All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Labeled cell injection into the brain organoid
NOTE: For this paper, cerebral organoids were produced using a commercial kit as per the manufacturer's instructions. This can be replaced with the cerebral organoid of interest. The materials used in this part of the protocol must be prechilled to avoid the gelling of the solubilized basement membrane matrix at temperatures above 4 °C.
- Place the insulin syringe(s), tips, and tubes that are going to be in contact with the solubilized basement membrane matrix at −20 °C to let them cool.
- Thaw a suitable-sized aliquot of solubilized basement membrane matrix on ice, depending on the number of injections that are going to be performed (~2 μL/injection). This takes approximately 30 minutes.
- While the aliquot is thawing, split, harvest, and count the cells.
- Calculate the volumes needed per injection. If doing multiple injections, place the total volume of cells in a 1.5 mL tube, and add up to 1 mL of DMEM/F12.
NOTE: A volume that allows for a few extra injections should be used to account for losses/pipetting errors. - Place the cells in ice while the next steps are being prepared.
- After thawing, dilute the solubilized basement membrane matrix to a final concentration of 3 mg/mL in ice-cold DMEM/F12.
- Take the single-cell suspension from the ice, and spin it down at 300 × g for 5 min at 4 °C.
NOTE: If a refrigerated centrifuge is not available, spinning at room temperature generally appears to be tolerated. - Gently remove the medium completely without disturbing the cell pellet. Resuspend the cell pellet by gentle pipetting in the diluted solubilized basement membrane matrix (3 mg/mL) to obtain a final volume of 2 μL per injection to be performed, and immediately place it back on ice until use.
NOTE: The resuspension of the cells needs to be performed slowly and with prechilled tips to avoid the formation of bubbles and gelling. - Transfer the prechilled syringe(s) from the −20 °C freezer into an ice bucket. Keep them there until use.
- Remove the plate with the brain organoids from the incubator. Use a wide-bore tip to transfer the organoid to be injected into a 35 mm dish. Remove all the medium possible without damaging the organoid to stabilize it and facilitate the injection.
NOTE: It is important to use wide-bore tips to avoid destroying the organoid. If they are not available, cut the end of a regular P1,000 tip with sterile scissors. - Place the 35 mm dish containing the organoid under a dissecting microscope to help guide and facilitate the injection.
NOTE: If a dissecting microscope is not available in a sterile area, it is possible to perform the injection without one, albeit with somewhat reduced control. Another alternative is the use of a magnification loop or glasses. - Once the organoid(s) are ready to be injected, gently resuspend the cells with a chilled P20 pipette tip, and move 2 μL (containing the desired number of cells to be injected) onto a prechilled sterile glass slide.
NOTE: For multiple injections, an additional 2 μL volume can be added across the glass slide. Keep it on ice, and take care to avoid evaporation. - Take a prechilled insulin syringe from the ice, and slowly draw up the 2 μL of cell suspension with the bevel of the needle facing down to allow all the cells/medium to be taken up.
NOTE: Make sure to go extremely slowly to avoid drawing in any air. - Open the lid of the dish containing the organoid, and focus the microscope on it. Hold the dish with one hand. Place the bevel of the needle up, and with the other hand, inject the cells slowly into the organoid surface.
NOTE: It is important to inject slowly to avoid damaging the tissue. - After the injection, place the lid back into the dish, and let the injected organoid sit for 1-2 min.
NOTE: This allows the diluted, solubilized basement membrane matrix to gel, holding the cells in place. If the medium is added too early, it can wash away the cells sitting at the surface. - Gently add 500 μL of the organoid medium, and transfer the organoid with a wide-bore tip into a well of a 24-well plate. Incubate the organoid at 37 °C and 5% CO2 overnight. Perform complete medium changes every second day.
NOTE: It is important to include a negative control organoid (mock-injected), which will be used for imaging adjustments. To do this, inject an organoid as previously described with diluted solubilized basement membrane matrix (3 mg/mL) to obtain a final volume of 2 μL without cells.