Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 exchange^6,7,8. 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 phenomenon^9,10, 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.

Protokół

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)

1. Collect 3 mL of peripheral blood by venipuncture with the informed consent of volunteers and patients.
2. Isolate peripheral blood mononuclear cells according to the Ficoll-Paque method¹⁵.
3. Establish iPSCs according to the protocol of the Sendai Reprogramming Kit (see Table of Materials).
   1. 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%.
   2. 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)

1. Remove the mTeSR1 medium from the iPSCs with a pipette and wash it 2x with 1 mL of 1x PBS.
2. 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.
3. 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).
4. 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.
5. 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.
6. Remove the anti-adherence rinsing solution, and rinse each well with 2 mL of EB-formation medium.
7. 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.
8. 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.
9. 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)

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.
   1. 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.

4. EB transfer and medium replacement (days 2-5)

1. Prepare a new 6-well low adhesion plate, and add 2 mL of EB-formation medium to each well.
2. 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.
3. 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.
   1. 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.

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.

1. Fix the EBs in 4% paraformaldehyde at 4 °C for 30 min, followed by washing in 1x PBS 3x for 10 min each time.
2. 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.
3. 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.
4. Dilute the respective secondary antibody (see Table of Materials) with 1x PBS at 1:500 and add 1 mL to the EBs.
5. After incubating at room temperature for 2 h, wash the cells in 1x PBS 3x for 10 min each time.
6. 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.
7. 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)

1. Prepare a new 6-well plate with low adhesion, and add 3 mL of Neural induction medium (Supplementary Table 2) to each well.
2. Turn on the lateral soft light (mentioned in step 3.) and turn off other indoor light sources.
3. 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).
4. 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.
5. Proceed to the next step.

7. Embedding in the basement membrane matrix (days 7​-10)

1. 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.
2. Turn on the lateral soft light and turn off the light source on the top of the console.
3. Keep the membrane matrix on ice to prevent solidification.
4. 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.
5. 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.
6. 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.
7. Place the 6-well plate into a 37 °C incubator for 30 min, and solidify the membrane matrix droplets containing EBs.
8. Add 3 mL of the Expansion medium to each well and gently blow up the matrix-embedded EBs to suspend them.
9. 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)

1. Gently remove the original medium, and add 3 mL of Maturation medium (Supplementary Table 4) to each well.
2. Place the organoid plate on a horizontal shaker in a 37 °C incubator.
3. 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 speed^17,18, 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.
4. Change the fresh Maturation medium every 2-3 days.
5. 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 sequencing^19,20,21.

9. Frozen sections and immunofluorescence of cerebral organoids

1. 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.
2. Remove the PBS, and immerse the organoids in 30% sucrose at 4 °C overnight.
3. Embed the organoids in 10%/7.5% gelatine/sucrose at 37 °C for 1 h and then quickly transfer them to the embedding mold.
4. Add dry ice to 100% ethanol to prepare a dry ice/ethanol slurry, and place the organoid samples in it for quick freezing.
5. Store the frozen samples in a −80 °C freezer. When required, make frozen sections with a thickness of 20 µm.
6. 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 cells^22,23,24, and DAPI for nuclear DNA staining.

Wyniki

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 (Figur...

Dyskusje

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

Materiały

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/