Basic Research On Culture Of Olfactory Ensheathing Cells And Related Cells

Part I Preliminary research on isolation, culture and morphology of olfactory ensheathing cells from miceObjective:To investigate different approaches to in vitro culturing olfactory ensheathing cells (OECs) from mice to obtain highly enriched population, better activity and appearance of OECs for certain experiments. Methods:Olfactory bulbs were obtained from4BALB/C mice aged2to3months, through foramen magnum for preparing cell suspension using two peripheral layers of cells from olfactory bulbs. The culturing included three groups, i.e., unpurified cells as control, cell purification using single differential adhesion with cytosine arabinoside(Ara-c) on poly-L-lysine-coated culture plate or purification with single differential adhesion with Ara-c on naked culture plate. Cell growth and morphology of primary and/or subcultured OECs were observed and identified using immunocytochemistry. Results: Cell growth and morphology of the purified OECs in naked culture plate was superior to poly-L-lysine-coated culture plate, and yet, only the cell number in control group was significantly better at early metaphase than that of two other groups. Conclusion: Single differential adhesion with Ara-c treatment of the uncoated culture plate appears efficient and time-saving in culturing OECs from mice. Figure8table2reference12 Part â…¡ Preliminary research on isolation, culture and morphology of olfactory ensheathing cells from ratsObjective:To investigate diverse approaches to in vitro culturing olfactory ensheathing cells(OECs) from rats to obtain highly enriched population, better activity and appearance of OECs for certain experiments. Methods:Olfactory bulbs were obtained from2Sprague Dawley (SD) rats aged2to3months, through foramen magnum for preparing cell suspension using two peripheral layers of cells from the olfactory bulbs. Culturing of OECs was managed by cell purification using single differential adhesion with cytosine arabinoside (Ara-c) on poly-L-lysine-coated culture plate or purification with single differential adhesion with Ara-c on naked culture plate. Cell growth and morphology of primary OECs and their sub-culturing were observed and identified using immunocytochemistry. Results:Cell growth and morphology of purified OECs in poly-L-lysine-coated culture plate was superior to that without treatment. Conclusion:Culturing of OECs requires necessary culture plate coating with Ara-c in poly-L-lysine. Figure1table1reference17 Partâ…¢ Isolation, culturing and identification of astrocytes from miceObjective:To probe into an approach to isolation, culturing and identification of astrocytes (AST) from newborn mice for analogous culturing of the olfactory ensheathing cells (OECs). Methods:The spinal cord was obtained from one neonatal BALB/c mouse by developing into cell suspension to culture ASTs as culturing protocol for OECs. Identification of ASTs was performed through immunocytochemical stain using glial fibrillary acidic protein (GFAP), neurofilament protein (N200) and S100protein, respectively. Results:Immunocytochemical staining of astrocytes treated by S100led to much better cell population and morphology than did with GFAP. Conclusion:Immunocytochemical staining with S100protein looks the most favorable method to identify ASTs. Although the culturing protocol is identical to that for OECs, yet, digestion with trypsin shall be removed from the procedure. Figure7table0reference8 PartIV Research of co-culturing olfactory ensheathing cells with spinal cord tissue cells from miceObjective:To investigate the effects of in vitro culturing olfactory ensheathing cells (OECs) with cells from spinal cord tissue of mice on the growth of spinal cord tissue cells. Methods:Both cells were obtained from the olfactory bulbs and spinal cord tissues of neonatal BALB/c mice and cultured in three groups:single culture of mouse OECs or spinal cord tissue and co-culturing OECs with spinal cord tissue. Inverted phase contrast microscope was used to observe the growth condition for each group of cells and immunocytochemical staining to examine and identify the cell count and cellular activities. Results:Co-culturing of OECs with the cells from spinal cord tissue resulted in better cell density as compared with the two control groups. Cell staining was more frequently seen in co-culturing treated with NF200and GAP43, leaving blank stain of OECs. Besides, OECs were found to have inhibitory effects on astroglia cells in culturing. Conclusion:OECs appear significantly to improve the growth of cells from spinal cord tissues of mice. Figure7table0reference7...