Measurement of LDL Receptor Activity in HepG2 Cells Grown on Microcarrier Beads

Dovey, Lynda Ann (1995). Measurement of LDL Receptor Activity in HepG2 Cells Grown on Microcarrier Beads. MPhil thesis The Open University.



Atherosclerosis- related conditions, coronary heart disease (CHD), stroke and peripheral arterial occlusions are the most important cause of death and invalidism in the Western world. A high plasma cholesterol level is a major risk factor for atherosclerosis. One of the main determinants of the plasma cholesterol level is the efficiency with which low density lipoprotein (LDL) is removed from the plasma via the hepatic LDL receptor. It is therefore important to study the factors controlling the expression of the receptor.

In the past, human hepatocytes have been grown in vitro as a monolayer culture in tissue culture dishes. This system permits the identification of substances that regulate the receptor activity but is limited as a research tool because substances secreted by the cells, such as bile acids and LDL itself, may cause feedback regulation of the pathway.

This thesis describes an alternative approach, in which HepG2 cells are grown on microcarrier beads which can then be packed into a column so that substances secreted by the cells can be removed by perfusion.

The study consists of four main sections:- i) Optimising conditions for establishing HepG2 cells on Cytodex microcarrier beads (discussed in Chapter 3); ii) Development of a column system for perfusion, either constant fresh medium (single pass) or by medium being constantly recycled (reperfusion) (discussed in Chapter 4); iii) Investigation of ligands which were available for the measurement of the LDL receptor to find the most suitable for use in this system (discussed in Chapter 5); iv) Perfusion column system,where some of the effects of single pass perfusion and re-perfusion were observed (discussed in Chapter 6).

HepG2 cells grown on Cytodex 2 microcarrier beads and perfused over various time periods were found to express lower LDL receptor activity when fed by re-perfusion compared to single pass perfusion. This did not appear to be due to depletion of essential nutrients, but to substances secreted by cells.

25 hydroxycholesterol used in this system was shown to regulate the LDL receptor. However, the extent of regulation was found to alter depending on whether the cells were fed by single pass perfusion or re-perfusion.

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