This work was supported by grant 075-15-2019-1669 from the Ministry of Science and Higher Education of the Russian Federation (RT-PCR analysis) and by grant No. 19-15-00425 from the Russian Science Foundation (for all other work). The authors also thank Pavel Belikov for his help with the video editing. Figures in the manuscript were created with BioRender.com.

NOTE : Use sterile technique throughout the protocol, excluding steps 1.2 and 1.3. Warm all culture media and solutions to 37 &#176;C before applying to cells or organoids. Cultivate cells in a CO2 incubator at 37 &#176;C in 5% CO2 upon 80% humidity. The protocol scheme is shown in Figure 1.
1. Transforming Petri dishes into mini bioreactors
<ol>
	<li>Cut sterile 15 mL centrifuge tubes in rings of 7-8 mm in height; autoclave the rings.</li>
	<li>Break low-adhesion, untreated or microbiological Petri dishes into crumbs. Dissolve about 1 g of plastic crumbs in 10 mL of chloroform overnight to prepare liquid plastic. 
	&#8203;CAUTION: Work in a fume hood.</li>
	<li>Check that the resulting liquid plastic is viscous enough for pipetting; its drop retains a spherical form and does not spread on the surface. If it is very liquid, add more plastic crumbs. If it is thick, then add chloroform.</li>
	<li>Make a plastic knob in the center of a sterile ultra-low adhesion 6-cm Petri dish. There are two equally suitable ways, as detailed below.
	<ol>
		<li>Put the autoclaved plastic ring on the center and drop the liquid plastic to the inside of the ring.</li>
		<li>Without any plastic ring, drop the liquid plastic on the center of the Petri dish.</li>
	</ol>
	</li>
	<li>Leave the dishes open for 2-3 h in a laminar flow hood until dried complete. Treat the dried dishes with ultraviolet radiation for 15-20 min.</li>
</ol>
2. Induction of neuronal differentiation of iPSCs
<ol>
	<li>Cultivate iPSCs in the medium for pluripotent stem cells up to 75-90% confluence in 35 mm Petri dishes precoated with a matrix consisting of extracellular proteins.</li>
	<li>Prepare medium A-SR. See Table 1 for details.</li>
	<li>Aspirate the cultivation medium and add 2 mL of A-SR medium at Day 0 of differentiation.</li>
	<li>Prepare medium A (see Table 2).</li>
	<li>Cultivate cells in medium A for two weeks from Days 2-14, refreshing medium in Petri dishes every other day.</li>
</ol>
3. The formation of spheroids from neuroepithelial precursor cells at Day 14
<ol>
	<li>At Day 14, make spheroids from neuroepithelial precursor cells using a special 24-well culture plate containing approximately 1,200 microwells in each well (Figure 2C). Follow the procedure given below. 
	NOTE: At this differentiation stage, a 35 mm Petri dish usually contains 3 - 3.5 x 106 neuroepithelial precursor cells. Thus, one 35 mm Petri dish with neuroepithelial precursor cells is sufficient for 3 - 4 wells of 24-well culture plate with microwells.</li>
	<li>Prepare a 24-well culture plate with microwells: To each well, add 1 mL of medium A. Centrifuge briefly at 1,300 x g for 5 min in swinging bucket rotor fitted with plate holder. Control under the microscope that there are no bubbles in microwells.</li>
	<li>Prepare the medium B (Table 3).</li>
	<li>Remove the medium from the Petri dish with neuroepithelial precursor cells; wash the cells with 2 mL of DMEM/F12. For the cell detachment, treat the cells with 1.5 mL of 0.48 mM EDTA solution prepared in PBS. Control the cell detachment under the microscope.</li>
	<li>Harvest the cells into a 15 mL tube. Add 5 mL of DMEM/F12 in the tube to wash the cells. Centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend cells in 2 mL of medium B.</li>
	<li>Check the cell concentration and viability by Trypan Blue staining and a hemocytometer. Calculate the volume of suspension needed to contain 1 x 106 viable cells in total.</li>
	<li>Transfer the cell suspension containing 1 x 106 cells into each well of a 24-well plate with microwells. Add medium B up to 2 mL into each well, gently pipette cells up and down several times, and centrifuge briefly 100 x g for 1 min to capture cells in the microwells. 
	NOTE: The number of cells per well should not exceed 1 x 106. Otherwise, spheroids from neighboring microwells fuse.</li>
	<li>Check under the microscope that cells are evenly distributed in microwells. Repeat pipetting and centrifugation if cells are distributed unevenly.</li>
	<li>Incubate the plate overnight to let cells aggregate in spheroids.</li>
</ol>
4. Obtaining and cultivation of organoids
<ol>
	<li>The next morning (Day 15), check the quality of spheroids under the microscope. Ensure that they are transparent and smooth, if healthy (Figure 2A,B). Carefully collect the spheroids from each well into a 15 mL tube, leave the spheroids to precipitate by gravity for 2-3 min, and then remove the supernatant.</li>
	<li>Add to the spheroids 2 mL of the matrix thawed during the same time on ice. Mix gently by pipetting and incubate at room temperature for 30 min.</li>
	<li>To wash excess of the matrix, add to the tube 8 mL of medium B. Pipette gently, then centrifuge the tube for 1 min at 100 x g. 
	NOTE: Do not exceed the time and the speed of centrifugation to avoid the irreversible aggregation of spheroids.</li>
	<li>Remove the supernatant. Add to the tube 2&#160;mL of medium B, pipette gently. Split the spheroid suspension between two mini bioreactors, each containing 4 mL of medium B. Place the mini bioreactors into a 15 cm Petri dish to prevent the evaporation of water and to avoid contamination.</li>
	<li>Put the Petri dish with mini bioreactors on an orbital shaker. Cultivate the organoids at a rotation rate of 70-75 rpm.</li>
	<li>On Day 16, prepare medium C (Table 4).</li>
	<li>Transfer the organoids into 15 mL tube. For 5 min, let them fall to the bottom, aspirate the supernatant, add 5 mL of medium C. Return the organoids into the mini bioreactors. 
	NOTE: Be careful not to lose the spheroids, which are transparent and barely visible.</li>
	<li>Cultivate spheroids in medium C for two weeks, refreshing the medium every two days. At the end of these two weeks, leave about 100 spheroids per mini bioreactor for the following cultivation. Freeze excessive spheroids in a freezing medium in liquid nitrogen.</li>
	<li>On Day 30, prepare medium D (Table 5).</li>
	<li>Change the cultivation medium to medium D, which is a maturation medium. Refresh cultivation medium every 2-3 days for three weeks, then use medium D without BDNF and GDNF.</li>
</ol>

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The authors have nothing to disclose.

The described protocol has two crucial steps allowing the generation of high-quality organoids of uniform size. First, the organoids grow from spheroids which are near identical in cell number and cell maturity. Second, the home-made bioreactors provide each organoid a uniform environment, where organoids do not crowd or stick together.
The cell quality and state of cell maturation are essential to perform the protocol. It is critical to start neuronal differentiation at 75-90% confluence of i...

Human iPSC-derived models are widely used in the studies of neurodevelopmental and neurodegenerative disorders1. Over the past decade, 3D brain tissue models, so-called brain organoids, essentially complemented traditional 2D neuronal cultures2. The organoids recapitulate to some extent the 3D architecture of the embryonic brain and allow more precise modeling. Many protocols are published for the generation of organoids representing different brain regions: cerebral cortex3,4,5, cerebellum6, midbrain, forebrain, hypothalamus7,8,9, and hippocampus10. There have been multiple examples of using organoids to study human nervous system diseases11. Also, the organoids were implemented in drug discoveries12 and used in studies of infectious diseases, including SARS-Cov-213,14.
The brain organoids can reach up to several millimeters in diameter. So, the inner zone of the organoid may suffer from hypoxia or malnutrition and eventually become necrotic. Therefore, many protocols include special bioreactors8, shakers, or microfluidic systems15. These devices may require large volumes of expensive cell culture media. Also, the cost of such equipment is usually high. Some bioreactors consist of many mechanical parts that make them difficult to sterilize for reuse.
Most protocols suffer from the &#34;batch effect&#34;16, which generates significant variability among organoids obtained from the identical iPSCs. This variability hinders drug testing or preclinical studies requiring uniformity. The high yield of organoids enough to select organoids of uniform size may partially solve this problem.
The time factor is also a significant problem. Matsui et al. (2018) showed that brain organoids require at least six months to reach maturity17. Trujillo et al. (2019) also demonstrated that electrophysiological activity occurred in organoids only after six months of cultivation18. Due to the long organoid maturation time, the researchers often launch new differentiation before completing the previous one. Multiple parallel processes of differentiation require additional expenses, equipment, and laboratory space.
We have recently developed a mini bioreactor that mainly solves the problems mentioned above19. This home-made bioreactor consists of an ultra-low adhesion or untreated Petri dish with a plastic knob in the center. This plastic knob prevents crowding of organoids and their conglutination in the center of the Petri dish, which is caused by the rotation of the shaker. This paper describes how this inexpensive and simple home-made mini bioreactor allows generating high-quality brain organoids in large quantities.

<table><tbody><tr><td>Advanced DMEM/F-12</td><td>Gibco</td><td>12634010</td><td>DMEM/F-12</td></tr><tr><td>AggreWell400</td><td>STEMCELL Technologies Inc</td><td>34425</td><td>24-well culture plate with microwells</td></tr><tr><td>B-27 Supplement</td><td>Gibco</td><td>17504044</td><td>Neuronal supplement B</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>200 mM L-alanyl-L-glutamine</td></tr><tr><td>Human BDNF</td><td>Miltenyi Biotec</td><td>130-096-285</td><td></td></tr><tr><td>Human FGF-2</td><td>Miltenyi Biotec</td><td>130-093-839</td><td></td></tr><tr><td>Human GDNF</td><td>Miltenyi Biotec</td><td>130-096-290</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828028</td><td>Serum replacement</td></tr><tr><td>mTESR1</td><td>STEMCELL Technologies Inc</td><td>85850</td><td>Pliripotent stem cell medium</td></tr><tr><td>N2 Supplement</td><td>Gibco</td><td>17502001</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td>Basal medium for neuronal cell maintenance</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Gibco</td><td>15140130</td><td></td></tr><tr><td>Plasmocin</td><td>InvivoGen</td><td>ant-mpt-1</td><td>Antimicrobials</td></tr><tr><td>Purmorphamine</td><td>EMD Millipore</td><td>540220</td><td></td></tr><tr><td>StemMACS Y27632</td><td>Miltenyi Biotec</td><td>130-106-538</td><td>Y27632</td></tr><tr><td>StemMACS Dorsomorphin</td><td>Miltenyi Biotec</td><td>130-104-466</td><td>Dorsomorphin</td></tr><tr><td>StemMACS LDN-193189</td><td>Miltenyi Biotec</td><td>130-106-540</td><td>LDN-193189</td></tr><tr><td>StemMACS SB431542</td><td>Miltenyi Biotec</td><td>130-106-543</td><td>SB431542</td></tr><tr><td>Trypan Blue Solution</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Versen solution</td><td>Gibco</td><td>15040066</td><td>0.48 mM EDTA in PBS</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr></tbody></table>

brain organoid generation from induced pluripotent stem cells in home-made mini bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

The protocol scheme is shown in Figure 1. The protocol included five media in which iPSCs differentiated into brain organoids during at least one month. The differentiation was started then iPSCs reached the 75-90% confluence (Figure 2A,B). The first signs of differentiation towards neurons were observed on days 10-11 of iPSC cultivation in medium A when cells began to cluster into &#34;rosettes&#34; (Figure 2C). At...

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Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

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5.4K Views.  Federal Medical Biological Agency. Tot op heden zijn de cerebrale organoïden die worden onderscheiden van pluripotente stamcellen een vitro 3D-model dat het dichtst bij het inheemse menselijke hersenweefsel staat. Bovendien zijn cerebrale organoïden even geschikt voor het modelleren van verschillende pathologieën van het centrale zenuwstelsel, het testen van farmacologisch actieve stoffen en ook voor gebruik in de regeneratieve geneeskunde. Ondertussen bevindt deze technologie zich nog in de beginfase van ontwikkeling.

Video: Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

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

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Biochemistry

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

Bioengineering

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

Biology

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Source: Koch, L. S., et al. <a href="https://app.jove.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

Generating Mature Cerebellar Organoids From Induced Pluripotent Stem Cells Using Single-Use Bioreactors

Source: Silva, T.P., et al. <a href="https://app.jove.com/v/61143">Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for generating mature cerebellar organoids from induced pluripotent stem cells (iPSCs) using a single-use 3D bioreactor. The bioreactor facilitates iPSC aggregate formation, which then undergoes treatment with a series of culture media enriched in growth factors and chemokines that induce the differentiation of the iPSC aggregates into mature cerebellar organoids. Subsequently, the organoids are harvested and cryopreserved for further analysis.

1. Seeding of human iPSCs in the bioreactor

	Incubate iPSCs grown as monolayers in mTeSR1 supplemented with 10 &#181;M of ROCK inhibitor Y-27632 (ROCKi). ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

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