TITLE: Establishment and Maintenance of Organotypic Cerebellar Slice Cultures (OCerSC) from Aged Mice
AUTHORS: Kaitlan S. Smith, Michael Fernandes de Almeida, Jonathan C Schisler

[DESCRIPTION]
This protocol outlines a method for culturing organotypic slices from aged mouse cerebellar tissue, maintaining the cerebellum’s complex three-dimensional structure and cellular composition ex vivo. This technique simulates various physiological and pathophysiological conditions to study neurodevelopmental and neurodegenerative disorders and synaptic plasticity mechanisms. Expected outcomes include detailed neuronal morphology and functional connectivity analyses at the cellular and circuit levels using advanced imaging, electrophysiological techniques, and biochemical analyses such as protein expression, gene expression, metabolic activity, oxidative stress markers, neurotransmitter levels, calcium imaging, and enzyme activity assays.

[BEFORE_START]
Before you begin the slicing process, ensure that all six-well plates are prepped and placed in the incubator at 37 ºC with 5% CO2 for at least 30 minutes. This step is crucial to ensure that the media and inserts are at the correct temperature and conditions, which helps maintain the viability and quality of the cerebellum slices.

Safety Precautions:
When handling the plates, incubator, animals, and equipment, wear appropriate personal protective equipment (PPE), such as gloves, safety eyewear, and a lab coat.
Ensure that dissection is performed on a laboratory bench or in a separate hood dedicated to dissection.
Use a different hood or area for cerebellum slicing to maintain a sterile environment.
Follow proper aseptic techniques to minimize the risk of contamination and maintain sterility.

[GUIDELINES]
When planning for this experiment assume that one six-well plate, with 4-6 slices per well, can generally accommodate cerebellum slices for 2 animals.  

[STEPS]
SECTION: Equipment
2. 
 

 

 

SECTION: Reagents
3.               
  
  
  

SECTION: Materials
1. Slice Culture Consumables: 
  
  
      
  
SECTION: Solutions
4. Slice Culture Media #1 
Neurobasal-A Medium supplemented with:
2% B27
1% N2 Supplement
1% Glutamax
200 g/L glucose
1.5% Penicillin/streptomycin
1.5% Amphotericin B
80 μM Indomethacin

Slice Culture Media #2 
Neurobasal-A Medium supplemented with:
2% B27
1% N2 Supplement
1% Glutamax
200 g/L glucose
1.5% Penicillin/streptomycin
1.5% Amphotericin B

Phosphate Buffered Saline
SECTION: Materials
1.1. Tools:
Scalpel
Forceps 
Razor blade 
Scissors 
Transfer pipettes
Paint brushes (recommended sizes no.3 and no.2)
SECTION: Preparation Steps
5. Before Starting Tissue Harvest and Slicing Steps
Only one cerebellum should be simultaneously sliced and harvested to minimize the time between cerebellum isolation and slice distribution to the inserted wells. This practice helps ensure the viability and quality of the cerebellum slices by reducing the time the tissue is exposed to non-optimal conditions.
 

SECTION: Preparation Steps
5.1. Sterilize Equipment:
Use 70% ethanol to wipe down all surfaces inside the biological safety cabinet thoroughly. Ensure all areas, including corners and hard-to-reach spots, are properly cleaned.
Apply 70% ethanol to a clean cloth or paper towel and wipe down the tissue slicer and all dissection instruments, ensuring that all surfaces are clean.
Allow the equipment to air dry completely before use.
SECTION: Preparation Steps
5.3. Prepare Tissue Slicer
Mount a new blade on the tissue slicer. 
Set the slice thickness  .
SECTION: Preparation Steps
5.2. Prepare Six-well Plates 
Place one cell culture insert per well and add 1mL of Slice Culture Media #1.
Ensure membranes are thoroughly wet and that there are no air bubbles.
Place the six-well plates in an incubator at 37 °C  with 5% CO2 for at least 30 minutes before starting the cerebellar slicing process.
SECTION: Preparation Steps
5.5. Prepare Hibernation Media Dishes
Place Hibernate-A Medium in a 37 °C water bath for at least 30 minutes before starting the cerebellar slicing process. Ensure the medium reaches the correct temperature to maintain tissue viability.    
Prepare one 60 mm petri dish per animal.
Add 4-5 mL of pre-warmed Hibernate-A Medium to each dish. 
SECTION: Preparation Steps
5.6. Prepare Euthanasia and Dissection Prep 
Set up the euthanasia chamber according to the approved animal protocol.
Ensure that all necessary components are in place and functioning correctly. 
Double-check that the sterile dissection tools are ready. 
Obtain a 66 mm sterile petri dish for use during the dissection process.
Prepare sterile PBS and set to chill on ice. 
Transport the mice to the dissection area. This protocol was developed using 22-92 week-old B6129F1/Tac mice.
 

SECTION: Cerebellar Slicing
6. 
 

SECTION: Cerebellar Slicing
6.1. Dissect the Brain: 
Following proper IACUC-approved animal euthanasia, decapitate the animal.
Carefully expose and remove the brain, taking special care not to damage the cerebellum.
Transfer the brain to an empty sterile petri dish.
Using a transfer pipette, wash the brain with cold, sterile PBS to remove any extraneous hair or blood (Figure 1).
Remove the forebrain and spinal cord to isolate the cerebellum (Figure 1).
Transfer the cerebellum to a sterile petri dish containing 4-5 ml of pre-warmed Hibernate media (Figure 1). 
SECTION: Cerebellar Slicing
6.2. Slicing the Cerebellum:
Transfer the cerebellum to the Whatman filter paper glued onto the chopping disc.
Critical Point: Orient the brain so the cerebellum faces upwards and the sagittal plane is aligned with the chopper blade (Figure 1). This ensures that the slices will be sagittal, maintaining the structural integrity necessary for subsequent analyses.
Using the manual setting on the chopper, slice the cerebellum into sections (Figure 1). 
Use a paintbrush to hydrate the cerebellar tissue throughout the slicing process. (Avoid overwetting the cerebellum and the filter paper).
After each cut, use a paintbrush to transfer the cerebellum slice to a petri dish filled with pre-warmed Hibernate media (Figure 1). 
Select the intact cerebellar slices and transfer 4 to 6 individual slices onto the insert membranes in the six-well plates (Figure 1)to distribute them evenly on the insert. 
Incubate at 37 °C with 5% CO2.

     
 

    

 

SECTION: Culture Conditions
7. 
 

Incubation and Media Changes
Initial Incubation: Once slices are securely placed onto the membrane, incubate plates at 37ºC with 5% CO2. 37 °C 
First Media Change (18-24 hours after slicing): Remove the slice culture media #1 and replace it with fresh slice culture media #1. 1440 min 
Subsequent Media Changes: After the first media change, change the culture media every 2-3 days.
Media Schedule:
DEV 1-5: Maintain the OCerSC in Slice Culture Media #1, following a scheduled media change every 2-3 days. 2880 min 
DEV6 - End of the experiment: Maintain the OCerSC in Slice Culture Media #2, with a scheduled media change every 2-3 days. 2880 min 

 


 

SECTION: Preparation Steps
5.4. Prepare the Chopping Disc
Using Elmer’s glue, attach Whatman filter paper onto the chopping disc.
Allow the glue to dry completely to ensure the filter paper is firmly attached.
Mount the chopping disc onto the tissue slicer.