Preparation of organotypic cerebellar cultures

Solutions

Stock solutions: 1Molarity (M) CaCl₂; 1Molarity (M) MgCl_2;

5% volume pentobarbital sodium in ddH₂0

Cutting solution composition: 130millimolar (mM) n-methyl-D-glucamine Cl (NMDG); 24millimolar (mM) NaHCO₃; 3.5millimolar (mM) KCl; 1.25millimolar (mM) NaH₂PO₄; 0.5millimolar (mM) CaCl2; 5millimolar (mM) MgCl₂; 10millimolar (mM) D-(+)-glucose; 1mg/mL ascorbic acid; 2mg/mL pyruvic acid.

Cutting solution preparation: To make 1L, pour 850mL double-distilled water into a volumetric flask. Add 25.38gNMDG, 2.017g NaHCO3, 261mg KCl, 172mg NaH₂PO₄, 1.80g D-(+)-glucose, 1g ascorbic acid, 2g pyruvic acid. After complete dissolution SLOWLY add 5mL MgCl₂ stock solution and 500µL CaCl₂ stock solution.7.2 to 7.4 with HCl.

Steril filter and store at 4°C . The solution is stable for several months. Discharge if it becomes turbid.

Culture medium

Culture medium composition: 50% Basal medium Eagle (BME), 25% horse serum; 25% Hank's balanced salt solution (HBSS); 0.5 % D-(+)-glucose; 0.5% L-glutamine (200 mM solution); 1% antibiotic antimycotic solution (100 x)

Culture medium preparation : Work under a laminar flow hood and use sterile glassware/plasticware. In a 100 mL cylinder add the components in the order indicated above. For 50 mL: 25mL BME, 12.5mL horse serum; 12.5mL HBSS; 250µL D-(+)-glucose; 250µL L-glutamine; 500µL antibiotic antimycotic solution. Transfer in a glass bottle and protect from light with aluminum foil. Store at 4°C . Medium is stable for at least six months. Discharge if color changes and/or it becomes turbid.

Have ready the following: ice-cooled cutting solution; 50mL sterile glass or plastic becker; 150 mm diameter sterile glass or plastic Petri dishes; sterile dissection/slice handling tools; sodium pentobarbital stock solution (Room temperature ); 500µL disposable insulin syringes; sterile razor blades; sterile glass/disposable Pasteur pipettes; sterile filter paper dishes.

Euthanize mice at the required post-natal age with an overdose of intraperitoneal sodium pentobarbital (60mg ⁄ 100g body weight).

Quickly remove the brain from the skull while the head is kept submerged in the ice-cooled cutting solution and isolate the cerebellum under the stereomicroscope.

Before cutting, completely remove the meninges with a pair of N.7 Dupont forceps. Place the cerebellum on the stage of the tissue chopper within a drop of the ice-cooled cutting solution. Operate the chopper and cut 350 μm-thick parasagittal slices. Once terminated slicing, collect slices with a curved spatula (they are usually stuck together) and place them in a sterile 50-mm Petri filled with the ice-cooled solution. Store at 4°Cuntil ready to separate slices. Separate individual slices under the stereomicroscope with a spatula and a needle, trying not to damage the tissue. During the entire procedure, slices must be submerged in the ice-cooled cutting solution. Discharge damaged slices.

Before starting to seed slices onto the Millicell inserts bring the culture medium at Room temperature and fill some sterile 35-mm plastic Petri dishes with 1.1mL medium. Work under sterile conditions.

Collect slices one by one and carefully lift them onto the dry Millicell membrane. Once the required number of slices has been plated in the insert, place it inside a 35-mm Petri dish containing the required amount of medium. Be careful to avoid air bubbles forming between the insert membrane and the medium, i.e. check that the membrane's lower surface is completely wet.

Incubate at 34°C in 5% volume CO₂ for up to 30 days in vitro (DIV). Medium has to be changed twice a week. Allow slices to equilibrate to the in vitro conditions for at least 4-6 DIV before treatments (if applicable).