Roche Applied Science's Xcelligence system has been used to monitor cholesterol extraction and repletion and cholesterol re-organisation in human keratinocytes in real time.
The study was initiated by Beiersdorf.
The Xcelligence system is said to offer a novel approach to non-invasive long-term observation of membrane cholesterol dynamics in primary human keratinocytes.
The real-time cell analysis system measures alterations in cellular analysis alterations in cellular impedance using micro-electrodes on the surface of a 96-well culture plate (E-Plate).
The researchers were able to analyse the effects of membrane cholesterol re-organisation on proliferation and differentiation, and their findings imply a strict controlled mechanism for the regulation of membrane cholesterol composition in both early and late keratinocyte differentiation.
Cholesterol within biological membranes is organised in distinct liquid-ordered micro-domains, known as lipid rafts.
As was recently suggested, these lipid rafts play a role in cellular processes such as early differentiation and apoptosis in human keratinocytes.
To date, monitoring the dynamics of cholesterol organisation in plasma membranes remains challenging - the techniques available are simply limited.
The researchers are now the first to use the impedance-based Xcelligence system to monitor non-invasively and in real time membrane cholesterol re-organisation and keratinocyte cell behaviour.
The authors note that the use of this real-time cell analysis system will further enhance understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.
As a first step, the researchers evaluated that the real-time cell analysis system was a viable tool to monitor normal cell growth of primary keratinocytes.
They then looked at cholesterol extraction and repletion and found that alterations in cellular impedance could be correlated with changes in membrane cholesterol.
Not only did decreasing and increasing cellular impedance values represent extraction and repletion of cholesterol, the researchers also described a correlation between cellular impedance and cholesterol-dependent lateral mobility in lipid rafts.
They were then able to analyse physiological effects of the re-organisation of membrane cholesterol in more detail and saw that the proliferative capacity of primary keratinocytes was increased upon cholesterol depletion.
During the late stage of differentiation, keratinocytes undergo major calcium-dependent morphological changes.
According to the Beiersdorf study, these differentiation-related effects could also be visualised using cellular impedance measurements and the Xcelligence system.
They were able to further analyse the role of membrane cholesterol in late keratinocyte differentiation and identified keratin 2 as a previously unreported differentiation marker that is regulated by membrane cholesterol organisation.
Lipid-raft mediated signalling might play an additional role in this regulatory process.
These results were complemented by observations of differentiation-dependent morphological changes using the Xcelligence system.
