Purification and culture of oligodendrocytes by oscillating shaker method

Summary

Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press

Operation method

basic program

Principle

Using the differences in the growth and survival time of oligodendrocytes, astrocytes and microglia, as well as the characteristics of the cell growth mode and cell adhesion to the culture layer, the oligodendrocyte pre-resting cells were isolated from cultured mixed glial cells by a constant temperature shaker at 37℃, and then the purified and contaminated astrocytes were removed by a plastic petri dish or culture flask by using the principle of differential apposition to the wall in order to obtain high purity oligodendrocytes. oligodendrocytes, and finally obtain mature oligodendrocytes under conditions of in vitro induced differentiation.

Materials and Instruments

Neonatal 1-2d SD rat littermates
DMEM culture medium (l1960).DMEM F12 culture medium Fetal bovine serum 3. Insulin (16634). Transferrin (T2252) Glutamine (G8540) Sodium pyruvate (P2256). Bovine serum albumin(A9647) Sodium selenate(S5261) Hydrocortisone(H0888) Triiodothyronine(T-2752) NAC(A8199), polyornithine(P042l) DNase l(D5025) Above Sigma
Culture bottle 37℃ constant temperature shaker

Move

Primary cultures of oligodendrocytes derived from the cortex of SD rats at 1-2 d of neonatality.

After obtaining the cell suspension according to the general method, the detailed steps of the method described in the literature (NatureProtocols,2007,2:I044-I051; Journalof Neuroscience Methods,2005,50-56) are as follows.

1. To the cell sediment obtained by centrifugation, 1.5 ml of culture medium was added and the cells were gently resuspended with a small-bore burette, then diluted with complete medium (DMEM + 10% fetal bovine serum + 1 mM sodium pyruvate + 4 mM glutamine + 50 U/ml penicillin) and inoculated into culture flasks with polylysine-coated T75 at a density of 1. ^0x105/cm2, mixed well and then Put into the incubator at 37℃, _5%CO2 for incubation.

2. Don't shake the culture flask within 2 days of inoculation, and start to change the full volume of liquid after 4 days of culture, and then change the full volume of liquid every 3 days and observe the cell fusion degree under the microscope.

3 When cultured to the 7th-9th day, the astrocytes have been spread all over the bottom of the bottle, and can be seen in the upper layer of the cytosol is small, refractive, small protrusions of oligodendrocyte precursor cells, at this time you can begin to purify the cells.

4 Firstly, remove the microglial cells in semi-suspension growth by shaking (200r/min, 37℃, about 20min), and then quickly change the whole life-saving solution.

5 After the fluid exchange was stabilized overnight, the oligodendrocyte precursor cells were continued to be isolated by shaking (280 r/min, 37°C), and the cell suspension was collected and passed through a 200-mesh cell sieve after 18-20 h. The mixed astrocytes and microglial cells were then removed by differential wall adherence (the filtered cell suspension was inoculated into 100-mm dishes [Corning, NY] and left to stand, with gentle shaking by hand at 5-min intervals). Every 5 min, gently shake by hand for 30 min), the remaining cells in the culture flask can be added with fresh complete culture medium to continue the culture for 3d, and then repeat the steps of 5.

6. Collect the cell supernatant from the culture dish and count, centrifuge (1000r/min, 5min) and discard the supernatant, resuspend the cells in oligodendrocyte precursor cell proliferation medium (serum-free medium Satomedium with 10ng/ml PDGF-AA and 10ng/ml bFGF) and inoculate them at a density of (3-4) ^x104/cm2 in a poly(guanine) coated culture plate or slide, and inoculate them at a density of (3-4) ^x104/cm2 in a culture dish or slide. coated petri dishes or slides and supplemented with half amount of proliferating factors (PDGF-AA and bFGF) daily

7. 2-3d later, when the cells proliferate to the full size of the dish, the cells can be digested with digestive isolation medium (DMEM/F12 with 0.01% EDTA, 0.2mg/ml DNase I, 5µg/ml insulin) (directly aspirate the culture medium and add a small amount of digestive medium, shake in the palm of your hand for 5-8min), terminate the digestion by using serum-containing culture medium, blow the bottom of the dish carefully to dislodge all the cells, and then blow the bottom of the dish carefully, so that all the cells are dislodged. Blow the bottom of the dish carefully to dislodge all the cells, and then collect the cell supernatant for centrifugation.

8. Cells that have been passaged for one time can be inoculated into proliferation medium, or inoculated into the differentiation medium [serum-free medium Satomedium with 15 nM T3, 10 ng/ml CNTF, and 5 µg/ml NAC] to start the induction of oligodendrocyte differentiation. Thereafter, half amount of pro-differentiation factors (T3, CNTF and NAC) were supplemented every Id, and immature oligodendrocytes could be obtained after 2 d of culture in differentiation medium, and mature oligodendrocytes could be obtained after 6 d of culture. Serum-free medium Satomedium was prepared by adding 4 mM glutamate, 0.1 mM sodium pyruvate, 0.1% BSA, 50 µg/ml transferrin, 5 µg/ml insulin, 30 nM sodium selenate, 10 nM vitamin H and 10 nM hydrocortisone to DMEM/F12. The cell bodies of oligodendrocytes were smaller than those of astrocytes and microglia, and those of oligodendrocyte precursor cells were bipolar protrusions that branched out into a spiderweb-like pattern as they matured with differentiation (Fig. I-4). A series of markers are specifically expressed during the process of oligodendrocyte differentiation and maturation, which can be used to identify oligodendrocytes and their stage of differentiation according to different markers.

Caveat

1. Select newborn mice of 1-2d to culture oligodendrocytes. It is not suitable to culture oligodendrocytes if there are too many astrocytes in slightly older littermates or too many neurons in younger littermates.

2. The density of inoculation in primary culture should not be too small (generally 2-3 cells from the cortex of neonatal mice are inoculated in a T75 culture flask), otherwise it is difficult to obtain a sufficient number of oligodendrocytes.3 The time of single purification of OPCs by shaking bed should not be more than 24h, otherwise it will easily lead to the decline of cell activity.

4. It is better not to add streptomycin to the serum-free medium for culturing oligodendrocytes, as it has been reported that streptomycin has a certain effect on the activity of oligodendrocytes. In addition, the serum-free medium containing various neurofactors should be used up within 2 weeks.5 Oligodendrocytes do not grow well in an alkaline environment, and attention should be paid to CO2pH in the incubator.

Common Problems

1. Shaking the cells overnight by shaking the bed usually starts in the afternoon or evening, and the cells should be observed first in the early morning of the next day to prevent the lower layer of cells from dehiscence for too long.

2. Oligodendrocyte culture in mice Oligodendrocyte precursor cells usually obtained by this method are not easy to be induced to differentiate into mature cells, and can be referred to other methods of culture (NatureProtocols,2007,2:1044-I051).

3. Oligodendrocytes are a kind of cells that are extremely vulnerable and not easy to survive, and special attention should be paid to every detail in the more complicated culture process. Currently, the serum-free medium used to induce oligodendrocyte differentiation includes DF12+Nl/N2+T3 (oligodendrocytes survive for a short time in this medium and require a high density of cell inoculation), Neuro-basal medium+B27+T3 (which facilitates cell survival, supports low-density inoculation of cells and can be co-cultivated with neurons but the maturation process of oligodendrocytes is slower) and Neuro-basal medium+B27+T3 (which facilitates cell survival, supports low-density inoculation of cells and can be co-cultured with neurons but oligodendrocytes mature slowly). (supports low-density inoculation and co-culture with neurons, but oligodendrocyte maturation is slower) and Satomedium (the most recognized medium for oligodendrocyte-independent culture, which is conducive to the maturation and differentiation of cells). Different culture methods can be selected according to the requirements of each experiment. In addition, oligodendrocytes are unable to form myelin sheaths after maturation in the absence of neuronal axonal stimulation and will gradually die rather than survive for long periods of time.

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