Ps-11: Controlling Directional Migration of Neural Stem Cells Electrically (Pages: 0-0)


Aumari AM *,

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Objective: Abstract embryonic stem cells from mouse strain (OCT4-GIP) was Cultured in medium (DMEM/F12) supplemented with leukaemia inhibiting factor (LIF) in Petri dishes treated with gelatin 0.1% and the cultures incubated in an incubator CO2- temperature 36.9-36.7°C , and CO2 partial pressure of 9.4 to 2.4%. Cultured cells responded to factors of culture and proliferated well, and dispersed new cultures new daily. Quoted cultures cellular processing of a special called electrotactic chamber contains the medium of culture and provided of circuit electrical particular, and provided chamber to control the PH, and also provided by installation of electrical control ensures the strong from 5.5V to 12V for the cells cultured. for these cultered cells the experiment revalated this cell (OCT4-GIP) move effectively and activity apparent when they exposed of the electric field in the electrotactic chamber, and that this movement much larger than the movement of the same cells cultured in the same conditions without electric field (control). this phenomenon is useful stimulating the migration of stem cells In vivo tissue towards the target of a terrorist (like wounds) to expedite and facilitate the healing, as well as directs the context of these cells to repair nerve tissue battered. Key words: Embryonic stem cells, Electric field, wound healing, Migration of embryonic stem cells. Materials and Methods: Cells were seeded into electrotactic chamber coated with either gelatine (0.001%), or poly-ornithin-laminin. METAMORPH system was used for the electric fields (EF) direct current (dc) experiment. Electric power used from 500 volts onwards and current ranges between 5.5V to 12V. Two wavelengths were used: Green Fluorescence Protein (GFP), and BrightFiled. Each electric field and control experiments were carried out for a duration of 3-5 hours. Materials: -Medium with HEPES buffer(GIBCO, cat.no.15630-049) & LIF -Agar bridges, Agar (sigma, cat.no. A7002) -Glass slips for chambers -Coating gelatine and poly - orithin - laminin chambers -Steinbergs soluition -High vacuum grease Equipment: -Direct current power supply (CONSERT E143. and cables. -LAMBDA 10-2.Sutter instrument CO. Floresentes model ebq 100 ISOLATED -MetaMorph Microscope model ZEISS Axiovert S100TV -temperature T36.9°C -Computer model DELL -Voltage meter -Standard glass slides -Cover glass, size 22 × 22mm2, 22 × 40mm2 and 48 × 64mm2 -Silver wire electrode (Advent,cat, no. AG549109) -Glass tubes (inner diameter approximately 7mm) -Glass-cuting diamond pen or glass saw -Tissue culture Petri dishes with 10 mm diameter electrical insulating compound silicone grease (DC4; Dow Corning) - Time-lapse imaging system, ideally with function of X/Y/Z multiple position recording and multiple wavelength recording, as well as a CO2-supplied temperature control chamber incorpoated onto the microscope using the MetaMorph imaging system (Molecular Devices, UK). For result used program imageJ-mtrackJ and MetaMorph. Results: Cells were cultured and seeded to passage 46 (P46). Cultured cells were very healthy and some were frozen and kept in liquid nitrogen tank. At one stage, cells were contaminated. Contaminated cells were sterilized with vortex and discarded. All culture media, chemicals and solutions for cell culture were changed to avoid any further contamination. Oct4 marker protein expresses pluripotency. Embryonic stem cells tagged with Oct4-GFP marker protein expresses pluripotency, once the ES cells differentiate, Oct-GFP expression disappear and the cells lose pluripotency. Pluripotent embryonic stem cells were seeded in electrotactic chamber precoated with poly-ornithine/laminin for direct current electric field application. During direct current electric field application, 11V current was applied to electrotactic chamber containing a monolayer of stem cells/ neural progenitor cells and left for 5 to 7 hours. This results in migration of pluripotent embryonic stem cells at a short extent and also a significant migration of neural progenitor cells. Conclusion: This experimental system may be used to simulate and study cellular and molecular responses to electric signals in the events of wound healing.