Year

2021

Degree Name

Master of Research

Department

School of Medicine

Abstract

Current methods associated with the culturing of cells (i.e. cell culture) do not supply adequate levels of polyunsaturated fatty acids (PUFA). The net result is the genesis of cells that are PUFA deficient and have a fatty acid (FA) composition unlike that of any natural cell. Cells in culture have twice the monounsaturated fatty acid (MUFA) and half the PUFA levels of cells in situ (Else, 2020). Cells in culture are often used as surrogates to reflect the functional characteristics of their primary counterpart (Kaur & Dufour, 2012), however, with a FA composition unlike that of any natural cell, this role is questionable. The aim of this study was to develop a lipid supplement that could be used in cell culture to increase the level of PUFA by supplying more essential fatty acids (EFA), linoleic acid (LA, 18:2n6) and α-linolenic acid (ALA, 18:3n3). In this study, human embryonic kidney cells (HEK293) were used, as kidney has a naturally high level of omega-6 (n6) fats. From the different combinations of fats that were trialed in this study, Soy Phosphatidylcholine (Soy PC) was most effective at changing the composition of HEK293 cells. This phospholipid provided both 18:2n6 (63%) and 18:3n3 (5.7%) in relative abundance. Optimised antioxidant protection using vitamins E and C (1:1 ratio) was implemented in the lipid supplement to prevent lipid peroxidation in PUFA-enriched culture media. Voltage-gated ion channels reside within the cell’s plasma membrane and are modulated by acute exposure to PUFAs (Elinder & Liin, 2017). HEK293 cells stably expressing the voltage gated sodium channel Nav1.5 (HEK.Nav1.5) were thus used to explore functional effects associated with fatty acid compositional changes. Supplementation with Soy PC in the presence of antioxidants, of HEK293 and HEK.Nav1.5 resulted in FA lipid profiles more indicative of kidney in situ (Martinez, 1992). These cells displayed a significant uptake in linoleic acid that is naturally high in kidney tissue, with a compensatory decrease in oleic acid (18:1n9, MUFA) without changes in saturated fatty acid composition upon lipid supplementation. Naïve (non-transfected) HEK293 cells were able to synthesise arachidonic acid (20:4n6, ARA) from 18:2n6, demonstrating the capacity of these cells to incorporate supplied PUFA, and further convert it into the long-chain n6 derivative, 20:4n6. HEK293.Nav1.5 cells followed the same trend as their non-transfected counterpart in regards to LA incorporation, but differed notably in their ability to increase their ARA level. Investigation of Nav1.5 channel function subsequent to lipid supplementation demonstrated a dose-dependent decrease in maximum conductance as well as a hyperpolarising shift in the voltage dependence of steady state inactivation consistent with previous reports (Bohannon et al., 2020; Kang & Leaf, 2000; Xiao et al., 1995). Therefore, in the presence of adequate EFA, HEK293 cells in culture revert to a fatty acid composition more indicative of their natural origin. The functional changes observed in Nav1.5 channel function highlight the importance of EFA remediation of cells in culture to provide a more accurate system to model membrane protein behaviour and ultimately cellular physiology.

FoR codes (2020)

3101 Biochemistry and cell biology

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.