Non-Invasive Real-time Monitoring of Membrane Cholesterol Dynamics in Human Keratinocytes with Roche's xCELLigence System

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In a recent study initiated by Beiersdorf AG (1), Florian Spörl and colleagues evaluated the use of Roche Applied Science's (SIX: RO, ROG; OTCQX: RHHBY) xCELLigence System as a viable system to monitor not only cholesterol extraction and repletion but also cholesterol reorganization in human keratinocytes in real time. The xCELLigence System offers a novel approach to noninvasive longterm observation of membrane cholesterol dynamics in primary human keratinocytes. This realtime cell analysis system measures alterations in cellular impedance using microelectrodes on the surface of a 96well culture plate (E-Plate). Spörl et al. were able to analyze the effects of membrane cholesterol reorganization on proliferation and differentiation, and their findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and late keratinocyte differentiation.

Cholesterol within biological membranes is organized in distinct liquidordered microdomains 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 organization in plasma membranes remains challenging - the techniques available are simply limited. Spörl and colleagues are now the first to use the impedancebased xCELLigence System to monitor noninvasively and in real time membrane cholesterol reorganization and keratinocyte cell behavior. The authors note that the use of this realtime 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 realtime 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, Spörl et al. also described a correlation between cellular impedance and cholesteroldependent lateral mobility in lipid rafts. They were then able to analyze physiological effects of the reorganization 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 calciumdependent morphological changes. According to the Beiersdorf study, these differentiationrelated effects could also be visualized using cellular impedance measurements and the xCELLigence System. They were able to further analyze 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 organization. Lipidraft mediated signaling might play an additional role in this regulatory process. Remarkably, these results were complemented by observations of differentiationdependent morphological changes using the xCELLigence System.

(1) Spörl F, Wunderskirchner M, Ullrich O, Bömke G, Breitenbach U, Blatt T, Wenck H, Wittern KP, Schrader A. Real-Time Monitoring of Membrane Cholesterol Reveals New Insights into Epidermal Differentiation. J Invest Dermatol 2009 Dec 31. [Epub ahead of print] PMID: 20043016

About Beiersdorf AG

Beiersdorf AG is a cosmetics company headquartered in Hamburg, Germany, that employs more than 20,000 people worldwide and that generated sales of €5.748 billion in 2009. The company has been listed in the DAX since December 2008. Its flagship Nivea brand is the world's largest skin and beauty care brand.* Other names in its internationally successful brand portfolio include Eucerin, La Prairie, Labello, 8x4, and Hansaplast/Elastoplast. The affiliate tesa SE is one of the world's leading manufacturers of selfadhesive product and system solutions for industry, trade, and consumers. Beiersdorf has more than 125 years' experience in skin and beauty care and stands for innovative and highquality products.

* Source: Euromonitor, "Skin and Beauty Care Products excluding Scents and Hair Dyes by Sales, 2008"
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