This article describes a method for visualizing embryoid bodies (EBs) using lateral soft light illumination. The technique facilitates the neural differentiation of EBs into neuroepithelial layers.
Take a lateral soft light illumination setup containing an acrylic board fitted with white pads and white LED lights installed on one edge.
Position a low-adhesion multi-well plate containing embryoid body formation medium in front.
Add the embryoid bodies, or EBs to the plate.
The LED light enters from the side of the acrylic board and emits parallel beams, laterally illuminating the EBs and enabling naked-eye visualization.
Rotate the plate to induce a swirl flow, converging the EBs to the center.
Replace the spent medium with fresh EB formation medium.
Collect the EBs using a wide-bore pipette tip.
Hold the pipette upright to allow the EBs to settle by gravity.
Transfer the EBs to another low-adhesion plate containing a neural induction medium.
Touch the tip to the medium. The EBs sink due to liquid surface tension.
Specific factors in the medium initiate neural differentiation in the EBs, forming a neuroepithelial layer.
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.
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