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

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

1. Preparation of the soft light lamp (day 1)

1. Use a transparent acrylic board with a thickness of 0.3-0.5 cm in the size of A5 paper. Paste white pads on the front and back of the acrylic plate.
2. 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 (Figure 1A-F, Figure 2B).
   NOTE: As the diameters of EBs in the early stage are approximately 200 µm to 300 µm, it is difficult to observe them clearly with the naked eye under the fluorescent lamp of clean benches. In contrast, due to the enhancement of diffuse reflection, we can detect the EBs clearly by using laterally illuminated soft light (Figure 2B, C). A 6-well plate is recommended for EB cultures in subsequent experiments. However, to better show the visual effect of soft light, dishes are sometimes used to take pictures and videos in this study instead of plates, so please do not misunderstand. The laterally illuminated soft light lamp can also be used for the 6-well plate.

2. EB transfer and medium replacement (days 2-5)

1. Prepare a new 6-well low adhesion plate and add 2 mL of embryoid body (EB)-formation medium to each well.
2. Remove the EBs together with the medium with a 1000 µL wide-bore pipette tip (see Table of Materials) and transfer them to the 6-well low adhesion plate (~100 EBs/well).
   NOTE: The operation process adopts a soft light lamp (mentioned in step 1) to make the EBs easier to observe. Turn off other indoor light sources to improve the visual effect of the soft light.
3. Replace with the same volume of fresh EB-formation medium every day. Use the secondary flow to gather the EBs to the center and change the medium.
   1. 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 medium to resuspend the EBs.
      NOTE: The principle secondary flow (Figure 2D). Induce a swirl flow by rotating the dish along a circular orbit. Due to the swirl flow, a secondary flow is induced and directed toward the center. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation, after which medium change or embryoid transfer can be executed readily.

3. Neural induction (days 5-7)

1. Prepare a new 6-well plate with low adhesion and add 3 mL of Neural induction medium (Table 1) to each well.
2. Turn on the lateral soft light (mentioned in step 1) and turn off other indoor light sources.
3. Transfer the EBs to the 6-well plate with added Neural induction medium (~100 EBs/well). Add as little as possible of the original medium to the new well.
   NOTE: Introduce simple skills of organoid transfer. Naturally, under gravity and with a relatively higher density than the medium, the resuspended EBs will gradually sink by applying the operation shown in Figure 2E. Hence, the EBs can be conveniently transferred. Since, compared to EBs, free cells and dead cell fragments sink more slowly, most of the free cells and dead cell fragments can thus be removed through this sedimentation method (Figure 2F).
4. Incubate the samples at 37 °C and 5% CO₂ for 24 h.
   NOTE: Under the microscope, the diameter of the EBs was approximately 500 µm, and the edge was translucent, indicating that a neuroepithelial layer formed.

Table 1. Composition of the Neural induction medium