Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Our field of research is primate brain development and evolution. We are trying to understand the factors underlying neocortical expansion in primates. To address this question in a practical and ethically justifiable way, we use brain organoids as our research model.

Differences in brain development between modern humans, us, and neanderthals have recently emerged. These differences are due to a small number of amino acid changes in proteins with key roles in brain development. Cerebral organoids have been a crucial model system in these studies.

The study of primate development from an evolutionary point of view conducting gain and loss of function studies is important. However, apes including humans naturally cannot be used for such experiments, so genetic modification of organoids plays a vital role in this field. While working as a postdoc in my lab, Michael Heide demonstrated that the human specific gene ARHGAP11B was a key player in increasing brain size during human evolution.

As a group leader at the German Primate Center now, Michael contributed crucial insight by comparing human versus chimpanzee cerebral organoids. Our protocol offers a targeted approach for genetic modifications by specific micro injecting ventricle-like structures instead of commercial electroporation cuvettes. This approach uses a cost efficient setup including square wave electroporator and Petri dish electrode chamber instead of expensive nuclear effector solutions.

While studying function and brain development in primates by genetic modification is technically possible, it is methodologically demanding, expensive, and not allowed for great apes. The electroporation of primate cerebral organoids provides a fast and cost efficient approach to introduce genetic modifications in a model close to primate brain development. In the future, we would like to focus on the characterization of differentially expressed genes in the cortex of different primate species.

We will study these genes by the electroporation of primate cerebral organoids and analyze their potential effects on cortical progenitor cells. To begin, wash the cultured iPSCs with Dulbecco PBS or DPBS and add one milliliter of proteolytic and collagenolytic mixture. Incubate the dish at 37 degrees Celsius for two minutes to detach the cells.

Next, add 1.5 milliliters of prewarmed iPSC culture medium to stop the reaction. Dissociate the cells from the cell culture dish by pipetting up and down seven to 10 times to obtain a single-cell suspension. Transfer the cell suspension to a 15-milliliter conical centrifuge tube.

Pellet the cells at 200 g for five minutes at room temperature and aspirate the supernatant. Resuspend the pellet in two milliliters of iPSC culture medium supplemented with 50 micromolar Y27632. Now use 10 microliters of the cell suspension to count the cells using a Neubauer chamber.

Adjust the concentration of the cell suspension to 9, 000 cells per 150 microliters using iPSC culture medium supplemented with 50 micromolar Y27632. To generate embryoid bodies or EBs, shake the cell suspension tube to prevent cell sedimentation before seeding 150 microliters of the cell suspension into each well of an ultra-low attachment 96-well plate. Culture the EBs in a humified atmosphere for 48 hours.

At the end of the incubation, remove 100 microliters of medium per well and add 150 microliters of prewarmed fresh medium without Y27632. Create a 4 x 4 dimple grid on the parafilm by placing the parafilm grid on the 0.2 milliliter tube rack with the paper-enveloped side facing up, and gently press a gloved finger against each rack hole to embed the EBs in the basement membrane matrix. Then, remove the paper and cut the dimple grid out of the parafilm square using scissors to adjust its size to fit into a 60-millimeter cell culture dish.

Place the dimple to parafilm back on the 0.2 milliliter tube rack to provide a basis for the basement membrane matrix droplet generation. Carefully transfer the EBs one after another from the well of the cultured dish to the parafilm dimples using a pipette with a cut 200 microliter pipette tip. After moving 16 EBs to the grid, take a new 200 microliters pipette tip and remove the remaining medium from the dimples.

Now pipette one drop of basement membrane matrix onto each dimple containing one EB.Take a 10-microliter pipette tip and quickly move the EBs into the center of each droplet without disturbing the droplet borders. Place the dimpled parafilm with the basement membrane matrix drops in a 60-millimeter cell culture dish and incubate for 15 to 30 minutes at 37 degrees Celsius to allow the matrix to polymerize. Further, to detach the matrix embedded EBs from the parafilm, add five milliliters of differentiation medium without vitamin A to the dish and turn the parafilm square upside down using forceps so that the side with the EBs is facing the bottom of the dish.

Carefully shake the dish to detach the basement membrane matrix drops containing the EBs from the parafilm. If some of them are still attached, take an edge of the parafilm square using forceps and rapidly roll it up toward the center of the dish multiple times. Then, culture the cerebral organoids on an orbital shaker at 55 rpm in a humidified atmosphere with 5%carbon dioxide and 95%air at 37 degrees Celsius.

Keep the organoids in DM without vitamin A with medium changes every other day. A bright-filled image of a 32-day post-seeding human cerebral organoid with ventricle-like structures on the periphery and a compact healthy morphology is shown. Begin by preparing a sufficient amount of electroporation mix for the control and gene of interest.

Connect the Petri dish electrode chamber to the electroporator. Then, using microloader tips, fill microinjection needles with eight microliters of each electroporation mix. Cut the tips of the needles before use to achieve a stable flow using fine scissors.

Ensure to remove only a small part of the tip as a blunt and wide tip can severely damage the organoids. Under a microscope, choose five cerebral organoids with smooth borders and visible ventricle-like structures. Then, move them to a 35-millimeter cell culture dish containing prewarmed DMEM/F12 using a cut 1000 microliter pipette tip.

Now carefully insert the needle through the wall of a ventricle-like structure and infuse it with the electroporation mix until visibly filled. Do not apply excessive pressure on ventricle-like structures to avoid bursting. Transfer one micro injected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F12.

Arrange the organoid so that the surfaces of the microinjected ventricle-like structures face toward the electrode connected to the positive pole of the electroporator. Now electroporate the cerebral organoids one by one using five pulses of 80 volts for 50 milliseconds each with an interval of one second. If the microinjection and electroporation were performed under non-sterile conditions, move the electroporated organoids under a laminar flow hood to a new 35-millimeter cell culture dish filled with prewarmed DMEM/F12.

Then, culture the electroporated organoids in DM with vitamin A on an orbital shaker at 55 rpm in a humified atmosphere of 5%carbon dioxide and 95%air at 37 degrees Celsius. The next day, after electroporation, check the cerebral organoids for successful electroporation under a conventional inverted fluorescence microscope. Transfer the electroporated organoids to a 15-milliliter conical centrifuge tube using a cut 1, 000 microliter pipette tip and remove excess medium.

Add a sufficient amount of 4%paraformaldehyde in DPBS and incubate for 30 minutes at room temperature. At the end of the incubation, aspirate the paraformaldehyde. Then, add five milliliters of DPBS, shake gently, and aspirate the DPBS.

Store the organoids in DPBS at four degrees Celsius until further use. Two days after electroporation, GFP-positive cells are almost exclusively localized in the ventricular zone and are positive for PAX6 indicating that these cells are epical progenitors or newborn basal progenitors. After 10 days, the GFP-positive cells are localized in the basal regions.

These cells are also positive for PAX6 or NeuN, which is indicative of basal progenitors or neurons respectively. 3D reconstruction of the electroporated cerebral organoids to obtain an impression of the 3D distribution of the GFP-positive cells is shown.