Skip to main content Accessibility help
×
Home
Hostname: page-component-cf9d5c678-5wlnc Total loading time: 0.274 Render date: 2021-08-01T19:38:14.916Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "metricsAbstractViews": false, "figures": true, "newCiteModal": false, "newCitedByModal": true, "newEcommerce": true, "newUsageEvents": true }

Serum Choice Influences Lipid Accumulation and Cell Viability in Fatty Acid Treated Immortalised Hepatocytes and Hepatic Stellate Cells

Published online by Cambridge University Press:  19 October 2020

Z. Zhang
Affiliation:
School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT
J.L. Thorne
Affiliation:
School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT
J.B. Moore
Affiliation:
School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT
Rights & Permissions[Opens in a new window]

Abstract

Type
Abstract
Copyright
Copyright © The Authors 2020

Non-alcoholic fatty liver disease (NAFLD) is now the most common cause of liver disease in developed countries(Reference Moore1). Previous research has shown that vitamin D may reduce the inflammatory and pro-fibrogenic activity of hepatic stellate cells (HSCs) in vitro (Reference Udomsinprasert and Jittikoon2). However, the mechanisms underpinning the role of vitamin D in NAFLD pathogenesis, especially in HSCs, are not fully understood. With an overall objective of establishing a lipid loading model in immortalised hepatocytes (HepG2 cells) and HSCs (LX-2 cells); the aim of these experiments was to test the cell's responses to different fatty acid doses when cultured in either serum-containing medium (SCM), serum-free medium (SFM) or charcoal-stripped serum-containing medium (CSM).

Cells were routinely cultured in SCM and seeded to reach approximately 80% confluence prior to experimental treatment. Then, to test if there was interference from lipophilic materials contained in the serum, the SCM was either replenished, replaced with CSM, or replaced with SFM, and cells cultured for 16hrs in the fresh media prior to 6 h or 24 h fatty acid treatment (0-500μM, 1:1 oleic acid: palmitic acid). Nile red was used to measure total intracellular lipid, while cell viability was measured by MTT assay. Data were quantified relative to the cells cultured in SCM at each concentration and analysed by two-way ANOVA with Holm-Sidak test.

A significant effect of medium choice on cell viability was observed, with reductions seen in both cell lines treated in SFM at both timepoints (HepG2 n = 3, 6 h P = 0.0304, 24 h P < 0.0001; LX-2 n = 5, 6 h P < 0.0001, 24 h P < 0.0001). Loss of viability was particularly pronounced in LX-2 cells cultured in SFM for 24 h, where an ~40% reduction was observed (P < 0.0001) independent of any effects of fatty acid dose on viability (P = 0.3533). On the other hand, no differences in viability were detected between cells cultured in CSM compared to SCM.Fatty acid treatment led to dose-dependent intracellular lipid accumulation in both cell lines, however, effects from serum were observed. After 6 h of fatty acid treatment, cells treated in SFM or CSM had accumulated less lipid relative to SCM cultured cells (HepG2 n = 3, LX-2 n = 5, both P < 0.0001). While this reduction remained at 24 h for cells treated in SFM, there was no difference in lipid loading observed between SCM and CSM at this time point (HepG2 n = 3 P = 0.0379; LX-2 n = 5 P < 0.0001).

In summary, these experiments show CSM is a better choice, resulting in optimal cell viability and higher lipid loading in both cell lines after 24 h fatty acid treatment. Our results underscore the importance of serum choice in for in vitro lipid loading experiments. Using CSM for vitamin D and fatty acid cotreatment, ongoing experiments are examining mRNA, protein, and microRNA expression in hepatocytes and HSCs in response to vitamin D and lipid loading cotreatment.

References

Moore, JB (2019) Proc Nutr Soc 78, 290304.10.1017/S0029665119000570CrossRefGoogle Scholar
Udomsinprasert, W & Jittikoon, J (2019) Biomed Pharmacother 109, 1351–60.10.1016/j.biopha.2018.10.140CrossRefGoogle Scholar
You have Access

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Serum Choice Influences Lipid Accumulation and Cell Viability in Fatty Acid Treated Immortalised Hepatocytes and Hepatic Stellate Cells
Available formats
×

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

Serum Choice Influences Lipid Accumulation and Cell Viability in Fatty Acid Treated Immortalised Hepatocytes and Hepatic Stellate Cells
Available formats
×

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

Serum Choice Influences Lipid Accumulation and Cell Viability in Fatty Acid Treated Immortalised Hepatocytes and Hepatic Stellate Cells
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *