nerve aggregates

Summary

Brains were removed from fetal rats at 17 d or 18 d of gestation (contact the local animal ethics committee) and the brains were made into single cell suspensions. Brain cells were cultured in multiwell plates precoated with agar to form aggregates. Over the course of 20 d of culture, the cells in the aggregates formed mature organoid brain structures.

Operation method

Program 25.5 Nerve aggregates

Principle

Brains were removed from fetal rats at 17 d or 18 d of gestation (contact the local animal ethics committee) and the brains were made into single cell suspensions. Brain cells were cultured in multiwell plates precoated with agar to form aggregates. Over the course of 20 d of culture, the cells in the aggregates formed mature organoid brain structures.

Materials and Instruments

PBSA Type II Trypsin
Dulbecco's Modified Eagle's Culture Solution
Agar Porous tissue culture plates Petri dishes Test tubes Scalpels Dissecting scissors Surgical forceps Conical flasks Water baths

Move

Coat the wells of a multiwell plate with agar culture solution as follows:

(a) Prepare a 3% agar storage solution in a conical flask (3 g agar per 100 ml D-PBSA);

(b) Heat the flask in boiling water until the agar is dissolved. Place an empty conical flask in the boiling water and fill this flask with 10 ml of hot agar solution;

(c) Slowly add pre-warmed complete growth medium to the flask until the medium reaches a concentration of 0.75% in the agar;

(d) Add 0.5 ml of warm medium-agar solution to each well of the multiwell culture plate;

(e) Allow the agar to cool and gelatinize.

These multi-well culture plates can be stored in a refrigerated box for up to one week.

1. Whole brains were excised from littermates of 17 d or 18 d gestation fetal rats under sterile conditions, and the brain tissue was placed in 10 cm Petri dishes containing D-PBSA.

2. The tissue was cut into small pieces of approximately 0.5 ^cm3 using a scalpel.

3. The tissue pieces were transferred into a test tube and washed 3 times with D-PBSA. Allow the tissue to settle to the bottom of the tube between each wash.

4. Add 5 ml of trypsin solution to the tissue and digest for 5 min at 37°C in a water bath.

5. Disperse the tissue by blowing with a Pasteur pipette about 20 times.

6. Allow the tissue to settle for 3 min, then transfer the mixed cell suspension, which is free of tissue clumps, into a test tube containing 5 ml of growth medium.

7. Add 5 ml of fresh trypsin solution to the undisperse tissue. Fresh trypsin solution is added to the undispersed tissue and the digestion and dispersion steps are repeated more than twice.

8. The cell suspension is centrifuged (200 g for 5 min ).

9. The supernatant is aspirated and the cells are suspended and collected in 10 ml of growth medium.

10. Cells are counted and 1 ml of cell suspension is added to each of the wells coated with agar ( ^3x106 cells).

11. The _we ll plates are placed in CO2 incubator for 48 h. 12. The cells are gathered into the plate and the plates are incubated for 2 hours.

12. Transfer the cell aggregates into sterile 10 cm Petri dishes and add 10 ml of growth medium.

13. Transfer the large aggregates to new agar-coated well plates individually using a Pasteur pipette.

14. Replace the culture medium every 3 d. Carefully pipette off the original culture medium. Carefully aspirate off the original culture medium and add fresh culture medium to cover the aggregates.

Over the course of 20 d of incubation, the cell aggregates may become spherical and develop into organoid structures.

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