Primary culture experiments with Schwann cells
Primary culture experiments with Schwann cells
Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press
Operation method
basic program
Principle
Schwann cells are myelinating glial cells of the peripheral nervous system that effectively promote peripheral nerve regeneration. Recent studies have shown that Schwann cells can also improve the microenvironment of central nerve regeneration, so it is imperative to culture Schwann cells in vitro. Currently, the easiest and fastest method to isolate and purify Schwann cells is the differential wall-application method, i.e., according to the difference in the wall-application speed of different cells on the culture vessel, contaminated cells such as fibroblasts are removed to obtain high-purity Schwann cells.
Materials and Instruments
Neonatal 1-3d rat and mouse littermates 2 months old adult rats and mice Move 1. Acquisition of neonatal rat Schwann cells After sterilizing the animals in accordance with the method of the General Introduction, the detailed steps were as follows. Dorsal root ganglia were isolated under sterile conditions and blown and dispersed into cell suspension with DMEM culture medium containing 10% fetal bovine serum (see the section on dorsal root ganglia cell culture for details). Alternatively, the sciatic nerves of animals were isolated and extracted bilaterally, rinsed 3 times in PBS, placed in D-Hanks, and stripped of connective tissue and nerve periphery. The obtained tissues were cut into about 1mm3 size, put into 0.25% trypsin and 0.06% collagenase complex digestive solution at 37℃ for 40 min. 10 drops of fetal bovine serum were added to terminate the digestion, and then centrifuged at 900r/min for 5 min, and the supernatant was removed. Then make single cell suspension in DMEM culture medium containing 10% fetal bovine serum according to the general method, adjust the appropriate cell concentration and inoculate the cells on polylysine-coated medium, and incubate them for 3-5d in an incubator at 37℃ with 5% CO2::: Adult rats and mice were cultured in the incubator at 37℃ for 3-5d. 2.Acquisition of adult rat and mouse Schwann cells One male 2-month-old mouse was taken, anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg/kg), and the distal ends of the sciatic nerves were cut off bilaterally under aseptic conditions. 7d later, 20-25mm of pre-denatured treated sciatic nerve was isolated and extracted, rinsed three times with PBS, placed in D-Hanks, and the outer membrane of the nerves was peeled off. The obtained tissues were cut into about 1mm3 size, 0.05% Collagenase-Dispase complex digestive solution was separated and digested at 37℃ for 3-5h, 2000r/min, centrifuged for 10min, and the supernatant was discarded. The supernatant was then resuspended with DMEM medium containing 10% FCS, centrifuged at 900r/min for 5min, and discarded. Finally, single cell suspension was made with DMEM medium containing 10% FCS and inoculated in Petri dishes coated with 10µg/ml Laminin (37°C, 2h). 37°C, 5% C02 for 3-5d. 3. Culture of Schwann cells The cells cultured in the above two steps were digested by 0.125% trypsin and made into single cell suspension, placed in uncoated dishes and incubated at 37°C, 5%CO2 incubator for 30min, then the uncoated cell suspension was transplated in another-uncoated vessel, and the above operation was repeated after 30min, and finally, the uncoated cells were inoculated according to the appropriate density in the The cells were then inoculated into polylysine or Laminin-coated culture vessels at a suitable density and cultured at 37℃ with 5% CO2 for 5-10 d. By 7 d, the typical Schwann cell morphology was a small ovoid or spindle-shaped cell, with a strong sense of three-dimensionality, and long, slender bipolar and tripolar protrusions interwoven with each other to form a network. With the prolongation of culture time, the longer protrusions were arranged in parallel to form bundles, and the cytosol grew "shoulder-to-shoulder" with good consistency. Caveat 1. Schwann cell culture methods are very different for animals of different ages and must be selected as needed. 2. Successful isolation and removal of fibroblasts from the nerve membrane components is the key to culture and purification. Since the sciatic nerve of fetal rats is very small, only by stripping the outer nerve membrane as much as possible can we effectively avoid excessive growth of fibroblasts in primary culture. 3. Schwann cells from adult rats are more difficult to culture in vitro, and their growth is absolutely dependent on the Laminin matrix, whereas neonatal rats grow well on polylysine overlay. Common Problems 1. The traditional classical differential wall affixation method is a simple, economical, rapid and effective method to isolate and purify adult rat Schwann cells. The other methods tried by the author, such as chemical drug inhibition and immunoaffinity adsorption, have the defects of drug inhibition of Schwann cell activity, low yield, need for special instruments and time-consuming. 2. The pre-denaturation time of adult rats should be 7-10d, otherwise the yield and purity of cells will be affected. 3. Several experiments have confirmed that the proliferation and viability of Schwann cells in prenatured peripheral nerves are stronger than those in unactivated nerves. For more product details, please visit Aladdin Scientific website.
DMEM medium with 10% fetal bovine serum 0.05% Collagenase-Dispase 10µg ml Laminin
Culture Bottle