scholarly journals Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components during Fibroblast Passage of Human Cytomegalovirus

2018 ◽  
Author(s):  
Amine Ourahmane ◽  
Xiaohong Cui ◽  
Li He ◽  
Dirk Dittmer ◽  
Mark Schleiss ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Culture Media ◽  
Adaptive Mutations ◽  
Culture Passage ◽  
Genes Encoding ◽  
Cell Culture Passage

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly during cell culture passage, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt expression of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As in vivo CMV replicates in the context of host antibodies, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and aquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. An epitheliotropic RL13+/ UL131A+ virus was isolated by limiting-dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than has been previously possible.

scholarly journals Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 221 ◽  
Author(s):  
Amine Ourahmane ◽  
Xiaohong Cui ◽  
Li He ◽  
Meaghan Catron ◽  
Dirk Dittmer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Cultured Cells ◽  
Culture Media ◽  
Single Amino Acid ◽  
Adaptive Mutations ◽  
Glycoprotein Complex ◽  
Genes Encoding

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.

scholarly journals The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

2013 ◽  
Vol 2013 ◽  
pp. 1-22 ◽  
Author(s):  
Ana Lúcia Vargas Arigony ◽  
Iuri Marques de Oliveira ◽  
Miriana Machado ◽  
Diana Lilian Bordin ◽  
Lothar Bergter ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Culture Media ◽  
Genomic Stability ◽  
Sole Source ◽  
Cell Culture Media ◽  
The Media ◽  
Fetal Bovine

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic thein vivoenvironment, providingin vitromodels used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previousin vitroexperiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

scholarly journals Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

1999 ◽  
Vol 340 (1) ◽  
pp. 309-320 ◽  
Author(s):  
Sikha Bettina MUKHERJEE ◽  
S. ARAVINDA ◽  
B. GOPALAKRISHNAN ◽  
Sushma NAGPAL ◽  
Dinakar M. SALUNKE ◽  
...  
Keyword(s):  
Cell Culture ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Culture Media ◽  
Glutathione S Transferase ◽  
Steroid Binding ◽  
Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

2007 ◽  
Vol 81 (23) ◽  
pp. 13168-13179 ◽  
Author(s):  
Artur Kaul ◽  
Ilka Woerz ◽  
Philip Meuleman ◽  
Geert Leroux-Roels ◽  
Ralf Bartenschlager
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Infectious Hepatitis ◽  
Adaptive Mutation ◽  
Viral Genomes ◽  
Adaptive Mutations ◽  
Spread Of Infection

ABSTRACT Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

scholarly journals Improving the metabolic fidelity of cancer models with a physiological cell culture medium

2019 ◽  
Vol 5 (1) ◽  
pp. eaau7314 ◽  
Author(s):  
Johan Vande Voorde ◽  
Tobias Ackermann ◽  
Nadja Pfetzer ◽  
David Sumpton ◽  
Gillian Mackay ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cancer Cells ◽  
Culture Media ◽  
Hypoxia Inducible Factor ◽  
Commercial Media ◽  
Physiological Medium ◽  
Cancer Models ◽  
Cancer Spheroids

Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models.

Sedimentation-Diffusion Behaviour of Nanoparticles

2020 ◽  
Author(s):  
francesco giorgi ◽  
Peter Macko ◽  
Judith M. Curran ◽  
Maurice Whelan ◽  
Andrew Worth ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gold Nanoparticles ◽  
Theoretical Model ◽  
Cultured Cells ◽  
Culture Media ◽  
Biological Response ◽  
Optical Microscope ◽  
Local Concentration ◽  
Nominal Concentration

Abstract Background The biological response of organisms exposed to nanoparticles is often studied in vitro using adherent monolayers of cultured cells. In order to derive accurate concentration-response relationships, it is important to determine the local concentration of nanoparticles to which the cells are actually exposed rather than the nominal concentration of nanoparticles in the cell-culture medium. In this study, we investigated the sedimentation–diffusion process of different sized and charged gold nanoparticles in vitro by evaluating their settling dynamics and by developing a theoretical model to predict the concentration depth-profile of nanoparticles in solution over time.Results Experiments were carried out in water and in cell-culture media at a range of controlled temperatures. The optical phenomenon of caustics was exploited to track nanoparticles in real-time in a conventional optical microscope without any requirement for fluorescent labelling that potentially affects the dynamics of the nanoparticles. The results obtained demonstrate that size, temperature, and the stability of the nanoparticles play a pivotal role in regulating settling dynamics of nanoparticles. For gold nanoparticles larger than 60 nm in diameter, the initial nominal concentration did not accurately represent the concentration of nanoparticles local to the cells.Conclusion Nanoparticles dynamics in solution regulate the amount of material at the cellular level and must be taken into account when evaluating the biological response of organisms. the theoretical model proposed in this study accurately described the settling dynamics of the nanoparticles and thus represents a promising tool to support the design of in vitro experiments and the study of concentration-response relationships.

scholarly journals Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2483
Author(s):  
Anik Karan ◽  
Elnaz Khezerlou ◽  
Farnaz Rezaei ◽  
Leon Iasemidis ◽  
Mark A. DeCoster
Keyword(s):  
Cell Culture ◽  
Regression Analysis ◽  
Multivariate Regression ◽  
Culture Media ◽  
Morphological Changes ◽  
Multivariate Regression Analysis ◽  
Cell Number ◽  
Polymerization Process

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

scholarly journals In vitro activities of azithromycin, clarithromycin, and other antibiotics against Chlamydia pneumoniae.

1996 ◽  
Vol 40 (11) ◽  
pp. 2669-2670 ◽  
Author(s):  
C C Kuo ◽  
L A Jackson ◽  
A Lee ◽  
J T Grayston
Keyword(s):  
Cell Culture ◽  
Chlamydia Pneumoniae ◽  
Complete Inhibition ◽  
Free Medium ◽  
Inclusion Formation ◽  
Culture Passage ◽  
Quinolone Antibiotics ◽  
Intracellular Concentrations ◽  
Cell Culture Passage

The in vitro susceptibilities of Chlamydia pneumoniae isolates to macrolide, tetracycline, and quinolone antibiotics were determined. Tetracycline, clarithromycin, and erythromycin had the lowest MICs in the first cell culture passage. Azithromycin required the lowest concentration for complete inhibition of inclusion formation on the second pass into antibiotic-free medium, likely reflecting its high intracellular concentrations.

scholarly journals In Vitro Bioavailability of the Hydrocarbon Fractions of Dimethyl Sulfoxide Extracts of Petroleum Substances

2020 ◽  
Vol 174 (2) ◽  
pp. 168-177 ◽  
Author(s):  
Yu-Syuan Luo ◽  
Kyle C Ferguson ◽  
Ivan Rusyn ◽  
Weihsueh A Chiu
Keyword(s):  
Cell Culture ◽  
Protein Binding ◽  
Aromatic Compounds ◽  
Culture Media ◽  
In Vitro Testing ◽  
Cell Culture Media ◽  
Chemical Profiles ◽  
In Vivo Extrapolation

Abstract Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

Sign in / Sign up

Export Citation Format

Share Document