Name: facilitating cerebral organoid culture via lateral soft light illumination
Uploaded: 2022-06-06T14:00:00
Description: Watch this Scientific Journal Video about Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination at JoVE.com

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).

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&#39;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 pre...

<ol>
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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</sup...

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 &#181;m to 300 &#181;m) 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Filter</td>
			<td>NEST Biotechnology, China</td>
			<td>331001</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L wide-bore pipette tip</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>9405163</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L wide-bore pipette tip</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>9405020</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Merck, Germany</td>
			<td>8057400005</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Servicebio, China</td>
			<td>G1101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well low adhesion plate</td>
			<td>NEST Biotechnology, China</td>
			<td>703011</td>
			<td>It is used for EBs suspension cultures</td>
		</tr>
		<tr>
			<td>Aaccute cell detachment solution</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>07920</td>
			<td>It is used to digest iPSC into single cells.</td>
		</tr>
		<tr>
			<td>AggreWell800 24-well</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>34811</td>
			<td>Microporous culture plate for EBs preparation.</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>07010</td>
			<td>Rinsing solution for cultureware to prevent cell adhesion</td>
		</tr>
		<tr>
			<td>B27-vit. A supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech, USA</td>
			<td>GMP100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beyotime Biotechnology, China</td>
			<td>ST025</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td>5810 R</td>
			<td>It can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates.</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Shitai Laboratory Equipment, China</td>
			<td>10212020C</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime Biotechnology, China</td>
			<td>C1002</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>ES-quality FBS</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque</td>
			<td>General Electric Company, USA</td>
			<td>17-5442-02</td>
			<td>Isolate the peripheral blood mononuclear cells according to Ficoll-Paque method.</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sangon Bioteach, China</td>
			<td>A609764</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Chicken IgY&#160; secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150171</td>
			<td>Goat anti-Chicken IgG. Conjugation: Alexa Fluor 647. Ex: 652 nm, Em: 668 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150120</td>
			<td>Goat anti-Mouse IgG. Conjugation: Alexa Fluor 594. Ex: 590 nm, Em: 617 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG secondary antibody</td>
			<td>Abcam, UK</td>
			<td>ab150077</td>
			<td>Goat Anti-Rabbit IgG. Conjugation: Alexa Fluor 488. Ex: 495 nm, Em: 519 nm. Use at 1:500 dilution.</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Merck, Germany</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>Servicebio, China</td>
			<td>DS-H200</td>
			<td>Relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer INFORS HT (Switzerland).</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Merck, Germany</td>
			<td>I9278-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning, USA</td>
			<td>354277</td>
			<td>Matrigel will solidify in the environment higher than 4 &#176;C, so it should be sub packed at low temperature.</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>85850</td>
			<td>iPSC culture medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab19857</td>
			<td>Host: Rabbit. Dissolve with 500 &#956;L PBS. Use at 1:200 dilution.</td>
		</tr>
		<tr>
			<td>Pathological frozen slicer</td>
			<td>Leica, Germany</td>
			<td>Leica CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX6 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab78545</td>
			<td>Host: Mouse. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>37350</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PSC dissociation solution</td>
			<td>Beijing Saibei Biotechnology, China</td>
			<td>CA3001500</td>
			<td>Enzyme free dissociation solution can be used for iPSC digestion and passage.</td>
		</tr>
		<tr>
			<td>Sendai Reprogramming Kit</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>A16518</td>
			<td>Establish iPSC according to the protocol of Sendai Reprogramming Kit.</td>
		</tr>
		<tr>
			<td>Slide Glass</td>
			<td>Shitai Laboratory Equipment, China</td>
			<td>188105W</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft light lamp</td>
			<td>NUT</td>
			<td>NUT</td>
			<td>A simple self made device, refer to supplementary figure 2 for preparation.</td>
		</tr>
		<tr>
			<td>STEMdiff Cerebral Organoid Kit</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>8570</td>
			<td>Contain: 1. EB Formation Medium; 2. Induction Medium; 3. Expansion Medium; 4. Maturation Medium.</td>
		</tr>
		<tr>
			<td>STEMdiff Cerebral Organoid Maturation Kit</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>8571</td>
			<td>Maturation Medium</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sangon Bioteach, China</td>
			<td>A502792</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck, Germany</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>TUJ1 primary antibody</td>
			<td>Abcam, UK</td>
			<td>ab41489</td>
			<td>Host: Chicken. Use at 1:1000 dilution.</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Sangon Bioteach, China</td>
			<td>A510146</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies, Canada</td>
			<td>72302</td>
			<td>Prepare a 5 mM stock solution in PBS, resuspend 1 mg in 624 &#181;L of PBS.</td>
		</tr>
		<tr>
			<td>Weblink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Raw sequencing data</td>
			<td>Genome Sequence Archive (Genomics, Proteomics &#38; Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences</td>
			<td>GSA-Human: HRA002430</td>
			<td>https://ngdc.cncb.ac.cn/gsa-human/</td>
		</tr>
	</tbody>
</table>

facilitating cerebral organoid culture via lateral soft light illumination

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.

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...

Watch this Scientific Journal Video about Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination at JoVE.com

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

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.

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

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 ...

The protocol facilitates operation of cerebral organoids culture and helps to promote the application of brain organoids. The earlier stage organoid can be seen with the naked eye by using a lateral soft light lamp which is convenient for many operations. The improved medium change methods and organoid transfer operations are also helpful for other types of organoid cultures and the design of automatic cultures machines.To begin, culture the iPSCs with mTeSR1 medium in a matrigel covered 35 millimeter Petri dish and change the medium every day. The iPSC growth density must not exceed 75%Then, pipette out the mTeSR1 medium from the iPSCs and wash it twice with one millimeter of PBS. After pipetting out the PBS, add 300 microliters of cell detachment solution to digest the iPSCs into single cells for three to four minutes at 37 degrees Celsius.Later, resuspend the cells in two millimeters of EB formation medium and centrifuge the sample for five minutes at 300 times g. Treat the specialized 24 well plate with 500 microliters per well of anti adherence rinsing solution for five minutes. Now, resuspend the cells in the EB formation medium containing Rho kinase inhibitor Y27632.After removing the anti adherence rinsing solution, rinse each well with two milliliters of EB formation medium. Next, add the cells to the specialized 24 well plates at a density of three times 10 to the sixth cells per well and centrifuge the plate at 100 times g for two minutes at room temperature. Uniformly distribute the cells in each chamber at the bottom of the plate.Incubate the samples at 37 degrees Celsius and 5%carbon dioxide for 24 hours. If centrifugation was not used in the previous step, incubate for 36 hours and keep the sample steady. Use a transparent acrylic board with a thickness of 0.3 to 0.5 centimeters in the size of A5 paper and paste white pads on the front and back of the acrylic plate.Next, 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. Now, prepare a new six well low adhesion plate and add two milliliters of EB formation medium to each well. After removing the EBs together with the medium using 1000 microliters wide bore pipette tip, transfer approximately 100 EBs per well to the six well low adhesion plate.To replace the EB formation medium every day with the same volume of fresh medium, induce a swirl flow by rotating the dish along a circular orbit. The EBs or organoids converge to the center of the well due to the secondary flow generated through rotation. Aspirate the old medium by pipeting to the edge of the well slowly.Do not suck too hard, otherwise the EBs will be removed together. Then, add fresh media to resuspend the EBs. For neural induction, prepare a new six well low adhesion plate with three milliliters of neural induction medium per well.Turn on the lateral soft light and turn off other indoor light sources. Now transfer the EBs to the six well plate with added neural induction medium by using a wide mouth pipette tip to suck both the EBs and the culture medium. Then, hold the pipette upright.The EBs will gradually sink under gravity and converge towards the mouth of the pipette tip. As the mouth of the pipette tip touches the liquid surface again, and due to liquid surface tension, the EBs quickly sink into the medium. Incubate the samples at 37 degrees Celsius and 5%carbon dioxide for 24 hours.Place the membrane matrix in a four degree Celsius refrigerator for 60 minutes to dissolve. After turning on the lateral soft light, turn off the light source on the top of the console and keep the membrane matrix on ice to prevent solidification. Transfer a small amount of EBs to a 60 millimeter dish containing a fresh expansion medium to make it easier to remove single EB.Using a 200 microliters wide bore pipette tip, suck a single EB containing approximately 10 microliters medium each time, and then make droplets by adding it to the bottom of the six well plate.Use five droplets per well. Add 15 microliters of the membrane matrix to each EB containing drop. Quickly mix and embed the EB ball in the center of the droplet.Allow the droplets containing EBs to solidify by placing the six well plate into a 37 degree Celsius incubator for 30 minutes. Then, add three milliliters of the expansion medium to each well and gently blow up the matrix embedded EBs to suspend them and incubate for three days. For organoid maturation, gently remove the original medium and add three milliliters of maturation medium to each well.Place the organoid plate on a horizontal shaker in a 37 degree Celsius incubator and continue to rotate the culture horizontally. Change the fresh maturation medium every two to three days. Due to gravity and relatively higher density than the medium, the resuspended EBs will gradually sink.Hence, the EBs can be conveniently transferred. As 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.The EBs cultivated in the early stage expressed the OCT4 marker which indicated good pluripotency. In the later stage, the EBs developed into mature cerebral organoids. The mature cerebral organoids were positive for apical progenitor cells marker PAX6 and neuron specific marker tubulin J1.The iPSCs from healthy individuals and SCA3 patients were cultivated to grow into organoids.The gene expression profiles of the normal cerebral organoid group and the SCA3 cerebral organoid group showed significant differences in pathways such as neurotransmitter transport, synapse formation, and regulation. When embedding the EB with basement membrane matrix the operation times should not be too long to avoid EBs drying out. It is recommended to protect rapidly.

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