Lesson learned: those things about cell culture

　　It’s harder to grow cells, it’s even harder to grow primary cells, and it’s even harder to grow primary neural stem cells! This is a fact recognized by the scientific research community. Cell culture is an indispensable experimental operation in the process of preparing monoclonal antibodies for hybridomas. A difficult thing, so many heroes hang themselves on cell culture!

　　Today, the editor of Hengyuan Biology will summarize the materials and culture of primary neural stem cells in detail, and teach you how to easily solve the problem of cell culture!

　　Neural stem cells refer to cell groups that have the ability to differentiate into neurons, astrocytes and oligodendrocytes, can self-renew and provide a large number of brain tissue cells, have self-renewal ability and multi-directional differentiation potential, and can damage Have the ability to respond to diseases.

　　Primary cultured single cells automatically aggregate into clusters within 24 hours, which is an important feature of neural stem cells in vitro culture.

　　Original materials and cultivation

　　Step 1: The original generation

　　1. Two-week-old pregnant mice were sacrificed by necking. After the abdomen of pregnant mice was disinfected with 75% alcohol, the abdominal cavity was opened with surgical scissors, the uterus was removed, the uterus was cut, and the fetal mice were taken out and placed in a 10 cm petri dish containing ice-cold PBS.

　　2. Decapitate the fetal rat, separate the skull and dura mater with ophthalmic scissors, take out the brain tissue, and fully take out the meninge and blood vessel tissue under a microscope.

　　3. Wash the brain tissue three times with PBS and cut it into 1mm3 size tissue pieces. As for the centrifuge tube containing DMEM/F12, pipette gently with the pipette 10 times, leave the centrifuge tube for 1~2min, and take the cell suspension. Repeat 2-3 times.

　　4. The cell suspension was centrifuged at 700 r/min for 6 min, and the supernatant was aspirated to obtain a cell pellet. After resuspension with (DMEM/F12+1%N2+2%B27+bFGF20mg/ml+EFG20mg/ml), pass through a 200 mesh screen and count.

　　5. Inoculate at a density of 5×105/ml, and incubate at 37°C with 5% CO2. Change the fluid every other day after 2 days. Usually, neurospheres grow out in about a week.

　　Take out certain tissue cells from the body in a sterile environment (depending on the purpose of the experiment), put them into a culture vessel after certain treatments (such as digestion and dispersion of cells, separation, etc.). This process is called material extraction. In the case of cell line expansion, there is no such process as obtaining materials. The first culture of tissue cells taken out of the body is called primary culture. In theory, all tissues in various animals and humans can be used for culture. In fact, larval tissues (especially embryonic tissues) are easier to cultivate than adult tissues. Tissues with low differentiation are easier to cultivate than those with high differentiation. Tumor tissues It is easier to cultivate than normal tissues. After taking the material, it should be processed immediately and cultivated as soon as possible. If it cannot be cultivated immediately, the tissue block can be cut into small pieces as large as soybeans and stored in the culture medium at 4°C. The tissues should be taken strictly to maintain sterility and avoid contact with other harmful substances. Pathological tissues and skin and digestive tract epithelial cells are easy to carry bacteria. Antibiotics can be used to reduce pollution. The method of organizing and separating scattered cells can be found in related literature.

　　Step 2: Subculture

　　1. Passage when observing the gray-black area in the center of the cell sphere under the light microscope, transfer the culture medium to a 15ml centrifuge, centrifuge at 800r/min for 5min, and discard the supernatant.

　　2. Add 0.125% pancreatin + 0.02% EDTA for digestion for 2-3 min, add trypsin inhibitor to terminate the digestion, gently pipette the neurosphere to the cell suspension, centrifuge at 800 r/min for 5 min, and discard the supernatant.

　　3. Add appropriate amount of stem cell culture fluid Neurobasal +1%N2+2%B27+20ng/ml bFGF, 20ng/ml EGF (addition of glutamine), adjust the cell concentration to 5×105/ml, and place it at 37℃, 5% CO2 continued subculture.

　　Experimental influence factors

　　1. The influence of donor age

　　Experimental studies have shown that the number of neural stems in the cerebral cortex of embryonic rats is significantly higher than that of neonatal rats, and the neural stems in mouse embryos with a gestational age of about 12 to 15 days have the strongest differentiation ability.

　　2. The effect of separation methods on cell purity and viability

　　The commonly used cell separation methods are mechanical separation method and enzyme digestion separation method. The disadvantage of the mechanical separation method is that the blowing force and frequency are not easy to control, and the mechanical cutting effect will reduce the cell activity; the disadvantage of the enzyme digestion method is that the digestion time is not easy to control, and the digestive enzymes used to separate neural stem cells have potential damage to the cells .

　　3. The effect of inoculation density on nerve trunk

　　The appropriate inoculation concentration of neural stem cells can generally be 1×10*8/L~1×10*9/L. If the inoculation cell density is too small, the cells will not form pellets, which is not conducive to cell proliferation; if the inoculation cell density is too large, after the first passage Soon there will be death, extra large, scattered, and rapid increase in the number of single cells. The half-volume exchange method can keep the cell living environment stable to the greatest extent, which is conducive to cell growth.

　　4. The influence of the timing of subculture

　　After the primary culture of neural stem cells, a large number of cells proliferate, which will inevitably lead to the death of most neural stem cells due to lack of nutrients. Therefore, timely passage is the key to prevent their death. According to literature reports, primary culture is generally used for 5 to 7 days. It is carried out at a time to avoid excessive cell proliferation and death, while maintaining the activity of cell proliferation.

　　Precautions

　　1. In order to maintain the cell viability in the process of sampling, the entire process of sampling should be carried out on ice and the meninges and vascular tissues should be stripped in a high-glucose medium containing 10% FBS, because the fetal rat's nerve trunk has a strong metabolism. Long-term damage to nerve trunks in a sugar-free environment.

　　2. In the process of collecting materials, do not take a cortex and peel off a meningeal blood vessel, but quickly remove all the fetal rat cortex and put it in a high-sugar medium.

　　3. Peeling meninges and blood vessels should be completely separated, otherwise, when making a single cell suspension, you will be forced to increase the force and frequency of pipetting, causing cell damage.

　　4. During the culture process, in order to prevent the cells from adhering to the wall, gently shake the medium the next day.

　　5. Avoid cell contamination. Strengthen the awareness of aseptic operation and do a good job in the disinfection of laboratories and experimental equipment.

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