The protocol facilitates operation of cerebral organoids culture and helps to promote the application of brain organoids. The earlier stage organoid can be seen with the naked eye by using a lateral soft light lamp which is convenient for many operations. The improved medium change methods and organoid transfer operations are also helpful for other types of organoid cultures and the design of automatic cultures machines.
To begin, culture the iPSCs with mTeSR1 medium in a matrigel covered 35 millimeter Petri dish and change the medium every day. The iPSC growth density must not exceed 75%Then, pipette out the mTeSR1 medium from the iPSCs and wash it twice with one millimeter of PBS. After pipetting out the PBS, add 300 microliters of cell detachment solution to digest the iPSCs into single cells for three to four minutes at 37 degrees Celsius.
Later, resuspend the cells in two millimeters of EB formation medium and centrifuge the sample for five minutes at 300 times g. Treat the specialized 24 well plate with 500 microliters per well of anti adherence rinsing solution for five minutes. Now, resuspend the cells in the EB formation medium containing Rho kinase inhibitor Y27632.
After removing the anti adherence rinsing solution, rinse each well with two milliliters of EB formation medium. Next, add the cells to the specialized 24 well plates at a density of three times 10 to the sixth cells per well and centrifuge the plate at 100 times g for two minutes at room temperature. Uniformly distribute the cells in each chamber at the bottom of the plate.
Incubate the samples at 37 degrees Celsius and 5%carbon dioxide for 24 hours. If centrifugation was not used in the previous step, incubate for 36 hours and keep the sample steady. 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 two 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 six 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 pipeting 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 six well low adhesion plate with three 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 six 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.
Place the membrane matrix in a four degree Celsius refrigerator for 60 minutes to dissolve. After turning on the lateral soft light, turn off the light source on the top of the console and keep the membrane matrix on ice to prevent solidification. Transfer a small amount of EBs to a 60 millimeter dish containing a fresh expansion medium to make it easier to remove single EB.Using a 200 microliters wide bore pipette tip, suck a single EB containing approximately 10 microliters medium each time, and then make droplets by adding it to the bottom of the six well plate.
Use five droplets per well. Add 15 microliters of the membrane matrix to each EB containing drop. Quickly mix and embed the EB ball in the center of the droplet.
Allow the droplets containing EBs to solidify by placing the six well plate into a 37 degree Celsius incubator for 30 minutes. Then, add three milliliters of the expansion medium to each well and gently blow up the matrix embedded EBs to suspend them and incubate for three days. For organoid maturation, gently remove the original medium and add three milliliters of maturation medium to each well.
Place the organoid plate on a horizontal shaker in a 37 degree Celsius incubator and continue to rotate the culture horizontally. Change the fresh maturation medium every two to three days. Due to gravity and relatively higher density than the medium, the resuspended EBs will gradually sink.
Hence, the EBs can be conveniently transferred. As 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.
The EBs cultivated in the early stage expressed the OCT4 marker which indicated good pluripotency. In the later stage, the EBs developed into mature cerebral organoids. The mature cerebral organoids were positive for apical progenitor cells marker PAX6 and neuron specific marker tubulin J1.The iPSCs from healthy individuals and SCA3 patients were cultivated to grow into organoids.
The gene expression profiles of the normal cerebral organoid group and the SCA3 cerebral organoid group showed significant differences in pathways such as neurotransmitter transport, synapse formation, and regulation. When embedding the EB with basement membrane matrix the operation times should not be too long to avoid EBs drying out. It is recommended to protect rapidly.
