Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

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 ....

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 pre.......

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

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/