Neural stem cell culture

Summary

Cultures of neural stem cells can be used (1) to enable their specific differentiation into neurons, astrocytes, and oligodendrocytes; and (2) their ability to self-renew and provide a large population of brain tissue cells. Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press

Operation method

neural stem cell culture

Principle

Since neural stem cells (Neural Stem Cells) have the potential for self-renewal and multidirectional differentiation, they can be obtained and studied by suspension neurosphere culture. That is, after the embryonic brain tissue is processed into individual cells, only cells with self-renewal ability can clone and proliferate into suspended neurospheres in the culture medium, and maintain the proliferation ability and the ability to differentiate to multiple neural progeny cells as the passaging proceeds.

Materials and Instruments

Neonatal SD rats
Paraformaldehyde Distilled water PBS DAB Serum medium Polylysine Fetal bovine serum Double-distilled water
Dissecting microscope Ophthalmic scissors Straws 24-well culture plates Ice maker Screamer

Move

1. obtaining specific brain tissue.

2. There are two usual methods for obtaining single cell suspensions. Mechanical separation method Procedure:

① Cover the tissue with a small amount of DF12 liquid, and then cut the tissue block into 1 mm³ size with iris scissors;

② Collect the tissue block into the neural stem cell culture medium;

(iii) Blow up the single cell suspension with a fire-polished pipette;

④The cell suspension was passed through a 200-mesh sieve;

⑤ Centrifuge at 800r/min for 3min and discard the supernatant.

The procedure of trypsin digestion + trypsin inhibitor termination method:

② Cut the tissue block into ^1mm3 size with iris scissors;

② Digest the tissue with 0.125% trypsin + 0.02% EDTA at 37℃ for 2min.

③ Add trypsin inhibitor to terminate digestion (the concentration used varies depending on the production company and the inhibitor activity, refer to the specific instructions);

④ Centrifuge at 800r/min for 3min, discard the supernatant.

3. Resuspend with neural stem cell culture medium, blow into single cell suspension with fire-polished pasteurized pipette, count the cells and inoculate with a density of ^1x105/ml.

4. Cultivate for 3-4d can 1/2 amount of fluid change, culture for 7-10d for passaging. Procedure: cut and collect neurosphere into centrifuge tube, centrifuge at 800r/min for 3min;.

(2) Discard the supernatant, add 0.125% trypsin + 0.02% EDTA to digest the tissues, incubate at 37℃ for 3-Smin;

@ Add equal volume of 1X trypsin inhibitor to terminate digestion;

@Collect the cells into a centrifuge tube, centrifuge at 800r/min for 3min, discard the supernatant;

@Add new neural stem cell culture medium and resuspend, use fire-polished pasteurized pipette to aspirate into single cell suspension, count and inoculate the cells.

5. Cultured neurospheres can be inoculated on the bottom of polylysine-coated dishes or slides, and differentiated and induced by adding neural stem cell differentiation solution to differentiate them to daughter cells. Since the method of suspension culture of neural kiloblasts is actually a retrospective judgment of the identity of the cells, that is, only the cells that can continue to proliferate and differentiate in multiple directions are true neural ubiquitous cells, the interpretation of the results should be combined with a variety of methods to make a comprehensive judgment. When conducting the experiment, it is also necessary to ensure the credibility of the experiment through the setting of the control. The specific methods are as follows:

1. Shape:

As shown in Figure 1-8, the spheres obtained from the clonal proliferation of neural stem cells are generally round and smooth in shape, with a tight texture, while the cell aggregates are generally loose and irregular.

2.Immunization. Cytochemical staining:

As in Figure I-9,Nestin is a marker for neural stem cells and can be identified by immunofluorescence staining for Nestin. :

3. self-renewal capacity:

Rigorous neural ubiquitous cell experiments can be performed by 3-5 passages to obtain NSC with high purity.

4.Differentiation potential: after neurosphere differentiation, immunofluorescence staining of progeny cells can be performed with neuronal markers, astrocyte markers (GFAP), oligodendrocyte markers (PDGFR), etc., to determine whether the cells obtained have multidirectional differentiation potential.

Caveat

1. Care should be taken to distinguish between cell aggregates and clonal spheroids, which can be done according to the method of interpretation of the results described above.2 The proliferative capacity of the cells is very importantly related to the age of the animal from which the tissue originated, as well as to the brain region from which it was taken. The younger the gestational age, the more naive the developmental state in which the stem cells are located, and the stronger the proliferative capacity of the cells; neural stem cells of cortical origin are more likely to obtain cells with better proliferative capacity than those of other brain regions.

3. Meningeal tissue has the effect of inducing wall attachment and differentiation of neural stem cells, so it is important to remove the meningeal tissue during tissue processing.4 Serum has an inducing effect on the differentiation of thousands of neural stem cells, so the culture medium with serum should not be used when terminating digestion.5 The time of transgeneration is generally about 7-10d, but if the volume of neurosphere is large, the center of the neurosphere has begun to appear black area under the light microscope (the reason for the black area may be due to the sphere volume is too large, which affects the transmission of light, but at this time the center of the sphere also tends to appear dead cells), it is necessary to transgenerate in a timely manner.

Common Problems

1. The cells obtained by mechanical separation are prone to cell aggregation, and the spheres obtained from culture may not be single-cell clones; while the concentration and time of the digesting solution of the digestion method should be adjusted according to the different ages of the animals and different regions of the tissue source to find out the optimal concentration and time of digestion.

2. Since neural stem cells also have a certain role in prompting differentiation after apposition, it is best to use new, scratch-free vessels for the dishes or flasks used to culture neural stem cells.

3 If wall-adherent cells are observed during culture, they can be salvaged by replacing the culture flasks, and the sooner they are found, the better. However, if the spheres have not yet formed, try not to change the vessels and touch the cells, otherwise the formation of neurospheres will be affected.

Reference "Practical Experimental Techniques in Neurobiology" Fourth Military Medical University Press

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/