TITLE: Preparation of organotypic cerebellar cultures
AUTHORS: Laura  Lossi, adalberto.merighi

[DESCRIPTION]
This protocol describes the basics to prepare organotypic cerebellar cultures using the membrane interface method.

[BEFORE_START]
Be sure to have all your tool and solutions ready. Working areas must be clean and sterilized with UV ligh or 70% ethanol.


[GUIDELINES]
All procedures must be carried out under sterile conditions.

[STEPS]
SECTION: Tissue sampling
4. Euthanize mice at the required post-natal age with an overdose of intraperitoneal sodium pentobarbital (60 mg ⁄ 100 g body weight). 

SECTION: Tissue sampling
5. 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.
 

SECTION: Tissue sampling
6. 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. 
 


SECTION: Slice seeding
8. 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.
SECTION: Slice seeding
9. 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).

 

SECTION: Preparation of solutions and culture media
None. Preparation of solutions
SECTION: Preparation of solutions and culture media
None. Preparation of cutting solution
SECTION: Preparation of solutions and culture media
None. 
SECTION: Preparation of solutions/culture medium
1. Solutions
SECTION: Preparation of solutions/culture medium
2. Culture medium 
SECTION: Preparation of solutions/culture medium
1.2. Cutting solution composition: 130 millimolar (mM) n-methyl-D-glucamine Cl (NMDG); 24 millimolar (mM)  NaHCO3; 3.5 millimolar (mM) KCl; 1.25 millimolar (mM) NaH2PO4; 0.5 millimolar (mM) CaCl2; 5 millimolar (mM) MgCl2; 10 millimolar (mM) D-(+)-glucose; 1 mg/mL ascorbic acid; 2 mg/mL pyruvic acid.

SECTION: Preparation of solutions/culture medium
2.1. 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) 
SECTION: Preparation of solutions/culture medium
2.2. 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: 25 mL BME, 12.5 mL horse serum; 12.5 mL  HBSS; 250 µL  D-(+)-glucose; 250 µL  L-glutamine;  500 µL 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.

SECTION: Preparation of solutions/culture medium
1.1. Stock solutions: 1 Molarity (M) CaCl2; 1 Molarity (M) MgCl2;
5 % volume pentobarbital sodium in ddH20

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

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

SECTION: Tissue sampling
3. Have ready the following: ice-cooled cutting solution;  50 mL 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 ); 500 µL disposable insulin syringes; sterile razor blades; sterile glass/disposable Pasteur pipettes; sterile filter paper dishes. 

 

SECTION: Slice seeding
7. 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 1.1 mL medium. Work under sterile conditions.