scholarly journals A DNAmCULTURE Epigenetic Fingerprint Recapitulates Physiological Aging

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 5-6
Author(s):  
Christopher Minteer ◽  
Marco Morselli ◽  
Margarita Meer ◽  
Jian Cao ◽  
Sabine Lang ◽  
...  
Keyword(s):  
Cultured Cells ◽  
In Vitro Models ◽  
Epigenetic Alterations ◽  
Physiological Aging ◽  
Biomarkers Of Aging ◽  
Epigenetic Aging ◽  
Passage Number ◽  
Aging Phenomena ◽  
Lung And Kidney

Abstract Aging elicits dramatic changes to DNA methylation (DNAm), however the causes and consequences of such alterations to the epigenome remain unclear. The utility of biomarkers of aging based on DNAm patterns would be greatly enhanced if in vitro models existed that recapitulated physiological phenotypes such that modulation could garnish mechanistic insights. Using DNAm from serially passaged mouse embryonic fibroblasts, we developed a marker of culture aging and asked if culture phenotypes, like exhaustive replication, are epigenetically analogous to physiological aging. Our measure, termed DNAmCULTURE, accurately estimated passage number and was shown to strongly increase with age when examined in multiple tissues. Furthermore, we observed epigenetic alterations indicative of early cultured cells in animals undergoing caloric restriction and in lung and kidney fibroblasts re-programmed to iPSCs. This study identifies culture-derived alterations to the methylome as physiologically relevant and implicates culture aging as an important feature in known epigenetic aging phenomena.

scholarly journals A DNAmRep epigenetic fingerprint for determining cellular replication age

2020 ◽  
Author(s):  
Christopher Minteer ◽  
Marco Morselli ◽  
Margarita Meer ◽  
Jian Cao ◽  
Sabine Lang ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Enrichment Analysis ◽  
T Antigen ◽  
Drug Induced ◽  
Chromatin Regulators ◽  
Biomarkers Of Aging ◽  
Epigenetic Aging ◽  
Passage Number ◽  
Cellular Replication ◽  
Aging Phenomena

AbstractAging is known to elicit dramatic changes to DNA methylation (DNAm). However, the causes and consequences of such alterations to the epigenome remain unclear. Therefore, the utility of biomarkers of aging based on DNAm patterns will depend on our ability to link them to cellular features, such as proliferation, differentiation, transcriptional repression/activation, senescence, and transformation. Using DNAm from serially passaged mouse embryonic fibroblasts (MEFs), we developed a predictor of replication that is able to accurately predict passage number in independent sample. Our measure, termed DNAmRep, was shown to strongly increase with age when examined in multiple tissues (blood, liver, kidney, lung and adipose). Furthermore, we observed replicative deceleration in animal undergoing caloric restriction. Upon reprogramming to iPSCs, cells derived from both lung and kidney fibroblasts exhibited resetting of our DNAmRep measure. This measure also increased in response to differentiation among B-cell populations. Enrichment analysis implicated CBX7, RNF2 and HDAC2 as potentially important transcription factors and chromatin regulators in the DNAm replication signature. Finally, DNAmRep correlated with beta-galactosidase activity in replicative senescent MEFs, but not in radiation or drug induced senescent MEFs, and was partially separated by immortalization with Large T antigen K1 mutant (LTK1), suggesting that it reflects mitotic history, rather than senescent state. Overall, this study identifies mitotically-derived alterations to the methylome, which partially underlie known epigenetic aging phenomena.

scholarly journals Reappraisal of Human HOG and MO3.13 Cell Lines as a Model to Study Oligodendrocyte Functioning

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1096 ◽  
Author(s):  
Kim M. A. De Kleijn ◽  
Wieteke A. Zuure ◽  
Jolien Peijnenborg ◽  
Josje M. Heuvelmans ◽  
Gerard J. M. Martens
Keyword(s):  
Schwann Cells ◽  
Cell Lines ◽  
Myelin Sheath ◽  
Cultured Cells ◽  
In Vitro Models ◽  
Brain Functioning ◽  
Disease Mechanisms ◽  
Sheath Formation ◽  
Mrna Expression Levels

Myelination of neuronal axons is essential for proper brain functioning and requires mature myelinating oligodendrocytes (myOLs). The human OL cell lines HOG and MO3.13 have been widely used as in vitro models to study OL (dys) functioning. Here we applied a number of protocols aimed at differentiating HOG and MO3.13 cells into myOLs. However, none of the differentiation protocols led to increased expression of terminal OL differentiation or myelin-sheath formation markers. Surprisingly, the applied protocols did cause changes in the expression of markers for early OLs, neurons, astrocytes and Schwann cells. Furthermore, we noticed that mRNA expression levels in HOG and MO3.13 cells may be affected by the density of the cultured cells. Finally, HOG and MO3.13 co-cultured with human neuronal SH-SY5Y cells did not show myelin formation under several pro-OL-differentiation and pro-myelinating conditions. Together, our results illustrate the difficulty of inducing maturation of HOG and MO3.13 cells into myOLs, implying that these oligodendrocytic cell lines may not represent an appropriate model to study the (dys)functioning of human (my)OLs and OL-linked disease mechanisms.

scholarly journals Cell-based in vitro models in environmental toxicology: a review

Biomonitoring ◽  
2017 ◽  
Vol 4 (1) ◽  
Author(s):  
Michael Poteser
Keyword(s):  
Cultured Cells ◽  
Biological Effects ◽  
Epidemiological Data ◽  
In Vitro Models ◽  
Environmental Toxicology ◽  
In Vitro Assays ◽  
Cellular Effects ◽  
Potential Risks ◽  
Cellular Pathways

AbstractAn analysis of biological effects induced by environmental toxins and exposure-related evaluation of potential risks for health and environment represent central tasks in classical biomonitoring. While epidemiological data and population surveys are clearly the methodological frontline of this scientific field, cellbased in vitro assays provide information on toxin-affected cellular pathways and mechanisms, and are important sources for the identification of relevant biomarkers. This review provides an overview on currently available in vitro methods based on cultured cells, as well as some limitations and considerations that are of specific interest in the context of environmental toxicology. Today, a large number of different endpoints can be determined to pinpoint basal and specific toxicological cellular effects. Technological progress and increasingly refined protocols are extending the possibilities of cell-based in vitro assays in environmental toxicology and promoting their increasingly important role in biomonitoring.

scholarly journals Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Michael J. Burgum ◽  
Martin J. D. Clift ◽  
Stephen J. Evans ◽  
Nicole Hondow ◽  
Afshin Tarat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cultured Cells ◽  
Alveolar Epithelial Cell ◽  
Health And Safety ◽  
In Vitro Models ◽  
Type I ◽  
Toxicological Evaluation ◽  
Layer Graphene ◽  
Few Layer Graphene

Abstract Background Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. Conclusions Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

Endothelial cell monolayers as a tool for studying microvascular pathophysiology

1997 ◽  
Vol 273 (6) ◽  
pp. G1189-G1199 ◽  
Author(s):  
Peter R. Kvietys ◽  
D. Neil Granger
Keyword(s):  
Endothelial Cell ◽  
In Vitro Model ◽  
Cultured Cells ◽  
Critical Role ◽  
In Vitro Models ◽  
Cell Monolayers ◽  
Biological Responses ◽  
Vitro Model

Endothelial cells contribute to a variety of biological responses that facilitate organ function. This critical role of the endothelial cell has resulted in the development of different in vitro models that utilize monolayers of cultured cells to simulate conditions that exist in the intact animal. This review focuses on endothelial cell monolayers as a model system for research on certain pathophysiological conditions affecting the gastrointestinal tract. The advantages and limitations of endothelial cell monolayers are addressed, along with evolving technologies and strategies that hold promise for extending the utility of this in vitro model for studies of gastrointestinal function and disease.

scholarly journals Cytotoxicity Studies on Naproxen and Piroxicam Nanoformulations

2020 ◽  
Vol 8 (3) ◽  
pp. 87-94
Author(s):  
Sandeep Patnaik ◽  
K Madhusudhana Rao ◽  
Vijay Sai
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Cultured Cells ◽  
In Vitro Models ◽  
In Vivo Studies ◽  
Tetrazolium Salt ◽  
Mucous Layer ◽  
Cytotoxicity Studies

Caco-2 cells were used as in vitro models to assess the cell viability characteristics of the carriers Soluplus®, Gelucire 50/13 and PVP K25 and the nanoformulations of Naproxen and Piroxicam. The assessment of cell viability was done using the tetrazolium salt based MTT assay. Gelucire 50/13 and its NFs were observed to have slightly higher cytotoxicity than PVP and Soluplus® and their respective NFs. All the NFs were observed to follow the cytotoxicity trend of the polymers. Our results show that no significant decrease in cell viability was seen until 0.01% concentration of Gelucire 50/13 for 12-h exposure. The NFs as well as the polymers alone had no significant effect on the viability of Caco-2 cells below 0.01% concentrations. The intestine has a protective mucous layer, whereas the cell culture monolayers do not. The intestinal tissues also have more capacity to recover from trauma than the cultured cells. Hence the present NFs can be expected to show lesser cytotoxicity when subjected to in vivo studies.  

scholarly journals Aging Biomarkers for Clinical Trials and Drug Discovery

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 4-5
Author(s):  
Margarita Meer ◽  
Raghav Sehgal ◽  
Morgan Levine
Keyword(s):  
Clinical Trials ◽  
Initial Step ◽  
Biological Aging ◽  
Working Definition ◽  
Biomarkers Of Aging ◽  
Epigenetic Aging ◽  
Aging Mechanisms ◽  
Definition Of

Abstract Developing targeted therapies first requires a working definition of the condition of interest. Unfortunately for aging, this very initial step poses a challenge since chronological age is often not indicative of biological age nor modifiable. This symposium will demonstrate the enormous progress being made towards developing more reliable and valid measures for quantifying biological aging. First, Dr. Albert T. Higgins Chen will show how inaccuracy caused by noise at individual CpG sites can lead to high technical variability in the most widely applied biomarkers of aging—epigenetic clocks. He will further discuss how this can be overcome through novel statistical techniques. Second, Dr. Benoit Lehallier, will discuss plasma proteomic clocks and share insights into their potential roles in Alzheimer's disease and utilization in clinical trials. Third, quantifying the multifactorial aging process can be facilitated by projects incorporating multimodal biomarker data. Pei-Lun Kuo from the Baltimore Longitudinal Study of Aging will present an analysis of longitudinal trajectories of more than 30 phenotypes, which when combined into a single summarized score yield important insights. Fourth, our ability to uncover aging mechanisms and perform drug screens, requires valid and reliable measures that can be applied in vitro. Christopher Minteer who developed in cellulo epigenetic markers will demonstarte how epigenetic aging changes that can be induced in culture shed light on aging in vivo. Finally, a summarizing discussion will be held by Dr. Nir Barzilai, an expert in the field, who is leading the Targeting Aging with Metformin (TAME) clinical trial.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Analysis and applicability of different in vitro models of Glioblastoma multiforme

2014 ◽  
Vol 226 (06) ◽  
Author(s):  
D William ◽  
M Linnebacher ◽  
CF Classen
Keyword(s):  
Glioblastoma Multiforme ◽  
In Vitro Models
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