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TY  - THES
AU  - Afanasenkau, Dzmitry
TI  - Supported lipid bilayer as a biomimetic platform for neuronal cell culture
VL  - 26
CY  - Jülich
PB  - Forschungszentrum Jülich, Zentralbibliothek
M1  - RWTH-CONV-143477
T2  - Schriften des Forschungszentrums Jülich : Reihe Information
SP  - XIV, 132 S. : Ill., graph. Darst.
PY  - 2013
N1  - Zsfassung in dt. und engl. Sprache
N1  - Zugl.: Aachen, Techn. Hochsch., Diss., 2012
AB  - The lipid bilayer is the basis of the cell membrane. It serves as a barrier that separates the cell from the environment and provides platform for membrane proteins helping them to fulfill their functions. To advance studies of the cell membrane, membrane proteins and cell-cell interactions, it would be favourable to create an artificial platform which can mimic the cell membrane. It can help to exclude various factors influencing the experiments carried out on real cells and provide a possibility to focus on the phenomena interesting for researchers. It would allow many advanced methods of investigation which can not be applied for living cells. Such a platform can also help to use some advanced properties of the cell membrane in biosensors and medical applications. Several systems that can mimic the cell membrane have been developed such as liposomes (lipid vesicles) or pore suspended lipid membranes (such as black lipid membranes – a classical membrane system). Although these systems have been used extensively for many years, they possess disadvantages which limit their applications. Such a system as supported lipid bilayer (SLB) can mimic the cell membrane and provides high stability and accessibility for measurement techniques. The SLB consist of a lipid bilayer placed on a solid surface (such as glass, Si, mica) covered with aqueous solution and separated from the substrate by very thin (around 1 nm) hydration layer. It gives a possibility for incorporation of membrane proteins providing natural environment for them. It can be immobilized on the surface of a sensor – optical, electrical or mechanical. There are also ways of patterning of the SLB which enhance possibilities of integration with recording devices and is especially useful in experiments with guiding cells in culture or studying cell adhesion. The SLB can provide a wide spectra of possibilities for studying cells in vitro. They include studying of cell adhesion, cell signaling, redistribution of molecules in the cell membrane. However, by now the SLB has been used for experiments with cells not very intensively. One reason for it is that lipid bilayers made of only natural lipids such as phosphatidylcholine are cell repellent: cells can not adhere to it. Thus, to maintain cells the SLB should be functionalized in a special way. There are many possibilities for it but they are mostly complicated and not well studied. The aim of the present work is to study possibilities of using SLB as a platform for neuronal cell culture. To reach this aim several tasks were considered:1. Development of methods to increase cell adhesion to the SLB2. Utilizing proteins incorporated into the SLB for investigation of cell signaling processes.3. Patterning of the SLBFor increasing adhesion of cells a simple approach was developed: bilayers contained positively charged lipids (DOTAP) were used. Such bilayers, which are very easy to prepare, were shown in this work to promote adhesion of neuronal cells most likely due to electrostatic interaction. A possibility for using these bilayers for long term experiments with neuronal cell was demonstrated. Fluidic properties of the prepared SLB were monitored using fluorescence recovery after photobleaching (FRAP). A MatLab based software was developed to analyse FRAP images and calculate diffusion coefficient. The diffusion coefficient appeared to decrease with the increase of the concentration of positively charged lipid, but even the SLB consisted of only positively charged molecules was found to be fluid with the diffusion coefficient three time lower than for the bilayer made of phosphatidylcholine. Possibilities of using the SLB for studying cell signalling processes were shown in this work. Ephrin A5, a synaptic adhesion protein, which performs also many signalling functions, was incorporated into the SLB. Patch clamp experiments showed that the probability for a neuronal cells growing on such bilayers to develop functional synapses is higher than for control cultures maintained on the usual cell-culture substrates such as poly-L-lysine. For creating patterned SLB the idea of using giant unilamellar vesicles (GUV) was utilized. This method allowed to form patches of the SLBs of different type on a surface without risk of exposing the surface to air (which would destroy SLB) that exist in usually used methods such as droplet deposition. In conclusion, this work demonstrates a possibility of using SLB as a platform for neuronal cell culture which can allow long term experiments directed into studying cell adhesion, cell signaling, and cell development processes.
KW  - Zellkultur (SWD)
KW  - Nervenzelle (SWD)
KW  - Lipide / Doppelschicht (SWD)
LB  - PUB:(DE-HGF)11 ; PUB:(DE-HGF)3
UR  - https://publications.rwth-aachen.de/record/209271
ER  -