scholarly journals Strategic Framework for Parameterization of Cell Culture Models

Processes ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 174
Author(s):  
Pavlos Kotidis ◽  
Cleo Kontoravdi
Keyword(s):  
Experimental Data ◽  
Cell Culture ◽  
Sensitivity Analysis ◽  
Goodness Of Fit ◽  
Sampling Methods ◽  
Model Parameters ◽  
Sensitivity Index ◽  
Cho Cell ◽  
Culture Models ◽  
Cell Culture Models

Global Sensitivity Analysis (GSA) is a technique that numerically evaluates the significance of model parameters with the aim of reducing the number of parameters that need to be estimated accurately from experimental data. In the work presented herein, we explore different methods and criteria in the sensitivity analysis of a recently developed mathematical model to describe Chinese hamster ovary (CHO) cell metabolism in order to establish a strategic, transferable framework for parameterizing mechanistic cell culture models. For that reason, several types of GSA employing different sampling methods (Sobol’, Pseudo-random and Scrambled-Sobol’), parameter deviations (10%, 30% and 50%) and sensitivity index significance thresholds (0.05, 0.1 and 0.2) were examined. The results were evaluated according to the goodness of fit between the simulation results and experimental data from fed-batch CHO cell cultures. Then, the predictive capability of the model was tested against four different feeding experiments. Parameter value deviation levels proved not to have a significant effect on the results of the sensitivity analysis, while the Sobol’ and Scrambled-Sobol’ sampling methods and a 0.1 significance threshold were found to be the optimum settings. The resulting framework was finally used to calibrate the model for another CHO cell line, resulting in a good overall fit. The results of this work set the basis for the use of a single mechanistic metabolic model that can be easily adapted through the proposed sensitivity analysis method to the behavior of different cell lines and therefore minimize the experimental cost of model development.

scholarly journals Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

2021 ◽  
Author(s):  
Terry Riss ◽  
O. Joseph Trask
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Fluorescent Imaging ◽  
Culture Models ◽  
Assay Methods ◽  
Diffusion Rates ◽  
Cell Culture Models ◽  
Dimensional Cell ◽  
Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

scholarly journals Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

2006 ◽  
Vol 26 (17) ◽  
pp. 6425-6434 ◽  
Author(s):  
O. Jameel Shah ◽  
Tony Hunter
Keyword(s):  
Cell Culture ◽  
Tuberous Sclerosis ◽  
High Speed ◽  
Serine Phosphorylation ◽  
Feedback Signal ◽  
Insulin Receptor Substrates ◽  
Igf I ◽  
Culture Models ◽  
Cell Culture Models

ABSTRACT The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

Cell culture models of the ocular barriers

2005 ◽  
Vol 60 (2) ◽  
pp. 207-225 ◽  
Author(s):  
Margit Hornof ◽  
Elisa Toropainen ◽  
Arto Urtti
Keyword(s):  
Cell Culture ◽  
Culture Models ◽  
Cell Culture Models

Abstract 15951: Palmitate, Not Oleate, is the Preferred Fatty Acid for Cardiac Triacylglycerol Synthesis

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Stephen C Kolwicz ◽  
Rong Tian
Keyword(s):  
Cell Culture ◽  
13C Nmr Spectroscopy ◽  
Culture Studies ◽  
Triacylglycerol Synthesis ◽  
Culture Models ◽  
Contracting Heart ◽  
And Control ◽  
The Relationship ◽  
Cell Culture Models ◽  
Insight Into

Introduction: Previous studies using cell culture models identified cyto-toxic effects of palmitate and that supplementation with oleate was protective by redirecting palmitate into triacylglycerol (TAG) stores. However, other cull culture studies reported that diacylglycerol transferase 1 (DGAT1), the last enzyme in TAG synthesis, demonstrated a preference for oleate. At present, it is not clear whether the supply of exogenous fatty acids (FA) to the heart is differentially allocated into the endogenous TAG pool. Therefore, the purpose of the present study is to examine the influence of palmitate and/or oleate on cardiac TAG incorporation. METHODS/RESULTS: Hearts were isolated from DGAT1-transgenic (DGAT) and control littermates (CON) and perfused in Langendorff mode with a mixed substrate buffer consisting of glucose, lactate, insulin, and FAs. The FA supply was varied with 0.2mM of both labeled (13C) and unlabeled (12C) FAs in 4 different experiments: 1) 13C/12C palmitate; 2) 13C/12C oleate; 3) 13C palmitate/12C oleate; 4) 13C oleate/12C palmitate. The incorporation of 13C palmitate or 13C oleate into the TAG pool was monitored by 13C NMR spectroscopy. In CON hearts (n=3), the incorporation of palmitate was ~65% higher than oleate when the perfusate contained a homogenous supply of FA. This was also observed in DGAT hearts (n=4) although the incorporation of both palmitate and oleate was ~75% higher compared to CON (P <0.05). In the presence of oleate, palmitate incorporation decreased 25-30% in both CON and DGAT hearts. In contrast, oleate incorporation was diminished by ~50% and ~100% in CON and DGAT hearts, respectively, in the presence of palmitate. CONCLUSIONS: These data suggest that when palmitate and oleate are provided in equal concentrations, palmitate is more readily utilized in the synthesis of endogenous TAG stores in the heart. Furthermore, although overexpression of DGAT increases both oleate and palmitate incorporation, the DGAT1 enzyme demonstrates a preference for palmitate. These findings provide insight into the relationship between exogenous FA supply and endogenous TAG dynamics in the contracting heart.

scholarly journals Conjunctival melanoma and electrochemotherapy: preliminary results using 2D and 3D cell culture models in vitro

2018 ◽  
Vol 97 (4) ◽  
pp. e632-e640 ◽  
Author(s):  
Miltiadis Fiorentzis ◽  
Periklis Katopodis ◽  
Helen Kalirai ◽  
Berthold Seitz ◽  
Arne Viestenz ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Conjunctival Melanoma ◽  
Preliminary Results ◽  
Culture Models ◽  
2D And 3D ◽  
Cell Culture Models

scholarly journals Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models

Hepatology ◽  
2010 ◽  
Vol 53 (1) ◽  
pp. 32-41 ◽  
Author(s):  
Emmanuel Thomas ◽  
Jordan J. Feld ◽  
Qisheng Li ◽  
Zongyi Hu ◽  
Michael W. Fried ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Gene Induction ◽  
Culture Models ◽  
Cell Culture Models

scholarly journals Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Pirmin H. Lakner ◽  
Michael G. Monaghan ◽  
Yvonne Möller ◽  
Monilola A. Olayioye ◽  
Katja Schenke-Layland
Keyword(s):  
Cell Culture ◽  
Metabolic Activity ◽  
Three Dimensional ◽  
In Vitro Cell Culture ◽  
Culture Models ◽  
Cell Culture Models
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