Geração de iPSC-derivado do cérebro Organóides Humanos para modelar alterações precoces do desenvolvimento neurológico

The overall goal of this protocol for generating iPSC-derived Human Brain Organoids is to model early human neurodevelopmental disorders. This method can help answer key questions in the human brain development field such as the role of synthesomes and cilia in novel neurogenesis and primary microcephaly. The main advantage of this technique is that it is a robust and quick model to obtain reproducible results from patient's iPSC-derived brain organites.

Demonstrating the procedure will be Elke Gabriel, a post-doc from my laboratory at the Center for Molecular Medicine, Cologne. To begin this procedure, collect the neurospheres with a 200 microliter micropipette using a two millimeter tip previously cut with sterile scissors. Next, place the neurospheres approximately five milliliters away from each other on a paraffin film in a 100 milliliter dish.

And carefully remove as much of the remaining medium as possible. Then, add a drop of EHS matrix onto each single neurosphere. Incubate the EHS matrix drops with the neurospheres for 15 minutes at 37 degrees celsius.

After that, wash the neurospheres carefully from the paraffin film by flushing them with neurosphere medium. Subsequently, incubate the neurospheres for the next four days and add two millimeters of fresh neurosphere medium on day two. In this procedure, add 100 milliliters of brain organoid medium to each spinner flask through its side arms.

And prewarm the medium in the incubator at 37 degrees celsius for at least 20 minutes. Then, make sure the neurospheres are all separated. If two or more are connected through EHS matrix, separate them by cutting the connecting matrix with a scalpel.

Set up a stirring program at 25 RPM. Carefully transfer the EHS matrix embedded neurospheres into the spinner flasks containing 100 milliliters of organoid medium. Following that, space the spinner flasks on a magnetic stirring platform in the incubator for 14 days at 37 degrees celsius.

This is day zero of the organoid culture. Change the medium once a week by removing half of the medium and adding the same amount of the fresh medium. In this step, collect the organoids from the spinner flask culture on day 14 with a pre-cut one milliliter micro pipette.

Put all of them in a 60 millimeter dish and wash them once with five milliliters of warm DMEM F12 for three minutes. Afterward, prepare a 1.5 milliliter tube with 500 microliters of warm, 4%PFA. Place each organoid separately in each tube and fix them for 30-60 minutes at room temperature.

Afterward, use an inoculation loop to removed the organoids. Then, remove PFA and wash the fixed organoids twice for 10 minutes each time with one milliliter of PBS. Subsequently, store the organoids in one milliliter of PBS at four degrees celsius for up to 7 days for further use.

To embed the organoids for cryosectioning, remove PBS and add 1 milliliter of 30%sucrose solution in each tube to dehydrate the organoids before cryofreezing them. After adding sucrose solution, the organoids should be floating at the surface. Store the organoids overnight in sucrose solution at four degrees celsius.

By the next day, the organoids should've sunk down to the bottom of the tube. Following that, fill a vinyl specimen mold with 400 milliliters of OCT compound. And use an inoculation loop to place an organoid at the center of the mold.

Label the rim of the mold with the sample name. Then, freeze the organoid-containing mold at negative 80 degrees celsius until cryosectioning. Subsequently, coat the glass cryoslides with 0.1%PLL and PBS for five minutes at room temperature.

Then, let the slides dry for three hours. Store the slides at four degrees celsius and warm them up to room temperature before use. Following that, section the cryo-frozen organoids into 20 to 50 micrometer thick slices on the PLL-coated glass cryoslides.

Let the sections dry from one hour at room temperature and store the sections at negative 80 degrees celsius until further processing. Immunofluorescent imaging of cryosectioned organoids displaying a typical VZ and the primitive cortical plate. The VZ spans from the apical side to the basil side.

Note that the cells within the VZ display paliside-like nucleide suggesting that they are radial glial cells. The right panel shows the immunofluorescent staining of nested positive NPCs within the VZ and TUJ1 positive neurons in the primitive cortical plate. Shown here are two examples of VZs stained with PVIM and ARL 13B.

The regions marked by white squares are magnified. Apical radial glial cells dividing at the apical side of the VZ are PVIM positive. The division plane of an anaphase cell is horizontal to the lumen surface line which is marked by ARL 13B staining.

Once mastered, this technique can be done in 23 days if it is performed properly. While attempting this procedure, it's important to remember to perform periodic quality check and microplasma check on cultures. After its development, this technique paved the way for researchers in the field of development in neurobiology to explore the mechanisms of neurodevelopment in human brain organoids.

After watching this video, you should have a good understanding of how to generate human brain organoids from various iPSC cell lines including patient cell lines with compromised cellular functions.