Name: Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification
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We apologize to all the researchers whose work could not be cited due to space limitations. We thank Ulrich Bleyer of the technical services at DPZ and Hartmut Wolf of the workshop at MPI-CBG for the construction of the Petri dish electrode chambers; Stoyan Petkov and R&#252;diger Behr for providing human (iLonza2.2), rhesus macaque (iRh33.1) and marmoset (cj_160419_5) iPSCs; Sabrina Heide for the cryosectioning and immunofluorescence staining; and Neringa Liutikaite and C&#233;sar Mateo Bastidas Betancourt for critically reading the manuscript. Work in the laboratory of W.B.H. was supported by an ERA-NET NEURON (MicroKin) grant. Work in the laboratory of M.H. was supported by an ERC starting grant (101039421).

1. Culture of primate iPSCs
NOTE: Due to its robustness, the method presented here can be applied to any primate iPSC line. In this article, we describe cerebral organoid production from human (iLonza2.2)29, chimpanzee (Sandra A)30, rhesus macaque (iRh33.1)29,&#160;and common marmoset (cj_160419_5)31 iPSC lines. The culture conditions are summarized in Table 1. See th...

Genetic Modification of Primate Cerebral Organoids

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The procedures described here represent a unified protocol for the generation of cerebral organoids from different primate species with a targeted electroporation approach. This allows the ectopic expression of a GOI in a model system that emulates primate (including human) (patho)physiological neocortex development. This unified protocol for the generation of primate cerebral organoids uses the same materials (e.g., media) and protocol steps for all four primate species presented. Developmental differences between these...

Investigating the (patho)physiological development and evolution of the cerebral cortex is a formidable task that is hampered by the lack of suitable model systems. Previously, such studies were confined to two-dimensional cell culture models (such as primary neural progenitor or neuronal cell cultures) and evolutionarily distant animal models (such as rodents)1,2. While these models are useful for addressing certain questions, they are limited in modeling the complexity, cell type composition, cellular architecture, and gene expression patterns of the developing human neocortex in healthy and diseased states. These limitations lead, for example, to the poor translatability of mouse models of human diseases to the human situation, as described for certain cases of microcephaly (e.g., Zhang et al.3). Recently, transgenic non-human primates, which are an evolutionarily, functionally, and morphologically closer model of human neocortex development, have come into focus4,5,6,7,8 as they overcome many limitations of cell culture- and rodent-based models. However, the use of non-human primates in research is not only highly expensive and time-consuming but also raises ethical concerns. More recently, the development of brain organoid technology9,10 has emerged as a promising alternative that solves many of the limitations of previous models11,12,13,14,15,16.
Brain organoids are three-dimensional (3D), multicellular structures that emulate the main features of the cytoarchitecture and cell-type composition of one or multiple brain regions for a defined developmental time window11,12,13,14,17. These 3D structures are generated either from induced pluripotent stem cells (iPSCs) or, if available for the species of interest, from embryonic stem cells (ESCs). In general, two types of brain organoids can be distinguished based on the methodology used: unguided and regionalized (guided) brain organoids18. In generating the latter type of organoids, small molecules or factors are provided that guide the differentiation of the pluripotent stem cells to organoids of a particular brain region (e.g., forebrain organoids)18. By contrast, in unguided organoids, the differentiation is not guided by the addition of small molecules but rather relies exclusively on the spontaneous differentiation of the iPSCs/ESCs. The resulting brain organoids consist of cell types representing different brain regions (e.g., cerebral organoids)18. Brain organoids combine many key features of brain development with relatively cost- and time-efficient generation from any species of interest for which iPSCs or ESCs are available11,12,13,14. This makes brain organoids an excellent model for many kinds of neurobiological studies, ranging from evolutionary and developmental questions to disease modeling and drug testing15,16. However, addressing such questions using brain organoids strongly depends on the availability of different methods for genetic modification.
One key aspect of studying neocortex (patho)physiological development and its evolution is the functional analysis of genes and gene variants. This is usually achieved by (ectopic) expression and/or by knock-down (KD) or knock-out (KO) of those genes. Such genetic modifications can be classified into stable and transient genetic modification, as well as into the modifications being temporally and spatially restricted or not restricted. Stable genetic modification is defined by the introduction of a genetic alteration into the host genome that is passed on to all subsequent cell generations. Depending on the time point of genetic modification, it can affect all the cells of an organoid or can be restricted to certain cell populations. Most frequently, stable genetic modification is achieved in brain organoids at the iPSC/ESC level by applying lentiviruses, transposon-like systems, and the CRISPR/Cas9 technology (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20). This has the advantage that all cells of the brain organoid carry the genetic modification and that it is not temporally or spatially restricted. However, the generation and characterization of these stable iPSC/ESC lines are very time-consuming, often taking several months until the first modified brain organoids can be analyzed (reviewed by e.g., Fischer et al.17, Kyrousi et al.19, or Teriyapirom et al.20).
In contrast, transient genetic modification is defined by the delivery of genetic cargo (e.g., a gene expression plasmid) that does not integrate into the host genome. While this modification can, in principle, be passed on to subsequent cell generations, the delivered genetic cargo will be progressively diluted with each cell division. Therefore, this type of genetic modification is usually temporally and spatially restricted. Transient genetic modification can be carried out in brain organoids by adeno-associated viruses or by electroporation (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20), with the latter being described in detail in this article. In contrast to stable genetic modification, this approach is very fast and cost-efficient. Indeed, electroporation can be performed within minutes, and, depending on the target cell population(s), electroporated organoids are ready for analysis within days (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19). However, gross morphological changes of the brain organoid, such as differences in size, cannot be detected using this method, as this type of genetic modification is temporally and spatially restricted. This restriction can also be an advantage, for example, in the case of studying individual cell populations within the organoid or the effects on brain organoids at specific developmental time points (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19).
A classical approach to study gene function during brain development and evolution is in utero electroporation. In utero electroporation is a well-known and useful technique for the delivery of gene expression constructs into rodent21,22,23 and ferret24,25 brains. First, a solution containing the expression construct(s) of interest is microinjected through the uterine wall into a certain ventricle of the embryonic brain, depending on the region to be targeted. In the second step, electric pulses are applied to transfect the cells directly lining the targeted ventricle. This approach is not only limited to ectopic expression or the overexpression of genes, as it can also be applied in KD or KO studies by microinjecting short hairpin (shRNA) or CRISPR/Cas9 (in the form of expression plasmids or ribonucleoproteins [RNPs]), respectively26,27. However, the in utero electroporation of mouse, rat, and ferret embryos has the same limitations as described above for these animal models.
Ideally, one would like to perform in utero electroporation directly in primates. While this is, in principle, technically possible, in utero electroporation is not conducted in primates due to ethical concerns, high animal maintenance costs, and small litter sizes. For certain primates, such as great apes (including humans), this is not possible at all. However, these primates have the greatest potential for the study of human (patho)physiological neocortex development and its evolution. One solution to this dilemma is to apply the electroporation technique to primate brain organoids28.
This paper presents a protocol for the electroporation of a subtype of primate brain organoids, primate cerebral organoids. This approach allows the fast and cost-efficient genetic modification of cell populations within the ventricle-like structures of the organoids. Specifically, we describe a unified protocol for the generation of primate cerebral organoids from human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and common marmoset (Callithrix jacchus) iPSCs. Moreover, we describe the microinjection and electroporation technique in detail and provide &#34;go&#34; and &#34;no-go&#34; criteria for performing primate cerebral organoid electroporation. This approach is an effective tool for studying (patho)physiological neocortex development and its evolution in a model especially close to the human situation.

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			<td>20 &#181;L Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Merck</td>
			<td>8.05740.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3900</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dishes</td>
			<td>CytoOne</td>
			<td>CC7682-3359</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3003</td>
			<td></td>
		</tr>
		<tr>
			<td>AOC1</td>
			<td>Selleckchem</td>
			<td>S7217</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Inverted Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>replacable by comparable fluorescent microscopes</td>
		</tr>
		<tr>
			<td>AZD0530</td>
			<td>Selleckchem&#160;</td>
			<td>S1006</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement with Vitamin A (retinoic acid, RA) (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement without Vitamin A (50x)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX ECM 830 Square Wave Electroporation System</td>
			<td>BTX</td>
			<td>45-2052</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP77675</td>
			<td>Sigma-Aldrich</td>
			<td>SML0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Chimpanzee induced pluripotent stem cell line Sandra A</td>
			<td></td>
			<td></td>
			<td>doi: 10.7554/elife.18683&#160;</td>
		</tr>
		<tr>
			<td>Common marmoset induced pluripotent stem cell line cj_160419_5</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9112422</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>pH 7.0&#8722;7.3; warm to room temperature before use</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Selleckchem</td>
			<td>2449</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>glutamine substitute supplement</td>
		</tr>
		<tr>
			<td>Heparin (1 mg/mL stock)</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Human induced pluripotent stem cell line iLonza2.2</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9061349</td>
		</tr>
		<tr>
			<td>Human Neurotrophin-3 (NT-3)</td>
			<td>PeproTech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>19278</td>
			<td></td>
		</tr>
		<tr>
			<td>IWR1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MS5 stereomicroscope (MDG 17 transmitted-light base)</td>
			<td>Leica</td>
			<td>10473849</td>
			<td>replacable by comparable stereomicroscopes</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354277/354234</td>
			<td>basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Sigma-Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>818715</td>
			<td>handle with causion due to cancerogenecity</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10,000 U/mL)</td>
			<td>PanBiotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish electrode chamber</td>
			<td>self-produced (see Supplemental File 1)</td>
			<td></td>
			<td>also commertially available</td>
		</tr>
		<tr>
			<td>Pre-Pulled Glass Pipettes</td>
			<td>WPI</td>
			<td>TIP10LT</td>
			<td>borosilicate glass pipettes with long taper, 10 &#181;m tip diameter</td>
		</tr>
		<tr>
			<td>Pro-Survival Compound</td>
			<td>MerckMillipore</td>
			<td>529659</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF)</td>
			<td>PeproTech</td>
			<td>AF-450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhesus macaque induced pluripotent stem cell line iRh33.1</td>
			<td></td>
			<td></td>
			<td>doi: 10.3390/cells9061349</td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyi Biotech</td>
			<td>130-104-368</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>proteolytic and collagenolytic enzyme mixture</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12604-013</td>
			<td>recombinant trypsin substitute; warm to room temperature before use</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 96-well plates</td>
			<td>Costar</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Stemcell Technologies</td>
			<td>72305</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted microinjection and electroporation of primate cerebral organoids for genetic modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

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.

The protocol described here allows the efficient generation of cerebral organoids from human, chimpanzee, rhesus macaque, and common marmoset iPSC lines with minimal timing alterations required between species (Figure 1A). These organoids can be electroporated in the range of 20 dps to 50 dps, depending on the accessibility of the ventricle-like structures and the abundance of the cell population(s) of interest. However, prior to electroporation, it is important to determine whether the cere...

Watch this Scientific Journal Video about Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification at JoVE.com

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

targeted-microinjection-electroporation-primate-cerebral-organoids

Research

JoVE Journal

Developmental Biology

Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells (iPSCs)

Generation of Cerebral Organoids from Primate-Induced Pluripotent Stem Cells (IPSCs)  

To begin, wash the cultured iPSCs with Dulbecco's 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 200xg 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 four by four 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-milliliter cell culture dish.Place the dimpled 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 culture 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 brightfield image of a 32-day post-seeding human cerebral organoid with ventricle-like structures on the periphery and a compact, healthy morphology is shown.

Electroporation of Primate Cerebral Organoids

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 8 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/F-12 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 microinjected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F-12. 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/F-12. 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 1000 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 5 milliliters of DPBS, shake gently, and aspirate the DPBS. Store the organoids in DPBS at 4 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 apical 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. 3-D reconstruction of the electroporated cerebral organoids to obtain an impression of the 3-D distribution of the GFP-positive cells is shown.