Summary
Cerebral organoids provide unprecedented opportunities for studying organ development and human disease pathology. Although great success has been achieved with cerebral organoid culture systems, there are still operational difficulties in applying this technology. The present protocol describes a cerebral organoid procedure that facilitates medium change and organoid transfer.
Abstract
At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.
Introduction
Compared to two-dimensional (2D) culture systems, three-dimensional (3D) culture systems have several advantages, including genuine replication and efficient reproduction of complex structures of certain organs1. Therefore, cerebral organoids are one of the important auxiliary methods for the research fields of human brain development and disease2, drug screening, and cell therapy.
Culturing cerebral organoids by the rotating suspension method is conducive to their development and maturation3. Although cerebral organoid culture systems have achieved great success, they still face critical challenges that limit their application. For example, manual cultivation involves complicated manipulation steps and introduces obstacles to achieving large-scale applications. Additionally, at each developmental stage in the culture of cerebral organoids, changes in different media and cytokines are needed4. However, in the early stage, the organoids or EBs have tiny sizes (approximately 200 µm to 300 µm) and are almost visually inaccessible without appropriate apparatus. Inevitably, a certain amount of precious organoid samples are flushed away when the medium is changed. Many techniques have been explored to overcome this in other kinds of organoid cultures, and some examples include immersing entire organoid chips in a culture medium for 3 days without intervention5; adding a fresh medium through the coverslip after the old medium is absorbed using absorbent paper5; or applying complex microfluidic pipelines for fluid exchange6,7,8. Another obstacle encountered in the early stage of organoid cultivation is the difficulty of achieving direct observations with the naked eye, which can cause poor operations that lead to organoid damage and loss during the organoid transfer steps. Therefore, it is necessary to establish a more feasible protocol that facilitates medium change and organoid transfer to generate organoids.
A corresponding optimized operation based on engineering principles is proposed to overcome these problems, which significantly and conveniently facilitates many organoid procedures. In nature, when the sun shines into a house through a window gap, the naked eye can see the dust dancing in the light beam. Due to the diffuse reflection of sunlight on dust, some light is refracted into the eyeball to produce a visual image. Inspired by the principle of this phenomenon9,10, this study made a soft light lamp and illuminated the EBs laterally. It was found that EBs could be visually clear without affecting the viewing scope. A secondary flow pointing to the center is generated in the liquid by rotating the culture plate due to eddy currents11. Originally dispersed EBs accumulate in the center of the plate. Based on this, and the phenomenon that the sedimentation velocity of organoids is faster than that of cells, an easy operation method of medium change and organoid transfer without centrifugation is proposed. The organoids in the culture medium can be effectively separated from free cells and dead cell fragments through this transfer operation.
Here, a protocol that is easy to operate is proposed to generate cerebral organoids from human pluripotent stem cells. The operation technology was optimized by applying the engineering principle, making operations in 3D culture as simple and feasible as those in 2D culture. The improved liquid exchange method and organoid transfer operation are also helpful for other types of organoid culture and the design of automatic culture machines.
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Protocol
The protocol was conducted following the Declaration of Helsinki. Approval was granted by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Medical and ethical review [2021] No. 022). Before the experiment, each medium was prepared according to Juergen A. Knoblich's formula12 (Supplementary Tables 1-4), or a commercially available Cerebral Organoid Kit was used (see Table of Materials). The iPSCs used in this study were previously established by our laboratory and have obtained an informed exemption. The SCA3-iPSCs were generated from a 31-year-old female spinocerebellar ataxia type 3 (SCA3) patient genotyped as harboring 26/78 CAG repeats in the ATXN3 gene13 (Supplementary Figure 1A). The normal human iPSCs mentioned in a previous article were selected as NC-iPSCs14, and the ATXN3 gene was identified as having 14/14 CAG repeats (Supplementary Figure 1B).
1. Preparation of induced pluripotent stem cells (iPSCs)
- Collect 3 mL of peripheral blood by venipuncture with the informed consent of volunteers and patients.
- Isolate peripheral blood mononuclear cells according to the Ficoll-Paque method15.
- Establish iPSCs according to the protocol of the Sendai Reprogramming Kit (see Table of Materials).
- Culture the iPSCs with mTeSR1 medium in 35 mm Petri dishes covered with Matrigel (a basement membrane matrix, see Table of Materials). Change the medium every day. The iPSC growth density must not exceed 75%.
- Digest the cells with PSC dissociation solution (see Table of Materials) and subculture them at a ratio of 1:5 every 3-5 days.
CAUTION: Sendai virus is used here. It must be operated in the biosafety cabinet, and self-protection measures must be taken. It should be clear whether it can be used legally in the local country. Local biosafety laws and regulations must be strictly followed when used.
2. EB preparation (days 0-1)
- Remove the mTeSR1 medium from the iPSCs with a pipette and wash it 2x with 1 mL of 1x PBS.
- Remove the PBS with a pipette, and add 300 µL of cell detachment solution (see Table of Materials) to digest the iPSCs into single cells for 3-4 min at 37 °C.
- Resuspend the cells in 2 mL of EB-formation medium (Supplementary Table 1) and then centrifuge the sample for 5 min at 300 x g (room temperature).
- Pretreat the specialized 24-well plate with anti-adherence rinsing solution (500 µL/well) for 5 min (see Table of Materials).
NOTE: Anti-adherence rinsing solution is essential for optimal EB and spheroid formation. - Resuspend the cells in EB-formation medium containing Y-27632 (final concentration, 10 µM, see Table of Materials), with a cell density of 1.5 x 106 cells/mL.
- Remove the anti-adherence rinsing solution, and rinse each well with 2 mL of EB-formation medium.
- Add the cells to the specialized 24-well plates at a density of 3 x 106 cells/well (Figure 1A, left).
NOTE: Normally, 300 EBs can be prepared in each well. This method can prepare large quantities of EBs of the same size. - Centrifuge the plate at 100 x g for 2 min at room temperature. Uniformly distribute the cells in each chamber at the bottom of the plate (Figure 1A, right).
NOTE: If there is no suitable centrifuge, the plate can also be directly placed into the incubator for static culture, but the formation time of the EB ball will be several hours later than that under normal centrifugation. - Incubate the samples at 37 °C and 5% CO2 for 24 h. If centrifugation was not used in the previous step, incubate for 36 h. Keep the sample steady, and do not take it out of the incubator during this period.
3. Preparation of the soft light lamp (day 1)
- Use a transparent acrylic board with a thickness of 0.3-0.5 cm in the size of A5 paper. Paste white pads on the front and back of the acrylic plate.
- Install a row of LED white lights on the edge of the plate so that the lights can enter from the side of the acrylic plate and then shoot out in parallel (Supplementary Figure 2A-F, Figure 1B).
NOTE: As the diameters of EBs in the early stage are approximately 200 µm to 300 µm, it is difficult to observe them clearly with the naked eye under the fluorescent lamp of clean benches. In contrast, due to the enhancement of diffuse reflection, we can detect the EBs clearly by using laterally illuminated soft light (Figure 1B, C). A 6-well plate is recommended for EB cultures in subsequent experiments. However, to better show the visual effect of soft light, dishes are sometimes used to take pictures and videos in this study instead of plates, so please do not misunderstand. The laterally illuminated soft light lamp can also be used for the 6-well plate.
- Install a row of LED white lights on the edge of the plate so that the lights can enter from the side of the acrylic plate and then shoot out in parallel (Supplementary Figure 2A-F, Figure 1B).
4. EB transfer and medium replacement (days 2-5)
- Prepare a new 6-well low adhesion plate, and add 2 mL of EB-formation medium to each well.
- Remove the EBs together with the medium with a 1000 µL wide-bore pipette tip (see Table of Materials) and transfer them to the 6-well low adhesion plate (~100 EBs/well).
NOTE: The operation process adopts a soft light lamp (mentioned in step 3.) to make the EBs easier to observe. Turn off other indoor light sources to get the visual effect of the soft light better. - Replace with the same volume of fresh EB-formation medium every day. Use the secondary flow to gather the EBs to the center and change the medium.
- Aspirate the old medium by pipetting to the edge of the well slowly. Do not suck too hard; otherwise, the EBs will be removed together. Then, add fresh medium to resuspend the EBs.
NOTE: The principle secondary flow (Figure 1D). Induce a swirl flow by rotating the dish along a circular orbit. Due to the swirl flow, a secondary flow is induced directed toward the center. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation, after which medium change or embryoid transfer can be executed readily.
- Aspirate the old medium by pipetting to the edge of the well slowly. Do not suck too hard; otherwise, the EBs will be removed together. Then, add fresh medium to resuspend the EBs.
5. Checking pluripotency by labeling with pluripotency marker OCT4 (day 4)
NOTE: When the diameter of the EBs is greater than 300 µm, take several EBs for OCT4 marker immunofluorescence staining to detect their pluripotency.
- Fix the EBs in 4% paraformaldehyde at 4 °C for 30 min, followed by washing in 1x PBS 3x for 10 min each time.
- Transfer the EBs to room temperature PBS containing 0.3% Triton X-100 and 3% BSA for 2 h, followed by washing in PBS 3x for 10 min each time.
NOTE: After being treated withTriton X-100, the EBs float on the liquid surface and can be easily sucked away with the liquid during cleaning. Operation under a stereomicroscope is recommended. - Dilute the OCT4 primary antibody (see Table of Materials) with 1 mL of 1x PBS containing 1% BSA at a ratio of 1:200 and add to the EBs for incubation at 4 °C overnight, then wash in PBS 3x for 10 min each time.
- Dilute the respective secondary antibody (see Table of Materials) with 1x PBS at 1:500 and add 1 mL to the EBs.
- After incubating at room temperature for 2 h, wash the cells in 1x PBS 3x for 10 min each time.
- Remove the PBS, add 2 mL of 1x PBS solution containing 1 µg/mL DAPI, incubate at room temperature for 10 min, and then wash in PBS 3x for 10 min each time.
- Move the EB containing a small amount of liquid onto the slide, seal the slide with a cover glass coated with commercially available petroleum jelly (see Table of Materials) on the edge, and then observe and collect the image under the fluorescence confocal microscope.
NOTE: OCT4 expression represents EB pluripotency12. When the expression of OCT4 is less than 90%, the EBs should be discarded, and EBs should be prepared again.
6. Neural induction (days 5-7)
- Prepare a new 6-well plate with low adhesion, and add 3 mL of Neural induction medium (Supplementary Table 2) to each well.
- Turn on the lateral soft light (mentioned in step 3.) and turn off other indoor light sources.
- Transfer the EBs to the 6-well plate with added Neural induction medium (~100 EBs/well). Add as little as possible of the original medium to the new well.
NOTE: Introduce simple skills oforganoid transfer. Naturally, under gravity and with a relatively higher density than the medium, the resuspended EBs will gradually sink by applying the operation shown in Figure 1E. Hence, the EBs can be conveniently transferred. Since, compared to EBs, free cells and dead cell fragments sink more slowly, most of the free cells and dead cell fragments can thus be removed through this sedimentation method (Figure 1F). - Incubate the samples at 37 °C and 5% CO2 for 24 h.
NOTE: Under the microscope, the diameter of the EBs was approximately 500 µm, and the edge was translucent, indicating that a neuroepithelial layer formed. - Proceed to the next step.
7. Embedding in the basement membrane matrix (days 7-10)
- Place the membrane matrix in a 4 °C refrigerator for 60 min in advance to dissolve. Calculate the amount of the required matrix in advance. Approximately 100 EBs were embedded in 1.5 mL of the membrane matrix.
- Turn on the lateral soft light and turn off the light source on the top of the console.
- Keep the membrane matrix on ice to prevent solidification.
- Transfer a small amount of EBs to a 60 mm dish containing fresh Expansion medium (Supplementary Table 3) each time. Reduce the number of EBs to make it easier to remove a single EB.
- Then, use a new 6-well low adhesion plate. Suck a single EB (containing approximately 10 µL medium) with a 200 µL wide-bore pipette tip (see Table of Materials) each time, and then add it to the bottom of the 6-well plate to make droplets. Use five droplets per well.
NOTE: The key is to adjust the range of the pipette gun to 50 µL and suck an EB, and then refer to the operation of Figure 1E. When the EB settles to the bore of the pipette tip, the tip quickly touches the well bottom of the plate and pushes out approximately 10 µL of liquid to form a droplet. - Add 15 µL of the membrane matrix to each drop containing EB and mix it quickly. Embed the EB ball in the center of the droplet.
NOTE: EBs must not be aspirated with a standard pipette tip, which damages the EB. The 200 µL wide-bore pipette tip needs to be used instead. - Place the 6-well plate into a 37 °C incubator for 30 min, and solidify the membrane matrix droplets containing EBs.
- Add 3 mL of the Expansion medium to each well and gently blow up the matrix-embedded EBs to suspend them.
- Incubate at 37 °C and 5% CO2 for 3 days.
NOTE: If budding on the surface of EB is observed, it means that an expanded neuroepithelium has been formed16.
8. Organoid maturation (days 10-40)
- Gently remove the original medium, and add 3 mL of Maturation medium (Supplementary Table 4) to each well.
- Place the organoid plate on a horizontal shaker in a 37 °C incubator.
- Continue to rotate the culture horizontally. Set the shaker to the appropriate speed.
NOTE: When culturing cerebral organoids on a 6-well plate, a relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer of the horizontal shaker (see Table of Materials). According to the conversion between the relative centrifugal force and rotating speed17,18, RCF = 1.118 x 10−5 × R × rpm2, where RCF = relative centrifugal force (g), rpm = revolutions per minute (r/min), and R = rotation radius (cm), also known as shaking throw. The shaking throw parameters of different shakers may be different. Therefore, it can be estimated that the rotating speed is rpm = 299 x (RCF/R)1/2. - Change the fresh Maturation medium every 2-3 days.
- After 20-30 days, gradually culture the cerebral organoids to maturity.
NOTE: During this period, organoids can be used for experiments and detection, such as neural marker detection and transcriptome sequencing19,20,21.
9. Frozen sections and immunofluorescence of cerebral organoids
- Fix the organoids in 4% paraformaldehyde at 4 °C for 16 h and then wash them 3x with 1x PBS for 10 min each time.
- Remove the PBS, and immerse the organoids in 30% sucrose at 4 °C overnight.
- Embed the organoids in 10%/7.5% gelatine/sucrose at 37 °C for 1 h and then quickly transfer them to the embedding mold.
- Add dry ice to 100% ethanol to prepare a dry ice/ethanol slurry, and place the organoid samples in it for quick freezing.
- Store the frozen samples in a −80 °C freezer. When required, make frozen sections with a thickness of 20 µm.
- Refer to Steps 5.3.-5.5. for immunofluorescence. Use the PAX6 antibody to label apical progenitor cells, the TUJ1 antibody (see Table of Materials) to label neuronal cells22,23,24, and DAPI for nuclear DNA staining.
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Representative Results
The present study induced iPSCs (Figure 2B) into cerebral organoids (Figure 2C). The EBs cultivated in the early stage expressed the OCT4 marker (Figure 2A), which indicated good pluripotency. In the later stage, the EBs developed into mature cerebral organoids (Figure 2D). The research cultivated iPSCs from normal healthy individuals and SCA3 patients into cerebral organoids (Figure 3A). SCA3, also known as Machado-Joseph disease (MJD), is a neurodegenerative disorder caused by a polyglutamine expansion in the ATXN3 gene25. The RNA-seq data (uploaded to a public repository, see Table of Materials)revealed significant differences in gene expression profiles between the normal cerebral organoid group and the SCA3-cerebral organoid group in pathways such as neurotransmitter transport, synapse formation, and regulation (Figure 3B). Cufflinks software was used to compute the gene expression level and difference26. DEseq2 was further used to analyze the data27.
Figure 1: Optimized medium change and transfer operations of EBs. (A) EB preparation. The iPSCs were digested and added to the specialized 24-well plates (left). The cells formed EBs (right). The scale bar is 400 µm. (B) Schematic diagram of laterally illuminated soft light to assist in observing organoids. (C) The EBs were observed with different light sources. Yellow arrow: laterally illuminated light; red arrow: dark background; green arrows: EBs. (D) A swirl flow is induced by rotating the dish along a circular orbit. Due to the swirl flow, a secondary flow directed toward the center is induced. The EBs converge to the center of the dish driven by the secondary flow generated through rotation. White arrows: EBs. (E) Organoid transfer. (I) First, a wide-mouth pipette tip is used to suck up the mixture solution, including both the EBs and the culture medium. (II) Then, while keeping the pipette upright, the EBs gradually sink under the gravity effect and converge towards the mouth of the pipette tip. (III) As the mouth of the pipette tip touches the liquid (usually the fresh medium) surface again, and due to liquid surface tension, (IV) the EBs quickly sink into the medium (no need for additional blowing out by pipetting manually). Red line: the liquid level; yellow arrows: EBs. (F) Since free cells and dead cell fragments sink more slowly than EBs, most of the free cells and dead cell fragments can thus be removed through the above sedimentation method to facilitate the embryoid transfer. The red box in the upper right corner is an enlarged image. The scale bar is 400 µm. Please click here to view a larger version of this figure.
Figure 2: Induction of iPSCs into cerebral organoids. (A) Immunofluorescence staining of EBs. OCT4: octamer-binding transcription factor-4; DAPI: DAPI dihydrochloride. The scale bar is 100 µm. (B) iPSCs. The scale bar is 400 µm. (C) Cerebral organoids. The scale bar is 200 µm. (D) Immunofluorescence staining of cerebral organoids. PAX6: paired box 6 is the marker of apical progenitor cells; TUJ1: neuron-specific class III β-tubulin is a neuron-specific marker. The scale bar is 50 µm. Please click here to view a larger version of this figure.
Figure 3: Cultivation of iPSCs from healthy individuals and SCA3 patients into cerebral organoids. (A) Different stages covering the maturation of cerebral organoids starting from EBs. NC: normal group; SCA3: SCA3/MJD group. Scale bars of low- and high-magnification images are 400 µm and 200 µm, respectively. (B) GO enrichment analysis chart of down-regulated, differentially expressed genes between normal organoids and SCA3 organoids. Please click here to view a larger version of this figure.
Supplementary Figure 1: Identification of ATXN3 CAG repeats by capillary electrophoresis. (A) The SCA3 patient iPSC (SCA3-iPSC) was identified as having 26/78 CAG repeats in the ATXN3 gene. (B) The normal human iPSC (NC-iPSC) was identified as having 14/14 CAG repeats in the ATXN3 gene. Please click here to download this File.
Supplementary Figure 2: Preparation of the soft light lamp. (A) The power supply and the LED lights were installed on one side of the acrylic board (as shown in the red dotted box). The LED soft light lamp's scattering wavelength is 450-470 nm, luminous flux is 1300-1800 lm, and color rendering index is 75-85 Ra. (B) It was checked whether the LED bulbs could light normally (as shown in the red dotted box). (C) A white pad was pasted on the front and back of the acrylic plate (as indicated by the red arrows). (D) The overall appearance of the soft light lamp. (E) The soft light lamp can also be replaced by a commercial LED painting light pad without a frame, which is usually used for tracking when copying sketches. A layer of white film needs to be pasted directly above the LED painting light pad. (F)The soft light lamp at work. Please click here to download this File.
Supplementary Table 1: Composition of the EB-formation medium. At the initial stage of EB formation (generally the first 2 days), Y-27632 (50 µM) and bFGF (4 ng/mL) need to be added to the EB-formation medium. The medium needs to be filtered with a 0.2 µm filter unit and stored at −20 °C. Please click here to download this Table.
Supplementary Table 2: Composition of the Neural induction medium. The medium needs to be filtered with a 0.2 µm filter unit and stored at −20 °C. Please click here to download this Table.
Supplementary Table 3: Composition of the Expansion medium. A 1:100 dilution of 2-Mercaptoethanol was prepared in DMEM-F12, and 87.5 µL of this was added to the Expansion medium. B27 must not contain vitamin A. The medium needs to be filtered with a 0.2 µm filter unit and stored at −20 °C. Please click here to download this Table.
Supplementary Table 4: Composition of the Maturation medium. A 1:100 dilution of 2-Mercaptoethanol in DMEM-F12 was prepared and 87.5 µL of this was added to the Maturation medium. B27 must not contain vitamin A. The medium needs to be filtered with a 0.2 µm filter unit and stored at −20 °C. Please click here to download this Table.
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Discussion
Cerebral organoids open new avenues for medical research. Many useful applications of this technology are only beginning to be explored28. This research found that the transcriptome sequencing results of genetically diseased cerebral organoids and normal cerebral organoids can reflect the differences between disease and health. For example, the RNA-seq data analysis results (Figure 3B) are consistent with many reported studies on SCA3 diseases29,30,31,32. Experimental operations, such as medium change, EB transfer, and wrapping, are crucial for cultivating cerebral organoids and determining whether organoids with good uniformity can be prepared33,34. In this study, a cerebral organoid protocol that facilitated medium change and organoid transfer is introduced. A lateral soft light lamp can greatly assist in the operation of organoid culture. However, it is not difficult to make, the LED painting light pad and simple processing can also replace it. The simple liquid exchange method and organoid transfer operation make the whole culture process easier. The application of cerebral organoids is often affected by the problem of poor uniformity35. The biggest difference between this study and other cerebral organoid culture guidelines is that the focus is on optimizing the operation to make the experiment more repeatable.
It is very important to maintain the stability of the culture system. If conditions permit, it is suggested to prioritize the commercial cerebral organoid medium, which can effectively reduce the great differences in experimental results in different batches due to the instability of the medium. In addition, cerebral organoid medium storage at 4 °C should not last more than 2 weeks; otherwise, it will affect the stability of the culture system. Of course, it is also important to ensure that the iPSCs used are pluripotent rather than differentiated. OCT4 immunofluorescence detection can be used. If OCT4-positive cells are less than 90%, it is recommended to replace them with higher-quality iPSCs and culture cerebral organoids again.
There are some limitations in the cerebral organoid culture technology introduced in this study. For example, cerebral organoids cannot be intensively cultured in the later stage of development, which is a waste of culture space. This is also an urgent problem to be solved in applying cerebral organoids. If many cerebral organoids exhibit hollow expansion, the number of organoids in each well is reduced, the medium exchange frequency increases, and the organoids are in a nonhypoxic state with sufficient nutrients.
This study is an operational supplement to many existing cerebral organoid culture methods12,36,37and even other types of organ culture methods38,39. This will help develop automated organoid culture systems in the future and promote the research and application of organoids. If the technology of enhancing the light diffuse reflection effect of organoids is used in the automatic intelligent cultivation system, it can theoretically replace the high-precision image amplification camera, and only an ordinary image recognition device needs to be installed. In addition, medium exchange and organoid transfer through secondary flow generated by rotation and natural sedimentation on the suction head are theoretically more feasible than centrifugation in future automatic culture equipment.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This study was supported by the Natural Science Foundation of Guangdong Province (Grant No. 2020A0505100062), the Guangzhou City Science and Technology Key Topics Project (Grant No. 201904020025), the National Natural Science Foundation of China (Grant Nos. 31872800, 32070582, 82101937), and the Guangzhou City Postdoctoral Research Grant project (to Bangzhu Chen).
Materials
| Name | Company | Catalog Number | Comments |
| 0.2 μm Filter | NEST Biotechnology, China | 331001 | |
| 1000 μL wide-bore pipette tip | Thermo Fisher Scientific, USA | 9405163 | |
| 200 μL wide-bore pipette tip | Thermo Fisher Scientific, USA | 9405020 | |
| 2-Mercaptoethanol | Merck, Germany | 8057400005 | |
| 4% Paraformaldehyde | Servicebio, China | G1101 | |
| 6-well low adhesion plate | NEST Biotechnology, China | 703011 | It is used for EBs suspension cultures |
| Aaccute cell detachment solution | STEMCELL Technologies, Canada | 07920 | It is used to digest iPSC into single cells. |
| AggreWell800 24-well | STEMCELL Technologies, Canada | 34811 | Microporous culture plate for EBs preparation. |
| Anti-Adherence Rinsing Solution | STEMCELL Technologies, Canada | 07010 | Rinsing solution for cultureware to prevent cell adhesion |
| B27-vit. A supplement | Thermo Fisher Scientific, USA | 12587010 | |
| bFGF | Peprotech, USA | GMP100-18B | |
| BSA | Beyotime Biotechnology, China | ST025 | |
| Centrifuge | Eppendorf, Germany | 5810 R | It can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates. |
| Cover glass | Shitai Laboratory Equipment, China | 10212020C | |
| DAPI | Beyotime Biotechnology, China | C1002 | Used for nuclear staining. After DAPI was combined with double stranded DNA, the maximum excitation wavelength was 364nm and the maximum emission wavelength was 454nm. |
| DMEM-F12 | Thermo Fisher Scientific, USA | 11330032 | |
| ES-quality FBS | Thermo Fisher Scientific, USA | 10270106 | |
| Ficoll Paque | General Electric Company, USA | 17-5442-02 | Isolate the peripheral blood mononuclear cells according to Ficoll-Paque method. |
| Gelatin | Sangon Bioteach, China | A609764 | |
| Glutamax supplement | Thermo Fisher Scientific, USA | 35050061 | |
| Glutamax supplement | Thermo Fisher Scientific, USA | 17504044 | |
| Goat anti-Chicken IgY secondary antibody | Abcam, UK | ab150171 | Goat anti-Chicken IgG. Conjugation: Alexa Fluor 647. Ex: 652 nm, Em: 668 nm. Use at 1:500 dilution. |
| Goat anti-Mouse IgG secondary antibody | Abcam, UK | ab150120 | Goat anti-Mouse IgG. Conjugation: Alexa Fluor 594. Ex: 590 nm, Em: 617 nm. Use at 1:500 dilution. |
| Goat anti-Rabbit IgG secondary antibody | Abcam, UK | ab150077 | Goat Anti-Rabbit IgG. Conjugation: Alexa Fluor 488. Ex: 495 nm, Em: 519 nm. Use at 1:500 dilution. |
| Heparin | Merck, Germany | H3149 | |
| Horizontal shaker | Servicebio, China | DS-H200 | Relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer INFORS HT (Switzerland). |
| Insulin | Merck, Germany | I9278-5ML | |
| KOSR | Thermo Fisher Scientific, USA | 10828028 | |
| Matrigel | Corning, USA | 354277 | Matrigel will solidify in the environment higher than 4 °C, so it should be sub packed at low temperature. |
| MEM-NEAA | Thermo Fisher Scientific, USA | 11140050 | |
| mTeSR1 | STEMCELL Technologies, Canada | 85850 | iPSC culture medium |
| N2 supplement | Thermo Fisher Scientific, USA | 17502048 | |
| Neurobasal | Thermo Fisher Scientific, USA | 21103049 | |
| OCT4 primary antibody | Abcam, UK | ab19857 | Host: Rabbit. Dissolve with 500 μL PBS. Use at 1:200 dilution. |
| Pathological frozen slicer | Leica, Germany | Leica CM1860 | |
| PAX6 primary antibody | Abcam, UK | ab78545 | Host: Mouse. Use at 1:100 dilution. |
| PBS | STEMCELL Technologies, Canada | 37350 | |
| Penicillin-Streptomycin | Thermo Fisher Scientific, USA | 15140122 | |
| PSC dissociation solution | Beijing Saibei Biotechnology, China | CA3001500 | Enzyme free dissociation solution can be used for iPSC digestion and passage. |
| Sendai Reprogramming Kit | Thermo Fisher Scientific, USA | A16518 | Establish iPSC according to the protocol of Sendai Reprogramming Kit. |
| Slide Glass | Shitai Laboratory Equipment, China | 188105W | |
| Soft light lamp | NUT | NUT | A simple self made device, refer to supplementary figure 2 for preparation. |
| STEMdiff Cerebral Organoid Kit | STEMCELL Technologies, Canada | 8570 | Contain: 1. EB Formation Medium; 2. Induction Medium; 3. Expansion Medium; 4. Maturation Medium. |
| STEMdiff Cerebral Organoid Maturation Kit | STEMCELL Technologies, Canada | 8571 | Maturation Medium |
| Sucrose | Sangon Bioteach, China | A502792 | |
| Triton X-100 | Merck, Germany | X100 | |
| TUJ1 primary antibody | Abcam, UK | ab41489 | Host: Chicken. Use at 1:1000 dilution. |
| Vaseline | Sangon Bioteach, China | A510146 | |
| Y-27632 | STEMCELL Technologies, Canada | 72302 | Prepare a 5 mM stock solution in PBS, resuspend 1 mg in 624 µL of PBS. |
| Weblink | |||
| Raw sequencing data | Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences | GSA-Human: HRA002430 | https://ngdc.cncb.ac.cn/gsa-human/ |
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