Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

This protocol addresses some key issue existing in organoid generation like robustness and reliability, and we were able to apply these improved organoids to innovative research on neuronal aging. Heterogeneity and reproducibility are significant issues when generating cortical brain organoids. To overcome these, we first differentiate hPSCs into neuroectodermal colonies and then neuro epithelial spheroids, which generate reproducible tissue architecture.

These cortical brain organoids start to exhibit typical signs of senescence after a prolonged in vitro culture, which make them a useful platform for studying aging related neuronal processes. To begin, plate the hPSC colonies on an hESC qualified basement membrane matrix from one well of a six well plate at 60%confluency into three wells to achieve 20 to 30%density and then proceed with induction of 2D neuroectodermal colonies. Add fresh N2 medium supplemented with dual SMAD inhibitors daily to each well for the next three days.

For generating 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies, remove two milliliters of N2 medium from the six well plate and wash one time with HBSS to ensure that all of the N2 medium is removed. Add one milliliter of dispase to each colony containing well. Incubate the plate for 20 to 25 minutes at 37 degrees Celsius and check for colony detachment regularly.

At the end of the incubation, to stop the activity of the dispase enzyme, add one milliliter of N2 medium to the well. Using a wide-bore P1000 pipette tip or a modified P1000 pipette tip cut with sterile scissors, transfer the colonies into a 15 milliliter tube and allow the colony clumps to sink to the bottom of the tube with gravity. Then with a standard P1000 pipette tip, carefully remove the supernatant.

Replace it with one milliliter of fresh N2 medium and repeat washing thrice to ensure complete removal of dispase. After resuspending the cell clumps and N2 medium, transfer the cell suspension to one well of a six well plate and add 40 nanograms per milliliter of bFGF. After cryosectioning the organoids, remove the excess mounting solution by transferring the slides into a slide staining container with a lid and wash the sectioned organoid tissue three times with PBS for 10 minutes at room temperature.

Then incubate the slides overnight at 37 degrees Celsius with freshly made beta-galactosidase staining solution. Wash the stained tissues three times with PBS for 10 minutes each at room temperature to remove the beta-galactosidase solution. Now mount the washed tissues with a glass antifade mountant and allow the mounting solution to solidify for 30 minutes at room temperature before viewing it under the microscope.

Before neuroectodermal differentiation, the hPSC colonies showed a tight flat monolayer morphology without differentiated cells contaminating the colonies. The expression of NANOG also confirmed the pluripotency of the hPSC colonies. After differentiation of hPSC colonies into neuroectodermal colonies, a longer columnal shaped morphology was observed, and they were negative for NANOG.

After dispase treatment and exposure to FGF2 in the N2, the neural stem cells in these spheroids proliferated and formed a significant number of neural rosettes that demonstrated the apical-basal polarity of the spheroid by expression of Z01 in the cells at the center and the outer edge of the spheroid. Once embedded, the spheroid proliferates rapidly and starts budding, indicated by the presence of the compact tissue nodes and their expansion outward from the main body between one to three weeks, which was further confirmed by quantitative analysis and the spheroid diameter significantly increased over three weeks. The immunofluorescence staining confirmed the presence of neuro progenitor cells and cortical layer markers in the organoids with clear layering, which was observable across different time points.

The effect of neuronal aging related processes on the brain was also studied. With time, a significant increase in the presence of senescence associated beta-galactosidase was observed and at week 13, the presence of p21 was detected, which indicates senescence. It is important to ensure that all of the excess dispase is removed and that you gently detach and reseed the intact neuroectodermal colonies to form healthy spheroids.

This platform of our unique opportunity to study the biology of a human brain aging, it also allows a research on critical brain development, provided that this organoids being cultured using a bioreactor.