Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors
December 11th, 2021
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.
To date, the cerebral organoids differentiated from pluripotent stem cells is an vitro 3D model closest to the native human brain tissue. Additionally, cerebral organoids are equally suitable for modeling various central nervous system pathologies, testing pharmacologically-active substances, and also for use in regenerative medicine. Meanwhile, this technology is still at the initial stage of development.
The protocol can be divided into two groups depending on the method of obtaining aggregates and the further cultivation with differentiation. The first includes protocol and schemes of differentiation, different in the cultivation time and differentiation inducers with subsequent maturation of organoid in the shaker in special low-adhesion devices like market tubes or plates. Another group includes using a special bioreactors.
Analysis of various protocols has shown that organoids should be cultivated under conditions with circulating nutrient medium. However, the absence of simple and inexpensive systems for obtaining organoid with a standard size and morphology require the development of such devices for scaling the process. In this work, we describe our method for obtaining and testing simple and inexpensive mini bioreactors that make it possible to obtain large quantities of high-quality organoids.
Cut the sterile 15 milliliters centrifuge tubes into the rings, 7 or 8 millimeters in height. Autoclave the rings. Break low-aggregations on treated or microbiological Petri dishes into crumbs.
Dissolve about 1 grams of plastic crumbs in 10 milliliters of chloroform overnight to prepare a liquid plastic. Make a plastic knob in the center of a sterile ultra low-adhesion 6 centimeters Petri dish. There are two equally suitable ways.
The first, put the autoclave in a plastic ring on a center and apply half milliliters of liquid plastic to the inside of the ring. The second, without any plastic rings drop half milliliters of liquid plastic on the center of the Petri dish. Leave the dishes open for 2 or 3 hours in a laminar flow hood until complete dryness.
Cultivate induced pluripotent stem cells in the medium for pluripotent stem cells, up to 75 or 90%confluence in the 35 millimeters Petri dishes, pre-coated with a matrix dissolved in cold Dulbecco Modified Eagle Medium, and Fisher-12 medium. Prepare medium A plus serum replacement. Cultivate induced pluripotent stem cells for 1 day in the medium with serum replacement.
Change medium for pluripotent stem cells to a serum replacement medium at the day zero of differentiation. Prepare medium A.Change the medium to medium A at day 2 of differentiation. Cultivate cells in medium A for 2 weeks, refreshing medium in Petri dishes every 2 day.
At day 14, start the formation of spheroid using a special 24-well culture plate which contains approximately 1, 200 microwells in each well. Prepare a 24-well culture plate with microwells. Add to each well 1 milliliter of medium A, centrifuge briefly at 1, 300g for 5 minutes in a swinging-bucket rotor fitted with plate holder.
Control under the microscope that there are no bubbles in microwells. Prepare the medium B.Remove the medium from the Petri dish. For the cell detachment, treat the cells with 1.5 milliliters EDTA solution prepared on PBS.
Control the cell detachment under the microscope. Harvest the cell into 15 milliliter tube. Add 5 milliliter mixed Dulbecco and Fisher medium in the tube towards the cells.
Centrifuge at 200g for 5 minutes. Remove the supernatant and re-suspend cells in 2 milliliter of medium B.Transfer the cells suspension containing 1 billion cells into each well of a 24-well plate with microwells. Gently pipette cells up to and down several times and centrifuge briefly 100g for 1 minute to capture cell in microwells.
Control under the microscope that the cell are evenly distributed in microwells. Incubate the plate overnight for cell aggregation in spheroids. The next morning, day 15, check under the microscopes the quality of spheroids, which are transparent and smooth if healthy.
Carefully collect the spheroids from each well into a 15 milliliter tube, leave the spheroids to precipitate by gravity for 2 and 3 minutes, and then remove the supernatant. Add to the spheroids 2 milliliters of the matrix, thawed and ex tempore on ice. Mix gently by pipetting and incubate at room temperature for 30 minutes.
To wash excess of the matrix, add to the tube 8 milliliters of medium B.Pipette gently, then centrifuge the tube for 1 minutes at 100g. Remove the supernatant. Add to the tube 20 milliliters of medium B, pipette gently, then split the spheroid suspension between mini bioreactors.
Then place the mini bioreactors into a 15 centimeter Petri dish, which prevents the evaporation of water and contamination. Put the Petri dish with mini bioreactors on a orbital shaker. Cultivate the organoids at rotation rate 70, 75rpm.
On day 16, prepare medium C.Transfer organoid into 50 milliliter tube. For 5 minutes, let them fall to the bottom. Aspirate the supernatant and add 5 milliliters of the medium C.Rejoin the organoids in the mini bioreactors.
Cultivate spheroids in medium C for 2 weeks, refreshing the medium every 2 days. During these 2 weeks, select about 100 spheroids per mini bioreactors for the following cultivation. On day 30, prepare medium D.Change cultivation medium to medium D, which is a maturation medium.
Refresh cultivation medium every 2 or 3 days for 3 weeks. Then use the medium D dissolved in F and GDNF. After 1 month of consecutive cultivation in medium A and B, organoids standardized in size and morphology are obtained.
As it grows, they need to change the medium more often, up to 4 times a week also increases. After 2 months of cultivation, they reach 4 and 5 millimeter in diameter. And after another month, 5 and 6 millimeter and the growth stops.
The figure shows the appearance of organoid enzyme, clear sections painted with hematoxylin and eosin. Immunohistochemical analysis for 1 month shows the presence of SOX2 positive clusters of progenitor cells, including inside central part. In the peripheral of the organoids, glial fibrillary acidic protein, microtubule-associated protein 2, and thiazine hydrolase positive.
Cells are concentrated, which corresponds to mature form of neurons. In some cases, clusters of whites on necrotic area are formed in the central part. This indicates the limitation of this protocol for obtaining larger organoids.
Most likely, the limiting factor of growth is the rate of diffusion of nutrient and oxygen. The various media and cell type of spheroids and testing our system for some kinds of organoids. Lots of brain and retina, combined organoids from liver carcinoma cells and mesenchymal stem cells, combined organoids from capital blast mesenchymal stem calls and the hematopoietic stem cells differentiation from induced pluripotent stem cells and intestine organoids.
Was it variable in the initial composition of cells? Differentiation factors, culture, and medium, extracellular matrix, and possibly also the gas mixture composition, the rate of the platform rotation, and other parameters. It will be possible to obtain organoid with the same shape, size, or morphology model in various organs and tissues.
The use of mini bioreactors proposed into this work.
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Overview
This article presents a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). These organoids serve as a 3D model that closely resembles native human brain tissue and are suitable for modeling central nervous system pathologies.
Key Study Components
Area of Science
- Neuroscience
- Stem Cell Biology
- Regenerative Medicine
Background
- Cerebral organoids are derived from pluripotent stem cells.
- They can be used for pharmacological testing and studying brain diseases.
- This technology is still in the early stages of development.
- Different methods exist for obtaining and cultivating organoids.
Purpose of Study
- To develop a reliable protocol for generating high-quality brain organoids.
- To explore the potential applications of organoids in research and medicine.
- To improve the efficiency of organoid production using bioreactors.
Methods Used
- Differentiation of iPSCs into cerebral organoids.
- Use of mini bioreactors for large-scale organoid production.
- Cultivation and maturation of organoids in low-adhesion devices.
- Comparison of different cultivation times and differentiation inducers.
Main Results
- Successful generation of brain organoids that mimic human brain tissue.
- Demonstrated the feasibility of using bioreactors for organoid cultivation.
- Identified optimal conditions for differentiation and maturation.
- Provided insights into the potential for modeling CNS diseases.
Conclusions
- The protocol enables the production of high-quality brain organoids.
- Organoids can be valuable tools for studying brain function and disease.
- Further development of this technology could enhance regenerative medicine applications.
Frequently Asked Questions
Executive Industry Relevance
iPSC-derived brain organoids produced in home-made mini bioreactors offer a scalable and cost-effective platform for modeling central nervous system pathologies and early-stage drug screening. This approach addresses key challenges in reproducibility, throughput, and standardization that limit the translational value of organoid models in discovery pipelines. By enabling the generation of high-quality, size-consistent organoids, this protocol supports more predictive and reliable preclinical research decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of disease-relevant neural pathways in a human-derived 3D context.
- Supports functional target validation by modeling CNS pathologies with greater physiological relevance.
- Facilitates mechanistic de-risking by providing a platform for hypothesis-driven studies on neural differentiation and maturation.
Screening & Assay Development
- Provides standardized organoid systems suitable for quantitative compound screening and pharmacological testing.
- Improves assay reproducibility and scalability through consistent organoid size and morphology.
- Enables preparation of validated biological models for downstream screening workflows.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by supporting the study of human neural cell types and maturation markers.
- Offers continuity from early discovery to preclinical validation by enabling long-term culture and maturation of organoids.
- Supports risk-adjusted advancement decisions by providing more predictive in vitro models of CNS disease.
Pipeline & Workflow Integration
This protocol positions brain organoid generation as a bridge from early discovery through lead identification and preclinical research, supporting iterative hypothesis testing and compound evaluation.
- Discovery Biology: Facilitates hypothesis-driven studies on neural differentiation and disease modeling.
- Screening: Delivers reproducible, quantitative outputs for compound efficacy and toxicity assessment.
- Analytics: Enables measurement of neural marker expression and morphological consistency across batches.
- Translational Research: Supports alignment with human disease biology and biomarker development.
- Enterprise Reuse: Provides a scalable, cost-effective platform adaptable to various organoid types and research needs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in CNS research.
- Operational Value: Enhances standardization, reproducibility, and throughput of organoid production.
- Strategic Value: Improves go/no-go decision quality and capital efficiency in early-stage CNS portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of CNS-targeted assets.
Implementation Considerations
- Requires expertise in iPSC culture, neural differentiation, and 3D cell biology.
- Needs access to basic cell culture infrastructure and orbital shakers for bioreactor operation.
- Demands cross-team standardization of differentiation protocols and quality control metrics.
- Adaptable to various organoid types with protocol modifications for cell composition and media.
- Limitations include necrotic core formation in larger organoids due to diffusion constraints.
