Effects of Succinic Acid Supplementation on Stable Cell Line Growth, Aggregation, and IgG and IgA Production

2020 ◽  
Vol 21 (10) ◽  
pp. 990-996
Author(s):  
Victoria Argentova ◽  
Teimur Aliev ◽  
Dmitry Dolgikh ◽  
Mikhail Kirpichnikov
Keyword(s):  
Cell Culture ◽  
Succinic Acid ◽  
Cell Lines ◽  
Recombinant Antibody ◽  
Lactate Production ◽  
Culture Method ◽  
Cell Aggregation ◽  
Stable Cell Line ◽  
Acid Supplementation ◽  
Stable Cell Lines

Background: Immunoglobulin (Ig) G is the most commonly used therapeutic antibodies. Recently, the interest in IgA antibodies to treat respiratory infectious diseases has been increasing. The reason for the inefficient use of IgA is recombinant antibody aggregation in cell culture, affecting the longevity and productivity of cell lines. Lactate is an important metabolite that affects the cultivation of stable cell lines producing monoclonal antibodies. Methods: In the present study, we investigated whether different combinations of succinic acid and micro-additives affect lactate production, which correlates with productivity. The effect of succinic acid substitution on productivity of cells producing IgG/IgA was analyzed using the static culture method in a six-well plate. Lactate was measured in supernatant of cell culture indirectly by using the activity of Lactate Dehydrogenase (LDH).A low lactate level was observed in cultivation medium supplemented with succinic acid or asparagine combined with some inorganic salts. Result: The results also demonstrated the effect of component supplementation on homogeneity, longevity, and productivity of cell culture. Supplementation of succinic acid eliminated cell aggregation and improved homogeneity of stable cell lines producing IgG and, especially, IgA. Conclusion: Overall, succinic acid supplementation to the culture medium has potential biotechnological applications in the production IgG and IgA.

scholarly journals Effect of Sialic Acid on Mammalian Cell Culture and Protein Expression: A Potential Productivity Enhancer for Biopharmaceutical Cell Culture Processes

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1449
Author(s):  
Xiangsong Chen ◽  
Shang Xiao ◽  
Jinyong Wu ◽  
Jianming Yao
Keyword(s):  
Cell Culture ◽  
Sialic Acid ◽  
Cell Lines ◽  
Hek293 Cell ◽  
Chinese Hamster ◽  
Lactate Production ◽  
Production Costs ◽  
Cell Culture Process ◽  
Cho Cell ◽  
Potential Productivity

Improved productivity of the two most commonly used cell lines in the biopharmaceutical industry, such as human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO), could reduce production costs and increase manufacturing capacity. One method for increasing protein productivity is the addition of antioxidants during the cell culture process. In this study, we examined the effect of sialic acid (SA) on one HEK293 cell line and two CHO cell lines. The addition of SA to HEK293 cell led to a higher viable cell density (VCD), viability (Via), and a lower lactate content in the later stage of cultures. Further results showed that SA reduced the reactive oxygen species (ROS), improved cell viability, reduced lactate production, and increased antibody expression by more than 20% in the later stage of the two CHO cell lines cultures. Besides, an optimized dose of SA had no significant effect on acidic variants level aggregation level, N-linked glycosylation pattern, and SA content on antibodies. These results suggest that the addition of SA can improve the productivity of biopharmaceutical cell culture processes.

scholarly journals Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

2017 ◽  
Author(s):  
Roman J. Szczesny ◽  
Katarzyna Kowalska ◽  
Kamila Klosowska-Kosicka ◽  
Aleksander Chlebowski ◽  
Ewelina P. Owczarek ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Efficiency ◽  
Protein Localization ◽  
Interaction Network ◽  
Bimolecular Fluorescence Complementation ◽  
Stable Cell Line ◽  
Dna Cloning ◽  
Stable Cell Lines ◽  
Mammalian Cell Lines ◽  
Recombination System

ABSTRACTDeciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

scholarly journals PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research

2021 ◽  
Vol 11 (9) ◽  
pp. 3729
Author(s):  
Katarzyna Balon ◽  
Benita Wiatrak
Keyword(s):  
Cell Culture ◽  
Dna Damage ◽  
Cell Lines ◽  
Inflammatory Factor ◽  
Research Model ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Primary Cell Lines ◽  
Neurobiological Research ◽  
The Impact

Models based on cell cultures have become a useful tool in modern scientific research. Since primary cell lines are difficult to obtain and handle, neoplasm-derived lines like PC12 and THP-1 offer a cheap and flexible solution for neurobiological studies but require prior differentiation to serve as a neuronal or microglia model. PC12 cells constitute a suitable research model only after differentiation by incubation with nerve growth factor (NGF) and THP-1 cells after administering a differentiation factor such as phorbol 12-myristate-13-acetate (PMA). Still, quite often, studies are performed on these cancer cells without differentiation. The study aimed to assess the impact of PC12 or THP-1 cell differentiation on sensitivity to harmful factors such as Aβ25-35 (0.001–5 µM) (considered as one of the major detrimental factors in the pathophysiology of Alzheimer’s disease) or lipopolysaccharide (1–100 µM) (LPS; a pro-inflammatory factor of bacterial origin). Results showed that in most of the tests performed, the response of PC12 and THP-1 cells induced to differentiation varied significantly from the effect in undifferentiated cells. In general, differentiated cells showed greater sensitivity to harmful factors in terms of metabolic activity and DNA damage, while in the case of the free radicals, the results were heterogeneous. Obtained data emphasize the importance of proper differentiation of cell lines of neoplastic origin in neurobiological research and standardization of cell culture handling protocols to ensure reliable results.

scholarly journals Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Bryan ◽  
Michael Henry ◽  
Ronan M. Kelly ◽  
Christopher C. Frye ◽  
Matthew D. Osborne ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Density ◽  
Cell Lines ◽  
Viable Cell ◽  
Viable Cell Density ◽  
High Peak ◽  
Proteomic Profiling ◽  
Cho Cell ◽  
Extended Culture ◽  
Key Pathways

Abstract Background The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. Results We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. Conclusion This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

scholarly journals Cultured Cell Sublines Highly Susceptible to Prion Infection

2000 ◽  
Vol 74 (9) ◽  
pp. 4377-4386 ◽  
Author(s):  
Patrick J. Bosque ◽  
Stanley B. Prusiner
Keyword(s):  
Cell Culture ◽  
Infected Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Uninfected Cell ◽  
Resistant Cell ◽  
Cultured Cell ◽  
Prion Infection ◽  
Blot Technique ◽  
Prion Propagation

ABSTRACT Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrPSc). In order to derive cell lines producing sufficient quantities of PrPSc for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrPSc. Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrPSc levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.

scholarly journals Isolation of a post-PKS C–C branching jadomycin from S. venezuelae ISP5230 in the presence of 8-aminooctanoic acid

2018 ◽  
Vol 96 (7) ◽  
pp. 760-764 ◽  
Author(s):  
Jeanna M. MacLeod ◽  
Stephanie M. Forget ◽  
Camilo F. Martinez-Farina ◽  
David L. Jakeman
Keyword(s):  
Natural Products ◽  
Amino Acid ◽  
Natural Product ◽  
Intramolecular Cyclization ◽  
Nucleophilic Addition ◽  
Culture Method ◽  
Amino Acid Supplementation ◽  
Acid Supplementation

The jadomycin family of natural products was first identified and characterized by Vining and co-workers at Dalhousie University in the 1990s. Herein, we report findings from a recently developed co-amino acid supplementation culture method with S. venezuelae ISP5230 using 8-aminooctanoic acid, where the major natural product was a jadomycin variant omitting an E-ring (1). These results reinforce that the 3a position is susceptible to nucleophilic addition by cellular metabolites in jadomycin biosynthesis when intramolecular cyclization is unfavorable. Further, the cytotoxicity data for several unsubstituted E-ring jadomycins are reported and discussed.

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