Primary culture experiments with nerve cells can be used to: acquire myelin sheaths and become nerve fibers after they have traveled some distance from the cell body.
Operation method
Neural cell primary culture experiment
Principle
Primary culture of neural cells is a method to create the most suitable in vitro environment for the growth of various types of neural cells, and to obtain cells in good condition and high purity. The culture of various types of neuronal cells from brain tissues follows a similar procedure and some common culture conditions.
Materials and Instruments
Animal tissue Move Except for animal sterilization, all other steps are carried out in the ultra-clean bench . 1. Sterilization of animals: According to the different types and requirements of cultured cells, fetal rats and neonatal rats are commonly used for neural cell culture. The sterilization methods are as follows. (1) Operation of pregnant rats After the pregnant rats are anesthetized with sodium pentobarbital, the abdominal fur is sterilized by wiping with a cotton ball dipped in iodophor. The wipe was performed from the center outward, and the cotton ball could be replaced 1-2 times, covering as large an area as possible, including the entire abdomen and the periphery of the external genitalia. The abdominal cavity of the animal was opened with the first set of dissecting instruments, and the uterus was separated and placed in a 100-mm glass dish containing cold D PBS with the second set of instruments, and the uterus was cut open at the interval of each litter with the third set of instruments, and the litter was peeled off. (2) Operation of littermates Littermates (within 7d of birth) were directly immersed in 75% alcohol on ice and sterilized at -20℃ for about 10min, and then the animals were unconscious to start the retrieval of materials. 2. Separation of cortical tissues: Neonates were decapitated using ophthalmic scissors, and the skin of the head was peeled off with forceps. The skin of the head was peeled backward and forward along the herringbone suture in ice D-PBS. Generally, the curved forceps are held in the left hand and fixed with appropriate force at both eyes of the animal, while the straight forceps are held in the right hand to complete the work of peeling off the skin, bones, and separating tissues. When separating the cortex or hippocampus under the microscope with fine instruments, the cortex is carefully slashed at a 45° angle along its edges and gradually peeled off, and the tissue is transferred into a 35-mm dish containing ice D-PBS; the meninges of each cortex or hippocampus are then carefully removed with fine instruments (the entire process is done on a cold ice pack). 3. Dispersion of the tissue; remove the D-PBS with the meninges by pipetting with a thick elbowed dropper, removing as much liquid as possible and avoiding sucking up the tissue; cut the tissue uniformly in handfuls with iris scissors to a size of lmm3 . Add the appropriate volume of digestive solution (generally over the tissue mass and can be shaken freely) and disperse the tissue evenly, and incubate at 37°C for about 15 min (gently shaking every 3-5 min, the younger the animal is, the easier it is to disperse the tissue, and the digestion time can be shortened accordingly). 4. Preparation of single-cell suspension! Use a thick elbow dropper to draw the digested tissue into a 10 ml centrifuge tube containing 3-5 ml of ice-cold complete medium and let it stand for about 5 min. Using the same burette, transfer the bottomed tissue to another 10 ml centrifuge tube containing about 5 ml of ice in complete medium and start blowing. Blow no more than 15 times at a time, first with a thick-bore elbow burette to disperse, allow to stand for a few moments, then transfer the supernatant with a thin-bore elbow burette to another centrifuge tube and blow about 15 times more. In the original centrifuge tube containing the precipitate, add complete medium and continue to blow, and collect the supernatant and repeat the process. After several blows, the tissue mass has almost disappeared, and the entire supernatant is passed through a 200-mesh sieve to remove the remaining tissue mass, collected and counted. Centrifuge (900r/min, 5min) to remove cell debris and resuspend the cells to a suitable volume for later use. Caveat 1. The preparation work must be done well before doing the primary generation, and the liquid should be prepared in advance. The whole operation is aseptic, and the culture fluid contains penicillin-streptomycin (double antibiotic) if not specified. 2. Sterilization of animals should be done in an area away from the ultra-clean bench, especially for adult animals. 3. Tissue extraction should be done in a low-temperature environment, which can greatly improve cell survival; and the whole process should not be too long, otherwise the cell activity will be significantly reduced. 4. In the process of tissue dispersion, the cortex from about 3 animal sources is generally sheared and digested in a small dish, and it is not advisable to process too much tissue in the same small dish. 5. The elbow burette used for blowing cells must be flame-polished beforehand, otherwise the cells will be easily injured and die; and try to avoid generating too many air bubbles when blowing cell suspension, which can improve the survival of cells. 6. Basically, all kinds of neural cells in primary culture need to be grown on culture medium treated with special substances, such as polylysine, laminin, and so on. Common Problems Experimental tips: 1. the method of culturing neuronal cells of rat and mouse origin is generally the same and, except for specific mentions, is essentially universal. 2. when removing the meninges, the left hand is generally held to gently hold the tissue in place, while the right hand is responsible for stripping it. After the entire meninges are stripped together, there is often less fibroblast contamination in the cell culture. 3. Tissue treated with tryptic digestive solution tends to be sticky, due to the release of DNA from the cells. This does not affect the digestion and can be removed later in the blowing process. For beginners, the final cell yield can be increased by adding DNAase to the digestion solution. 4. The addition of EDTA to the tryptic digest facilitates the dispersion of tissue cell clusters into single cells, and termination of EDTA relies heavily on dilution to reduce its concentration. For more product details, please visit Aladdin Scientific website.
Digestive solution (0.25% trypsin + O.04% EDTA) Complete culture medium (DMEM + 10% fetal bovine serum + double antibody) D-PBS solution Polylysine (encapsulated concentration 1OOµg ml) Sterilized ultrapure water Funeral ambulance 75% alcohol
Stereomicroscope Phase contrast microscope Fine instruments (curved tweezers, straight tweezers, iris scissors, sterilized in 75% alcohol) Ophthalmic instruments (curved tweezers, straight tweezers, scissors in 3 sets High-pressure sterilized) Various sizes of cell culture flasks and dishes (all from Nunc) Curved-tip burette 1 ml graduated burette 15 ml centrifugal tubes 100 mm glass dishes 200-mesh cell sieve mesh Cryogenic ice packs
Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press