A Technique to Optimize Cerebral Organoid Culture Using Lateral Soft Light Illumination

Use a transparent acrylic board with a thickness of 0.3 to 0.5 centimeters in the size of A5 paper, and paste white pads on the front and back of the acrylic plate. Next, install a row of LED white lights on the edge of the plate so that the lights can enter from the side of the acrylic plate and then shoot out in parallel.

Now, prepare a new six-well low-adhesion plate, and add 2 milliliters of EB formation medium to each well. After removing the EBs together with the medium, using 1000-microliters wide-bore pipette tip, transfer approximately 100 EBs per well to the 6-well low-adhesion plate.

To replace the EB formation medium every day with the same volume of fresh medium, induce a swirl flow by rotating the dish along a circular orbit. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation. Aspirate the old medium by pipetting to the edge of the well slowly. Do not suck too hard, otherwise, the EBs will be removed together. Then, add fresh media to resuspend the EBs.

For neural induction, prepare a new 6-well low-adhesion plate with 3 milliliters of neural induction medium per well. Turn on the lateral soft light and turn off other indoor light sources. Now, transfer the EBs to the 6-well plate with added neural induction medium by using a wide-mouth pipette tip to suck both the EBs and the culture medium. Then, hold the pipette upright. The EBs will gradually sink under gravity, and converge towards the mouth of the pipette tip.

As the mouth of the pipette tip touches the liquid surface again, and due to liquid surface tension, the EBs quickly sink into the medium. Incubate the samples at 37 degrees Celsius and 5% carbon dioxide for 24 hours.