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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

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 (Figur.......

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

Acknowledgements

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

NameCompanyCatalog NumberComments
0.2 μm FilterNEST Biotechnology, China331001
1000 μL wide-bore pipette tipThermo Fisher Scientific, USA9405163
200 μL wide-bore pipette tipThermo Fisher Scientific, USA9405020
2-MercaptoethanolMerck, Germany8057400005
4% ParaformaldehydeServicebio, ChinaG1101
6-well low adhesion plateNEST Biotechnology, China703011It is used for EBs suspension cultures
Aaccute cell detachment solutionSTEMCELL Technologies, Canada07920It is used to digest iPSC into single cells.
AggreWell800 24-wellSTEMCELL Technologies, Canada34811Microporous culture plate for EBs preparation.
Anti-Adherence Rinsing SolutionSTEMCELL Technologies, Canada07010Rinsing solution for cultureware to prevent cell adhesion
B27-vit. A supplementThermo Fisher Scientific, USA12587010
bFGFPeprotech, USAGMP100-18B
BSABeyotime Biotechnology, ChinaST025
CentrifugeEppendorf, Germany5810 RIt can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates.
Cover glassShitai Laboratory Equipment, China10212020C
DAPIBeyotime Biotechnology, ChinaC1002Used 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-F12Thermo Fisher Scientific, USA11330032
ES-quality FBSThermo Fisher Scientific, USA10270106
Ficoll PaqueGeneral Electric Company, USA17-5442-02Isolate the peripheral blood mononuclear cells according to Ficoll-Paque method.
GelatinSangon Bioteach, ChinaA609764
Glutamax supplementThermo Fisher Scientific, USA35050061
Glutamax supplementThermo Fisher Scientific, USA17504044
Goat anti-Chicken IgY  secondary antibodyAbcam, UKab150171Goat anti-Chicken IgG. Conjugation: Alexa Fluor 647. Ex: 652 nm, Em: 668 nm. Use at 1:500 dilution.
Goat anti-Mouse IgG secondary antibodyAbcam, UKab150120Goat anti-Mouse IgG. Conjugation: Alexa Fluor 594. Ex: 590 nm, Em: 617 nm. Use at 1:500 dilution.
Goat anti-Rabbit IgG secondary antibodyAbcam, UKab150077Goat Anti-Rabbit IgG. Conjugation: Alexa Fluor 488. Ex: 495 nm, Em: 519 nm. Use at 1:500 dilution.
HeparinMerck, GermanyH3149
Horizontal shakerServicebio, ChinaDS-H200Relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer INFORS HT (Switzerland).
InsulinMerck, GermanyI9278-5ML
KOSRThermo Fisher Scientific, USA10828028
MatrigelCorning, USA354277Matrigel will solidify in the environment higher than 4 °C, so it should be sub packed at low temperature.
MEM-NEAAThermo Fisher Scientific, USA11140050
mTeSR1STEMCELL Technologies, Canada85850iPSC culture medium
N2 supplementThermo Fisher Scientific, USA17502048
NeurobasalThermo Fisher Scientific, USA21103049
OCT4 primary antibodyAbcam, UKab19857Host: Rabbit. Dissolve with 500 μL PBS. Use at 1:200 dilution.
Pathological frozen slicerLeica, GermanyLeica CM1860
PAX6 primary antibodyAbcam, UKab78545Host: Mouse. Use at 1:100 dilution.
PBSSTEMCELL Technologies, Canada37350
Penicillin-StreptomycinThermo Fisher Scientific, USA15140122
PSC dissociation solutionBeijing Saibei Biotechnology, ChinaCA3001500Enzyme free dissociation solution can be used for iPSC digestion and passage.
Sendai Reprogramming KitThermo Fisher Scientific, USAA16518Establish iPSC according to the protocol of Sendai Reprogramming Kit.
Slide GlassShitai Laboratory Equipment, China188105W
Soft light lampNUTNUTA simple self made device, refer to supplementary figure 2 for preparation.
STEMdiff Cerebral Organoid KitSTEMCELL Technologies, Canada8570Contain: 1. EB Formation Medium; 2. Induction Medium; 3. Expansion Medium; 4. Maturation Medium.
STEMdiff Cerebral Organoid Maturation KitSTEMCELL Technologies, Canada8571Maturation Medium
SucroseSangon Bioteach, ChinaA502792
Triton X-100Merck, GermanyX100
TUJ1 primary antibodyAbcam, UKab41489Host: Chicken. Use at 1:1000 dilution.
VaselineSangon Bioteach, ChinaA510146
Y-27632STEMCELL Technologies, Canada72302Prepare a 5 mM stock solution in PBS, resuspend 1 mg in 624 µL of PBS.
Weblink
Raw sequencing dataGenome 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 SciencesGSA-Human: HRA002430https://ngdc.cncb.ac.cn/gsa-human/

References

  1. Jensen, C., Teng, Y. Is it time to start transitioning from 2D to 3D cell culture. Frontiers in Molecular Biosciences. 7 (33), (2020).
  2. Quadrato, G., et al. Cell diversity and network dynamics in photosensitive human ....

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