent development of site-specific integration cell lines.

Background and Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine. Materials and Methods: The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants. Results: Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2. Conclusion: Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising “humanised” in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.

Biophysical models are useful tools for predicting the biological effects of ionizing radiation. From a practical point of view, these models can help clinicians to optimize the radiation absorbed dose delivered to patients in particle therapy. The biophysical model NanOx was recently developed to predict cell survival fractions in the context of radiotherapy. The model takes into account the stochastic nature of radiation at different levels and considers as well the accumulation of radio-induced oxidative stress in cells caused by reactive chemical species. We show in this work how the general formalism of NanOx is adapted to hadrontherapy applications. We then use NanOx to compute the cell survival fractions for three cell lines (V79, CHO-K1 and HSG) in response to carbon ions of different energies, and benchmark the predictions against experimental data. The results attest that NanOx provides a good description of both the overkill effect and the evolution of the shoulders of cell survival curves with linear energy transfer.

Previous studies have shown that differences in experimental design factors may alter the potency of genotoxic compounds in in vitro genotoxicity tests. Most of these studies used traditional statistical methods based on the lowest observed genotoxic effect levels, whereas more appropriate methods, such as the benchmark dose (BMD) approach, are now available to compare genotoxic potencies under different test conditions. We therefore investigated the influence of two parameters, i.e. cell type and exposure duration, on the potencies of two known genotoxicants [aflatoxin B1 and ethyl methanesulfonate (EMS)] in the in vitro micronucleus (MN) assay and comet assay (CA). Both compounds were tested in the two assays using two cell types (i.e. CHO-K1 and TK6 cells). To evaluate the effect of exposure duration, the genotoxicity of EMS was assessed after 3 and 24 h of exposure. Results were analyzed using the BMD covariate approach, also referred to as BMD potency ranking, and the outcome was compared with that of more traditional statistical methods based on lowest observed genotoxic effect levels. When comparing the in vitro MN results obtained in both cell lines with the BMD covariate approach, a difference in potency was detected only when EMS exposures were conducted for 24 h, with TK6 cells being more sensitive. No difference was observed in the potency of both EMS and aflatoxin B1 in the in vitro CA using both cell lines. In contrast, EMS was more potent after 24 h exposure compared with a 3 h exposure under all tested conditions, i.e. in the in vitro MN assay and CA in both cell lines. Importantly, for several of the investigated factors, the BMD covariate method could not be used to confirm the differences in potencies detected with the traditional statistical methods, thus highlighting the need to evaluate the impact of experimental design factors with adequate approaches. [ABSTRACT FROM AUTHOR]

The human peptide transporter hPepT1 (SLC15A1), physiologically transporting dipeptides and tripeptides generated during food digestion, also plays a role in the uptake of small bioactive peptides and peptide-like drugs. Moreover, it might be addressed in prodrug strategies of poorly absorbed drugs. We hypothesised that the cyclic drug peptides octreotide and pasireotide could be substrates of this transporter because their diameter can resemble the size of dipeptides or tripeptides due to their strong structural curvature and because they reach the systemic circulation in Beagle dogs. For investigating possible hPepT1 substrate characteristics, we generated and characterised a CHO-K1 cell line overexpressing SLC15A1 by transfection and selection via magnetic beads. Possible inhibition of the uptake of the prototypical substrate Gly-Sar by octreotide and pasireotide was screened, followed by quantifying the uptake of the cyclic peptides in cells overexpressing SLC15A1 compared with the parental cell line. Although inhibition of Gly-Sar uptake was observed, uptake of octreotide and pasireotide was not increased in SLC15A1 overexpressing cells, indicating a lack of transport by hPepT1. Our data clearly indicate that octreotide and pasireotide are nonsubstrate inhibitors of hPepT1 and that their oral bioavailability cannot be explained by absorption via hPepT1. [ABSTRACT FROM AUTHOR]

Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly transient permeability of the targeted cell membrane. In this work, we present the electrotransfection of CHO-K1, 4T1 cell lines, and primary murine DCs with detectable protein-encoding plasmids in the sub-microsecond range. Microsecond (µs)- and nanosecond (ns)-range pulsed electric field transfection protocols were used. The efficiency of electrotransfection was evaluated using green fluorescent protein (GFP)-encoding plasmids (4.7 kbp; p-EGFP-N1) and plasmids expressing a firefly luciferase and red fluorescent protein (tdTomato) (8.5 kbp; pcDNA3.1(+)/Luc2 = tdT)). It was shown that the used nsPEFs protocol (7 kV/cm × 300 ns × 100, 1 MHz) ensured a better transfection efficiency than µsPEFs (1.2 kV/cm × 100 µs × 8, 1 Hz). Plasmid size and concentration had a strong impact on the cell transfection efficiency too. We also showed that there were no significant differences in transfection efficiency between immature and mature DCs. Finally, the nsPEF protocols were successfully applied for the stable transfection of the CHO-K1 cell line with the linearized pcDNA3.1(+)/Luc2 = tdT plasmid. The results of the study are applicable in gene therapy and DNA vaccination studies for the derivation of optimal electrotransfection conditions. [ABSTRACT FROM AUTHOR]

Chinese Hamster Ovary (CHO) cells are widely used for the high‐level production of recombinant proteins. We created a multiauxotrophic mutant of CHO‐K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA‐based genes for the eight missing activities. CHO8A cells permit: (1) selection of transfectant clones that have incorporated genes for eight or more different polypeptides, suitable for engineering complex proteins, or pathways; and (2) the single‐step selection of high producers of a particular protein. The latter is achieved by simultaneous use of eight vectors, each bearing one of the eight rescue genes and a cargo protein gene. Screening as few as 10 surviving colonies yielded high producers secreting mAbs at 84 picograms per cell per day or more. CHO8A was isolated by CRISPR‐Cas9 knockout of 10 genes in the pathways to pyrimidines (Dhodh, Umps, Ctps1, Ctps2, and Tyms) and purines (Paics, Atic, Impdh1, Impdh2, and Gmps). [ABSTRACT FROM AUTHOR]

Background: Antibiotic-resistance in E. coli is a global issue affecting humans especially the pediatric population. Antibiotic-resistant E. coli is a pathogen frequently pediatric population as well as healthy adults. Methods: This study aimed to examine the antibiotic resistance of E. coli causing pediatric diarrhea and its drug-resistant rates, its adhering abilities to cell line in vitro, and inhibition efficiency of a few selected chemical compounds. Clinical strains were isolated from both the healthy and infected pediatric population of Mizoram. Results: Adhesion is a significant pathogenic process during bacterial infections, which has been employed for pathotyping of DEC by comparing adhesion efficiency in both normal (CHO-k1) and cancer (HeLa) cell lines. E. coli adherent pathotypes were identified by both PCR assay and in-vitro cell adhesion assays; the study also evaluated the adhesion inhibition ability of human skimmed milk, gentamicin, and cephalexin in-vitro. Of all isolates, 20.05% of adherent DEC (EPEC, DAEC, and EIEC) and 11.39 % of non-adherent DEC (STEC and ETEC) were found to be associated with pediatric diarrhoea in Mizoram. Human skimmed milk has a high potential adhesion inhibition against EAEC (50.25/90.90 μg/mL), EPEC (53.42/259.70 μg/mL), and EIEC (59.13/30.30 μg/mL) in both cell lines in comparison with gentamicin and cephalexin. Conclusion: This study concludes that as a dietary supplement-human skimmed milk has high potential to prevent adhesion of DEC pathotypes in cells in-vitro thus in in-vivo.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

Erythropoietin (EPO) is a therapeutic protein that is widely used to increase red blood cell production in chronic kidney failure. EPO protein can be produced quickly with a transient gene expression system (TGE). However, the titer produced using TGE is usually lower than the stable gene expression system (SGE). It has been known that TGE can be improved by histone deacetylase inhibitors (iHDACs) such as valproic acid (VPA). This study was conducted to examine the VPA effect on EPO protein expression in CHO‐K1 suspension adapted cells and to find the optimum concentration of VPA on transient EPO protein production. EPO proteins was quantified using the enzyme‐linked immunosorbent assay (ELISA) method. The optimization of VPA concentrations showed that VPA increased the EPO protein yield by up to 2‐fold in transient EPO production, and the optimum concentration of VPA was 4 mM. VPA optimization was very helpful to obtain the maximum increase in the transiently expressed protein. Furthermore, this study can be used as a model to produce EPO proteins or other recombinant proteins rapidly with TGE of CHO‐K1 suspension adapted cells.

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Gene transfer into primary immune cells as well as into cell lines is essential for scientific and therapeutical applications. One of the methods used for gene transfer is electroporation (EP). EP is a method where a pulsed electric field (PEF) causes a highly transient permeability of the targeted cell membrane. In this work, we present the electrotransfection of CHO-K1, 4T1 cell lines, and primary murine DCs with detectable protein-encoding plasmids in the sub-microsecond range. Microsecond (µs)- and nanosecond (ns)-range pulsed electric field transfection protocols were used. The efficiency of electrotransfection was evaluated using green fluorescent protein (GFP)-encoding plasmids (4.7 kbp; p-EGFP-N1) and plasmids expressing a firefly luciferase and red fluorescent protein (tdTomato) (8.5 kbp; pcDNA3.1(+)/Luc2 = tdT)). It was shown that the used nsPEFs protocol (7 kV/cm × 300 ns × 100, 1 MHz) ensured a better transfection efficiency than µsPEFs (1.2 kV/cm × 100 µs × 8, 1 Hz). Plasmid size and concentration had a strong impact on the cell transfection efficiency too. We also showed that there were no significant differences in transfection efficiency between immature and mature DCs. Finally, the nsPEF protocols were successfully applied for the stable transfection of the CHO-K1 cell line with the linearized pcDNA3.1(+)/Luc2 = tdT plasmid. The results of the study are applicable in gene therapy and DNA vaccination studies for the derivation of optimal electrotransfection conditions.

Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf) protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNA-rpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile, recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral immune response in mice model.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO‐K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR‐Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor‐α receptor fusion protein etanercept and (b) the heavy and light chains of the anti‐Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine‐ and pyrimidine‐free commercial media. [ABSTRACT FROM AUTHOR]

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells. [ABSTRACT FROM AUTHOR]

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol–AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol–AmB structures at the cell surface, preventing AmB cytotoxicity. [ABSTRACT FROM AUTHOR]

Site-specific integration is an important approach used to address the problem of unstable cell lines in industry. In this study, we observed a reduction in the gene copy number and antibody production in a CHOK1 cell line BA03 capable of high antibody expression. We identified a new integration site named locus 7 in the intron region of the parva gene through sequencing, FISH, and genome walking. We demonstrate that the integration of the exogenous gene at this locus does not affect the transcription of the parva and, therefore, has a minimal impact on cell growth. We designed sgRNA and donor vectors to integrate the etanercept-coding gene into locus 7 and obtained a cell line, SSI-4. We performed a passaged stability study on SSI-4 and proved the possibility of the stable, site-specific integration of exogenous genes at this locus in terms of integration site, copy number, expression level, and cell growth. In summary, our study has identified a new integration site suitable for site-specific integration, which lays the foundation for the subsequent development of site-specific integration cell lines.

Most commercially available fluorescent dyes used for cellular images are toxic and their luminescence is often outshined by the intrinsic autofluorescence of the biological medium, the use of long emission lifetime inherent of the lanthanide compounds is a viable alternative. This paper reports the development of a luminescent hybrid based on silica nanoparticles decorated with Tb3+ complexes suitable for use in exploratory tests as biomarkers in the visualization of CHO-k1 cells by microscopy techniques. To achieve this aim, spherical/spheroidal (∼125 nm) functionalized silica particles were prepared by the sol-gel method and had their surface functionalized covalently with Schiff base ligands. Tb3+ Complexes were bonded onto the particles surface by a step-by-step methodology approach. By zeta potential, it was possible to investigate the particles surface charge after each synthesis step, reaching a negative value for the final hybrid, which showed intense luminescence in the green region, where the Tb3+5D 4 → 7F J transitions were observed in the emission spectrum. After the benzoic acid ligand (BzAc) addition, there was an intensification of Tb3+ emission, as well as an increase in the emission lifetime. Concerning the biological application of the material, the CHO-k1 cell viability was evaluated by the MTT method in the presence of the SiO 2 -[Tb(SB)(BzAc) y ] hybrid, and high biocompatibility was observed. The absorption process of particles by the cells was investigated by TEM, suggesting an endocytosis mechanism as a probable uptake process. The confocal images indicated a green emission of the biomarker due to the Tb3+, distributed through the cytoplasm, mainly closely to the cell nuclei. Therefore, the silica particles decorated with Tb3+ complexes proved to be suitable to act as biomarkers in biological assays and cellular imaging. • Step-by-step approach to decorate silica with Tb3+-complexes obtained via sol-gel. • Schiff base and benzoic acid ligands as sensitizers for Tb3+ ions. • Intense Tb3+ green emission. • Longer lifetime emission of the Tb3+-complexes after anchoring to the silica matrix. • Endocytosis mechanism for particles uptake process by CHO-k1 cells. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf) protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNArpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile, recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral immune response in mice model. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIVAIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional Nlinked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 Nlinked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation.

The diterpene kaurenoic acid (KA) has vasorelaxant, antimicrobial, anti-tumoural and anti-leishmanial effects. Semi-synthetic derivatives were obtained to achieve more satisfactory responses. The assessment of genotoxicity is part of the toxicological evaluation of therapeutic compound candidates. The present study investigated the cytotoxicity and genotoxicity of KA and its semi-synthetic derivatives methoxy kaurenoic acid (MKA) and kaurenol (KRN) using the CHO-K1 cell line. The cytotoxicity evaluation demonstrated that treatments with 200 and 400 μM KA reduced cellular proliferation to 36.5 and 4.43%, respectively, and that 100 and 200 μM KA reduced the survival fraction (SF) to 48.1 and 5.5%, respectively. MKA and KRN at concentrations of 400 μM reduced proliferation to 81 and 86.8%, respectively, while 100 and 200 μM KRN reduced the SF to 50%, and 200 μM MKA reduced the SF to 74%. No genotoxicity was observed for KA or MKA. However, 100 μM KRN increased the DNA damage index, as detected by comet assay, although a micronucleus assay did not confirm these data. The results demonstrated that KA and its semi-synthetic derivative MKA were not genotoxic when tested at noncytotoxic concentrations, but KRN was genotoxic at the highest concentration that was tested, as demonstrated by the comet assay. [ABSTRACT FROM AUTHOR]

miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation. [ABSTRACT FROM AUTHOR]

An aqueous extract of Rhizophora mangle L. bark is used as raw material in pottery making in the State of Espirito Santo, Brazil. This extract presents large quantities of tannins, compounds possessing antioxidant properties. Tannin antioxidant activity, as a plant chemical defense mechanism in the process of stabilizing free radicals, has been an incentive to studies on anti-mutagenicity. The present work aimed to evaluate possible antimutagenic activity of a R. mangle aqueous extract, using the Allium cepa test-system and micronuclear (MN) assay with blockage of cytokinesis in Chinese hamster ovary cells (CHO-K1). The Allium cepa test-system indicated antimutagenic activity against the damage induced by the mutagenic agent methyl methanesulfonate. A reduction in both MN cell frequency and chromosome breaks occurred in both the pre and post-treatment protocols. The MN testing of CHO-K1 cells revealed anti-mutagenic activity of the R. mangle extract against methyl methanesulfonate and doxorubicin in pre, simultaneous and post-treatment protocols. These results suggest the presence of phyto-constituents in the extract presenting demutagenic and bio-antimutagenic activities. Since the chemical constitution of Rhizophora mangle species presents elevated tannin content, it is highly probable that these compounds are the antimutagenic promoters themselves.

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd 3+ ), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend. [ABSTRACT FROM AUTHOR]

Magnetic resonance (MR) reporter genes have the potential for tracking the biodistribution and fate of cells in vivo, thus allowing the safety, efficacy and mechanisms of action of cell-based therapies to be comprehensively assessed. In this study, we evaluate the effectiveness of the iron importer transferrin receptor-1 ( TfR1) as an MR reporter gene in the model cell line CHO-K1. Overexpression of the TfR1 transgene led to a reduction in the levels of endogenous TfR1 mRNA, but to a 60-fold increase in total TfR1 protein levels. Although the mRNA levels of ferritin heavy chain-1 ( Fth1) did not change, Fth1 protein levels increased 13-fold. The concentration of intracellular iron increased significantly, even when cells were cultured in medium that was not supplemented with iron and the amount of iron in the extracellular environment was thus at physiological levels. However, we found that, by supplementing the cell culture medium with ferric citrate, a comparable degree of iron uptake and MR contrast could be achieved in control cells that did not express the TfR1 transgene. Sufficient MR contrast to enable the cells to be detected in vivo following their administration into the midbrain of chick embryos was obtained irrespective of the reporter gene. We conclude that TfR1 is not an effective reporter and that, to track the biodistribution of cells with MR imaging in the short term, it is sufficient to simply culture cells in the presence of ferric citrate. Copyright © 2016 The Authors Contrast Media & Molecular Imaging Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the potential of hemolymph utilization as a culture medium supplement to cultivate the animal cell CHO-K1. For this purpose 1% v/v of hemolymph was added to DMEM medium containing 10% v/v of FBS and 1 or 4.5 g/L of glucose. The culture was grown in spinner flasks incubated in a 10% v/v CO2 environment, at 37ºC, with the Cytodex 1 microcarrier. Comparing the results obtained from the culture with hemolymph against those without hemolymph, a positive influence of the hemolymph was observed, as the experiment with hemolymph presented a 52% higher cell concentration and a higher productivity of up to 40%.Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB) é uma das grandes prioridades de pesquisa em desenvolvimento de processos com célula animal. O objetivo do presente trabalho foi realizar uma análise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da célula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37ºC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influência positiva da hemolinfa no cultivo, já que o ensaio com hemolinfa apresentou uma concentração máxima de células 52% maior e uma produtividade máxima de até 40% maior.

Antibiotic-resistance in E. coli is a global issue affecting humans especially the pediatric population. Antibiotic-resistant E. coli is a pathogen frequently pediatric population as well as healthy adults. This study aimed to examine the antibiotic resistance of E. coli causing pediatric diarrhea and its drug-resistant rates, its adhering abilities to cell line in vitro, and inhibition efficiency of a few selected chemical compounds. Clinical strains were isolated from both the healthy and infected pediatric population of Mizoram. Adhesion is a significant pathogenic process during bacterial infections, which has been employed for pathotyping of DEC by comparing adhesion efficiency in both normal (CHO-k1) and cancer (HeLa) cell lines. E. coli adherent pathotypes were identified by both PCR assay and in-vitro cell adhesion assays; the study also evaluated the adhesion inhibition ability of human skimmed milk, gentamicin, and cephalexin in-vitro. Of all isolates, 20.05% of adherent DEC (EPEC, DAEC, and EIEC) and 11.39 % of non-adherent DEC (STEC and ETEC) were found to be associated with pediatric diarrhoea in Mizoram. Human skimmed milk has a high potential adhesion inhibition against EAEC (50.25/90.90 μg/mL), EPEC (53.42/259.70 μg/mL), and EIEC (59.13/30.30 μg/mL) in both cell lines in comparison with gentamicin and cephalexin. This study concludes that as a dietary supplement-human skimmed milk has high potential to prevent adhesion of DEC pathotypes in cells in-vitro thus in in-vivo. [ABSTRACT FROM AUTHOR]

The b2 auxiliary subunit of voltage gated sodium channels (VGSCs) appears at early stages of brain development. It is abundantly expressed in the mammalian central nervous system where it forms complexes with different channel isoforms, including Nav1.2. From the structural point of view, b2 is a transmembrane protein: at its extracellular N-terminus an Ig-like type C2 domain mediates the binding to the pore forming alpha subunit with disulfide bonds and the interactions with the extracellular matrix. Given this structural versatility, b2 has been suggested to play multiple functions ranging from channel targeting to the plasma membrane and gating modulation to control of cell adhesion. We report that, when expressed in Chinese Hamster Ovary cells CHO-K1, the subunit accumulates at the perimetral region of adhesion and particularly in large lamellipodia-like membrane processes where it induces formation of filopodia-like structures. When overexpressed in developing embryonic rat hippocampal neurons in vitro, b2 specifically promotes formation of filopodia-like processes in dendrites leading to expansion of the arborisation tree, while axonal branching remains unaltered. In contrast to this striking and highly specific effect on dendritic morphology, the targeting of functional sodium channels to the plasma membrane, including the preferential localization of Nav1.2 at the axon, and their gating properties are only minimally affected. From these and previously reported observations it is suggested that b2, among its multiple functions, may contribute to promote dendritic outgrowth and to regulate neuronal wiring at specific stages of neuronal development.

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. [ABSTRACT FROM AUTHOR]

Microcarriers provide large adhesion area allowing high cell densities in bioreactor systems. This study focused on the investigation of cell adhesion and cell growth characteristics of both anchorage-dependent CHO-K1 and anchorage-independent Ag8 myeloma cell lines cultivated on four different microcarriers (Biosilon®, Microhex®, Cytodex 3®, Cytoline 2®) by considering the cell kinetics and physiological data. Experiments were performed in both static and agitated cell culture systems by using 24-well tissue culture plates and then 50-ml spinner flasks. In agitated cultures, the highest specific growth rates (0.026 h for CHO-K1 and 0.061 h for Ag8 cell line) were obtained with Cytodex 3® and Cytoline 2® microcarriers for CHO-K1 and Ag8 cell line, respectively. Metabolic characteristics showed some variation among the cultures with the four microcarriers. The most significant being the higher production of lactate with microcarriers with CHO-K1 cells relative to the Ag8 cells. SEM analyses revealed the differences in the morphology of the cells along with microcarriers. On Cytodex 3® and Cytoline 2®, CHO-K1 cells attached to the substratum through long, slender filopodia, whereas the cells showed a flat morphology by covering the substratum on the Biosilon® and Microhex®. Ag8 cells maintained their spherical shapes throughout the culture for all types of microcarriers. In an attempt to scale-up, productions were carried out in 50-ml spinner flasks. Cytodex 3® (for CHO-K1 cells) and Cytoline 2® (for Ag8 cells) were evaluated. The results demonstrate that high yield of biomass could be achieved through the immobilization of the cells in each culture system. And cell cultures on microcarriers, especially on Cytodex 3® and Cytoline 2®, represented a good potential as microcarriers for larger scale cultures of CHO-K1 and Ag8, respectively. Moreover, owing to the fact that the cell lines and culture media are specific, outcomes will be applicable for other clones derived from the same host cell lines. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-a and epoetin-ß, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIVAIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-a (DARB) which possesses two additional Nlinked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 Nlinked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation. [ABSTRACT FROM AUTHOR]