Detection of Neuronal Aging in a Cortical Organoid Section

Abstract

Source: Shaker, M. R., et al. Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro. J. Vis. Exp. (2022).

This video demonstrates the method of detecting aging neurons in cortical organoid sections by staining for beta-galactosidase activity, which is elevated in senescent cells. The enzyme cleaves a substrate to produce a blue product, followed by microscopic observation of the stained neurons.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Characterization of neuronal aging in Cortical organoids

1. Process cortical organoids for cryosections:
   NOTE: The steps were performed in a Class 2 biosafety hood.
   1. Prepare 2 mL tubes, each filled with 1.5 mL of 4% paraformaldehyde (PFA).
   2. Cut the end of a P1000 pipette tip (to make it a wide-bore) and gently transfer each organoid to one of the 2 mL tubes prepared above (one organoid per tube).
      NOTE: To prevent excess Differentiation Medium (DM) media mixing with the PFA, allow the organoid to sink toward the opening of the P1000 pipette tip and rest the tip just above the top of the PFA in the tube before pipetting the organoid out. This will enable the researcher to transfer just the organoid and very minimal media.
   3. Allow the fixation process to take place at 4°C for 1 h.
   4. Using an uncut P1000 pipette tip, carefully aspirate excess PFA and add 1.5 mL of cold 1x PBS (Phosphate buffered saline).
   5. Transfer the tubes to an orbital shaker set at 70 rpm for 10 min at room temperature (RT).
   6. Repeat the washing process with cold 1x PBS three times to ensure all PFA has been thoroughly removed.
      NOTE: Do not dispose of PFA in regular waste containers; instead, prepare a specific chemical waste disposal container for this, as PFA is a hazard.
   7. Immerse the organoids in 1x PBS containing 30% sucrose and incubate at 4 °C until all organoids have sunk to the bottom of the tube.
      NOTE: The time required for the organoids to sink depends on their size and age. Three-month-old organoids may take up to 5 h.
   8. Using a cut, wide-bore P1000 pipette tip, gently transfer three to five organoids into a mounting mold containing a mounting solution made of 30% sucrose and 100% optimal cutting temperature (OCT) medium at a ratio of 3:2.
   9. Use a 10 µL pipette tip with the aid of a stereomicroscope to orientate and position organoids in a grid-like pattern.
   10. Place the mold on dry ice to solidify the sucrose OCT solution before proceeding with cryo-sectioning (16-20 µm) using a cryostat.
       NOTE: For senescence-associated beta-galactosidase, all tissues must be processed for sectioning once they have sunk. For immunofluorescence, tissues can be processed for sectioning the next day. If not stained immediately, all the slides containing sections must be stored at -20 °C before subsequent immunofluorescence or beta-galactosidase.
2. Process for analysis of senescence in the cortical brain organoids:
   NOTE: The following steps can be performed on a regular lab bench.
   1. Transfer the slides into a microscope slide staining container with a lid and wash the sectioned organoid tissue three times with 1x PBS for 10 min at RT to remove any excess mounting solution.
   2. Following this, incubate the washed tissue with freshly made beta-galactosidase staining solution overnight at 37 °C.
      NOTE: The beta-galactosidase staining solution is made of phosphate buffer (for 10 mL of phosphate buffer: 8.15 mL of 1 M NaH₂PO₄, 1.85 mL of 1M Na₂HPO₄) adjusted pH = 6, 100 mM of potassium hexacyanoferrate (III), 100 mM of potassium hexacyanoferrate (II) trihydrate, 5 M of NaCl, 1 M of MgCl₂, 20 mg/mL of X-Gal. Avoid using a standard cell culture incubator containing CO₂ as the CO₂ will alter the pH of the beta-galactosidase staining solution.
   3. To remove the beta-galactosidase solution, wash the stained tissues with 1x PBS three times for 10 min each at RT.
   4. Mount the washed tissues with a glass antifade mountant and allow the mounting solution to solidify for 30 min at RT before viewing under the microscope.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Formaldehyde (W/V) Methanol-free	Thermo Fisher Scientific	28908	4% of PFA are diluted in 1x PBS
CKX53 microscope with SC50 camera	Olympus
Dulbecco's Phosphate Buffered Saline	Sigma Aldrich Pty Ltd	D1408-500ML
Neurobasal Medium	Thermo Fisher Scientific	21103049	Used in DM media
OCT Embedding Compound Sakura Clear (118 mL/Bottle)	Tissue Tek	4583
Potassium Hexacyanoferrate (II) Trihydrate	Sigma Aldrich Pty Ltd	CP1087
Potassium hexacyanoferrate(III)	Sigma Aldrich Pty Ltd	455946
Prolong Glass Antifade Mountant	Life Technologies Australia (TFS)	P36980
Sucrose	Sigma Aldrich Pty Ltd	PHR1001-1G	30% of sucrose are diluted in 1x PBS