A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids are a significant tool to study disease in a system that is easily manipulated and incorporates a human setting and the appropriate interaction of multiple cell types. This technique can be applied to study numerous diseases of the brain, including neurodevelopmental disorders, toxic insults, and the growth and treatment of cancer in the brain. The main advantage of this protocol is the ability to generate highly reproducible brain organoids with a broad variety of neuronal cell types.

This is done using a very simplistic workflow that could be accomplished by most laboratories. And these are produced in a temporary appropriate manner without the influence from specialized growth factors or undefined matrices making it a very simple and cost effective system. To begin, combine 100 microliters of matrix with 5.9 milliliters of ice cold Dulbecco's Modified Eagle Medium or F12 media in a 15 milliliter conical tube.

Coat each well in a six-well plate with one milliliter of the membrane matrix. Wrap the plate in paraffin film and store overnight at four degrees Celsius. The next day, aspirate excess media from each well, rinse the wells with F12, and add H9 hESCS in a total volume of two milliliters of mTeSR1 media per well.

Culture the cells from week to week. Supply daily with two milliliters of mTeSR1 media and place the plate in a 37 degree Celsius low oxygen incubator with 5%oxygen and 5%carbon dioxide. Use glass tools to weed out differentiating cells from the culture between passages.

Refresh the media daily. Cells should be passaged four to six days prior to utilizing the H9 cells for organoid production. To passage the cells, start by rinsing them with DMEM F12 media and aspirating the excess liquid.

After rinsing, add protease solution and incubate the cells for 40 minutes in order for colonies to float within the well. Next, wash the cells three times using DMEM F12 and if necessary titrate to make the pieces smaller. Then distribute the cells at an approximate one-to-eight ratio over four six-well plates and place the plates in the incubator and feed daily.

After four to six days, the cells reach approximately 80%confluency. Transition them to a regular incubator. Dilute the protease stock solution to a working concentration by adding one milliliter of the stock solution to five milliliters of DMEM or F12 medium per each six-well plate of hESCS.

Aspirate and remove the cell culture media, then cover the hESCS with the protease solution. Place the plates in the incubator for 10 to 15 minutes or until the edges of the colonies round up and begin to separate from the matrix but before they round up completely. Tilt the plate, aspirate the protease solution, and gently wash the cells with two milliliters of DMEM or F12 for each well three times.

Make sure the colonies stay attached to the matrix. So it's critical that the pieces of ES cells are in the appropriate size so make sure that they're in dispase for the appropriate amount of time. If they're in there for too short of a time, it can be very difficult to flush them off and break them apart.

And if they're in there for too long, they can actually just come off during the wash steps. Add back about one to 1.5 milliliters of fresh mTeSR media to each well and flush the cells off the plate into a 50 milliliter conical tube using gentle pipetting. Aspirate and dispense hESCS within the plate until they reach approximately 1/30th of their original size.

Now the colony clusters resemble 250 to 350 micrometer sized pieces. Transfer the cells into a single ultra low attachment T75 flask containing 30 milliliters of mTeSR media without bFGF. The next day, tilt the flask such that the live cells pool in the corner.

If there are a large number of cells that have adhered to the bottom of the flask, use a 10 milliliter pipette to transfer the cells to a new flask. Expect to have a high population of dead cells for the first few days. Once the cells settle, aspirate off the media and dead cells leaving about 10 milliliters of media containing the live cells and add 20 milliliters of low bFGF media supplemented with 30 nanograms per milliliter of bFGF.

After two days, check the cells. Most of the cells should look healthy and bright. However, if more than a third of the cells appear dark, tilt the flask and replace 20 milliliters of media with 20 milliliters of low bFGF media supplemented with 20 nanograms per milliliter of bFGF.

On day three, remove half of the media and replace with 15 milliliters of hESC media supplemented with 10 nanograms per milliliter of bFGF. On day five, replace half of the media with 15 milliliters of neural induction media. After that, every other day, replace half of the medium with neural induction media.

After three weeks in culture, add 100X Penicillin-Streptomycin to the media at a final concentration of 1X if desired. Refresh half the media every other day. In this protocol, the formed brain organoids looked bright and similar in size without dark dying cells in the centers of these clusters.

The cells were gradually weaned off bFGF. On day five, they were placed into neural induction media and they remained in this media throughout the culture period. Although the organoids got larger and thus darker over time, the neural rosette-like structures expanded which indicates the initiation of neural differentiation and contains features of the embryonic neural tube.

To take a more in-depth look at the gene expression within the cells, qPCR analysis was performed. The glutamate transporter VLGUT1 was expressed at two and a half weeks, increased at five weeks, and remained consistent through five months of culture. A forebrain marker Foxg1 was expressed at low levels until five weeks in culture.

The deep layer marker Tbr1 peaked around five weeks and decreased subsequently. Whereas the expression of the upper layer marker Satb2, the ventral marker Eng1, the hindbrain spinal cord marker Hoxb4, as well as the oligodendrocyte marker Olig2 all increased over time. In contrast, the stem cell marker Sox2 decreased over time.

The glial marker GFAP peaked at five weeks and remained relatively constant subsequently. Remember to take the utmost care when handling ES cells in early stage organoids to avoid contamination as they're grown without antibiotics and also remember to feed according to schedule. Following this procedure, you can evaluate the organoids using techniques such as quantitative RT-PCR for gene expression and immunofluorescence for protein expression and localization.

Using this technique, we were able to study the ability of a small molecule to alleviate aspects of human neonatal hypoxic injury as modeled in a hypoxia chamber. It is significant that these human brain organoids behaved similarly to in vivo mouse experiments.