Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press
Operation method
basic program
Principle
Olfactory sheath cell is a specialized glial cell functionally intermediate between Schwann cells and oligodendrocytes, and is a key determinant of lifelong axonal regeneration in olfactory neurons. It has the roles of neurotrophic, inhibiting gliosis, scar formation, and sheath formation, which provides a suitable microenvironment for axon growth and strong migration properties, and has become one of the ideal candidate cells to promote CNS regeneration. Based on its difference in wall-application ability with fibroblasts, currently we often use differential wall-application method to obtain a large number of high-purity olfactory sheath cells.
Materials and Instruments
2 months old adult rats and mice Move 1After the animals were severed and executed, the olfactory bulbs were exposed, and the bilateral olfactory bulbs were extracted under aseptic conditions, the soft meninges were discarded, and the olfactory nerve fiber layer and olfactory bulblet layer were carefully stripped under the microscope. The obtained tissues were cut into about 1mm3 size, washed twice with D-hanks solution, digested separately with 0.125% trypsin at 37℃ for 25min, and then immersed into culture medium containing 20% serum to terminate the digestion for 8min, and then the tissues were aspirated and washed once with serum-free DF12 medium. Finally, single cell suspension was made with DF12 medium containing 20% fetal bovine serum according to the general method. 2. Place the blown single-cell suspension in an untreated culture flask and incubate at 37℃, 5% CO2 for 18h, then transfer the unaffixed cell suspension to another uncoated culture dish and continue incubation at 37℃, 5% CO2 for 36h, then repeat the above operation, and finally inoculate the unaffixed cells at the appropriate density in a culture dish coated with 25µg/ml poly-lysine. The cells were inoculated into 25µg/ml polylysine-coated culture medium at a suitable density. 3. In the first 3 d, change the liquid in 1/3 amount, and then change the liquid in 1/2 amount every 2 d (DF12 culture medium containing 10% fetal bovine serum), and incubate at 37℃, 5% CO2 for 5-10 d. Most of the olfactory sheath cells were already attached to the wall 24 h after inoculation, and appeared in the shape of irregular granules, and the refractive property of the cytosol was poor. After 3d of culture, there were occasional cells with conical or spindle-shaped cytosol, emitting short protrusions and webbed growth cone-like structures, and the pericellular debris material was reduced (Figure I-7A). Routine culture 5d, most of the cell cytosol is flat, cytoplasm extends outward, send out short, web-like protrusions. 10d after a few for the multipolar astrocyte-like, most of the cells are bipolar, tripolar Schwann cell-like, cytosol three-dimensional sense is strong, extend out the long and slender, soft protrusions interwoven with each other to form a network (Figure L-7B, C). Some other cells have large, flat bodies with irregular edges and a poor sense of three-dimensionality. Caveat 1 Olfactory sheath cells are located in two layers of the surface of the olfactory bulb, i.e., the olfactory nerve layer and the glomerular layer, and the differences between the structures of each layer of the olfactory bulb are not very obvious, and it is difficult to isolate them with absolute completeness even when the material is taken under the dissecting microscope. Therefore, the culture may be mixed with some miscellaneous cells, mainly fibroblasts from blood vessels, soft meninges and other connective tissues, but also a small number of astrocytes, microglia, neurons and so on. Microscopic sampling should be as careful as possible to remove the outer layer of the soft meninges, which can significantly reduce the number of fibroblasts. 2. The initial morphology of the olfactory sheath cells of adult animals cultured in primary culture is quite special, which is irregular and granular, resembling the shape of "fried egg" or flower. Beginners are often mistaken as culture failure, and it is recommended to observe patiently for 5-7d, and then the cells will gradually show the typical morphology. 3. At present, the purification of olfactory sheath cells mostly adopts the classical differential walling method, which is different from the differential purification of Schwann cells. Schwann cells are differentiated and matured in vitro before purification, while olfactory sheath cells are purified and separated by taking advantage of the difference in the wall-adhesion ability of various cells in the early stage of primary sampling and culture. Common Problems 1 Because the astrocytes in the olfactory bulb of adult rats have fewer components and are difficult to survive under these culture conditions, the author usually chooses a single differential walling, i.e., a single cell suspension is placed in untreated culture flasks and cultured for 18-24h, and after removing the main contaminating fibroblasts, the olfactory sheath cells can be obtained with both a higher yield and a higher degree of purity. 2. The morphology of olfactory sheath cells cultured in vitro varies depending on the age of the donor animal, the site of extraction and the culture conditions. At the same time, I observed that the olfactory bulb olfactory sheath cells of adult mice were slightly smaller than those of rats, and the protrusions emitted from the olfactory sheath cells of rats were thick and straight, while those of mice were slender and soft, suggesting that this may be related to the differences in species. For more product details, please visit Aladdin Scientific website.
DF12 medium with 20% fetal bovine serum D-hanks liquid Digestive solution (0.25% trypsin + 0.04% EDTA) 25µg ml polylysine
Culture bottle