scholarly journals Dnmt3a-Mediated DNA Methylation Changes Regulate Osteogenic Differentiation of hMSCs Cultivated in the 3D Scaffolds under Oxidative Stress

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Liangping Li ◽  
Zemin Ling ◽  
Wenwu Dong ◽  
Xiaoying Chen ◽  
Corina Vater ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Methylation ◽  
Cell Culture ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
3D Culture ◽  
Type I ◽  
3D Scaffolds ◽  
Culture Systems ◽  
Cell Culture Systems

Oxidative stress (OS) caused by multiple factors occurs after the implantation of bone repair materials. DNA methylation plays an important role in the regulation of osteogenic differentiation. Moreover, recent studies suggest that DNA methyltransferases (Dnmts) are involved in bone formation and resorption. However, the effect and mechanism of DNA methylation changes induced by OS on bone formation after implantation still remain unknown. Three-dimensional (3D) cell culture systems are much closer to the real situation than traditional monolayer cell culture systems in mimicking the in vivo microenvironment. We have developed porous 3D scaffolds composed of mineralized collagen type I, which mimics the composition of the extracellular matrix of human bone. Here, we first established a 3D culture model of human mesenchymal stem cells (hMSCs) seeded in the biomimetic scaffolds using 160 μM H2O2 to simulate the microenvironment of osteogenesis after implantation. Our results showed that decreased methylation levels of ALP and RUNX2 were induced by H2O2 treatment in hMSCs cultivated in the 3D scaffolds. Furthermore, we found that Dnmt3a was significantly downregulated in a porcine anterior lumbar interbody fusion model and was confirmed to be reduced by H2O2 treatment using the 3D in vitro model. The hypomethylation of ALP and RUNX2 induced by H2O2 treatment was abolished by Dnmt3a overexpression. Moreover, our findings demonstrated that the Dnmt inhibitor 5-AZA can enhance osteogenic differentiation of hMSCs under OS, evidenced by the increased expression of ALP and RUNX2 accompanied by the decreased DNA methylation of ALP and RUNX2. Taken together, these results suggest that Dnmt3a-mediated DNA methylation changes regulate osteogenic differentiation and 5-AZA can enhance osteogenic differentiation via the hypomethylation of ALP and RUNX2 under OS. The biomimetic 3D scaffolds combined with 5-AZA and antioxidants may serve as a promising novel strategy to improve osteogenesis after implantation.

scholarly journals Propagation of Astrovirus VA1, a Neurotropic Human Astrovirus, in Cell Culture

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Andrew B. Janowski ◽  
Irma K. Bauer ◽  
Lori R. Holtz ◽  
David Wang
Keyword(s):  
Cell Culture ◽  
Fold Increase ◽  
Northern Blotting ◽  
Type I ◽  
Initial Inoculum ◽  
Culture Systems ◽  
Cell Culture Systems ◽  
Human Astroviruses ◽  
Human Astrovirus

ABSTRACT Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or in vivo models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that led to the discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 cells yielded increasing copies of VA1 RNA, and multistep growth curves demonstrated a >100-fold increase in VA1 RNA 72 h after inoculation. The full-length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ∼26-nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems, which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon (IFN-I) response, as VA1 RNA levels were reduced by pretreatment of Caco-2 cells with IFN-β1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1. IMPORTANCE Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.

Recent Progress in Prediction Systems for Drug-induced Liver Injury Using in vitro Cell Culture

2020 ◽  
Vol 14 ◽  
Author(s):  
Shogo Ozawa ◽  
Toshitaka Miura ◽  
Jun Terashima ◽  
Wataru Habano ◽  
Seiichi Ishida
Keyword(s):  
Cell Culture ◽  
Recent Progress ◽  
Inflammatory Cells ◽  
Protein Adducts ◽  
In Vitro Assays ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Culture Systems ◽  
Cell Culture Systems

Background: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. Objective: In this study, the recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as for the limitations of in vitro cell culture systems for DILI research. Methods: Information related to DILI was collected through a literature search of the PubMed database. Results: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4+ helper T lymphocytes and recognition by CD8+ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells, or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILIinducing drugs with approximately 50% sensitivity and 90% specificity. Conclusion: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.

scholarly journals Mammary gland 3D cell culture systems in farm animals

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Laurence Finot ◽  
Eric Chanat ◽  
Frederic Dessauge
Keyword(s):  
Cell Culture ◽  
Mammary Gland ◽  
Bacterial Infections ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Farm Animals ◽  
Culture Systems ◽  
Cell Culture Systems

AbstractIn vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

In vitro cytotoxicity of macromolecules in different cell culture systems

1995 ◽  
Vol 34 (3) ◽  
pp. 233-241 ◽  
Author(s):  
Suthummar Choksakulnimitr ◽  
Sada Masuda ◽  
Hideaki Tokuda ◽  
Yoshinobu Takakura ◽  
Mitsuru Hashida
Keyword(s):  
Cell Culture ◽  
In Vitro Cytotoxicity ◽  
Culture Systems ◽  
Cell Culture Systems

scholarly journals Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

2004 ◽  
Vol 78 (17) ◽  
pp. 9257-9269 ◽  
Author(s):  
Kevin C. Klein ◽  
Stephen J. Polyak ◽  
Jaisri R. Lingappa
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Signal Sequence ◽  
Buoyant Density ◽  
Capsid Assembly ◽  
Culture Systems ◽  
Capsid Formation ◽  
Cell Culture Systems

ABSTRACT The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of ≈100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.

A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions

2021 ◽  
Author(s):  
Mattia Saggioro ◽  
Stefania D'Agostino ◽  
Anna Gallo ◽  
Sara Crotti ◽  
Sara D'Aronco ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Matrix ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Culture Systems ◽  
Matrix Interactions ◽  
In Vitro Systems ◽  
Extracellular Matrix Interactions

Three-dimensional (3D) culture systems are progressively getting attention given their potential in overcoming limitations of the classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs)...

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