Neural organoid and preparation method thereof

A technology of organoids and nerves, applied in the field of preparing the neural organoids, which can solve the problem of not being able to capture the complete continuum of regional diversity

Active Publication Date: 2021-10-12
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But organoids obtained through fusion do not capture the full continuum of regional diversity that the human brain encompasses

Method used

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  • Neural organoid and preparation method thereof
  • Neural organoid and preparation method thereof
  • Neural organoid and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1: Preparation of organoids

[0163] Differentiation of early organoids

[0164] D0: Digest overgrown human embryonic stem cells into single cells with digestive enzymes, inoculate them into v-shaped 96well plates at a density of 104 / well, add maintenance medium to each well for culture, and the maintenance medium is supplemented with 10 μM Y- The E8 medium of 27632 was placed in incucyteS3 for real-time fluorescence observation and photography;

[0165] D1: N2B27 medium containing 100nM LDN193189 and 10μM SB431542 was added to each well; the N2B27 medium contained: 48% (v / v) CTS-KnockOut DMEM / F-12, 48% (v / v) CTS-Neurobasal Medium , 1%(v / v) N2-CTS, 2%(v / v) B27-CTS, 1%(v / v) CTS-GlutaMAX;

[0166] D3: D1 medium was discarded, and N2B27 medium containing 100 nM LDN193189+10 μM SB431542+0.5 μM CHIR99021+0.1 μM SAG was added to each well;

[0167] D4: D3 medium was discarded, and N2B27 medium containing 100 nM LDN193189+10 μM SB431542+0.1 μM SAG was added to each ...

Embodiment 2

[0180] Example 2: Identification of organoids

[0181] Organoids were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (4P) for 30 minutes at room temperature (RT). Washed 3 times with PBS, then dehydrated overnight in 30% sucrose solution. Organoids were embedded in OCT medium, frozen on the surface of liquid LN2, and sectioned with a Leica SM2010R microtome. For immunostaining, the slices were washed 3 times with PBS, 10 minutes each time. Incubate at RT for 2h in permeation blocking solution (TBS: 3% TX-100 + 1% BSA with PBS), and incubate overnight at 4°C with the first strain of Ab diluted in TBS. Rinse 3 times with PBS, 10 minutes each time. Incubate with 2ab reagent diluted in TBS for 2 hours at RT, wash with PBS 3 times, 10 minutes each time. Finally stained with hoechst33342. All images were taken by a confocal microscope (Zeiss LSM 780 / 880).

[0182] Antibodies and their information are as follows:

[0183] chicken anti-GFP (Abcam, ab139...

Embodiment 3

[0187] Example 3: Organoids simulate the pathogenesis of PD

[0188] The organoid cultured to about 70 days was observed in tissue slices, and the results were as follows: Figure 4 As shown in A, organoids generate nerve bundles similar to mature neurons in vivo, providing a basis for studying neuronal projections. Subsequently, 1 mM MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Sigma D048) was added to the organoid medium cultured for about 70 days. MPTP is a neurotoxin, It can cause symptoms similar to Parkinson's disease by destroying dopamine-producing nerve cells in the substantia nigra to simulate the pathogenesis of PD. Tissue sections were then tested for caspase-3 (rabbit anti- caspase-3 (cell signaling, 9661S, 1:400)), TH (mouse anti-Tyrosine hydroxylase (TH) (Immunostar Systems, 22941, 1:2000)) and DARPP32 (mouse anti-DARPP32 (Santa Cruz, sc-271111, 1:300)) staining, the results are as follows Figure 4 As shown in B-4D, dopamine neurons (GFP and GFP / MCHER...

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Abstract

The invention provides a neural organoid comprising a multi-module structure capable of simulating a substantia nigra-striatum pathway in vitro, and a method for preparing the neural organoid. The invention further provides application of the neural organoid in disease research, drug screening, intracerebral transplantation and the like.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention provides a neural organoid comprising a multi-module structure capable of simulating the substantia nigra-striatum pathway in vitro, and a method for preparing the neural organoid. The present invention further provides the application of the neural organoids in disease research, drug screening, intracerebral transplantation and the like. Background technique [0002] The study of human brain development is limited by the lack of tissue samples and suitable in vitro models. Brain organoids are 3D cell culture tissues that simulate human brain development and disease pathogenesis in vitro, providing a good platform for studying organoids in vitro. Since Lancaster obtained human brain organoids for the first time in 2013, scientists have obtained organoids of the forebrain, cortex-midbrain, thalamus, and spinal cord in vitro. These organoids can be used to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12Q1/02
CPCC12N5/0619G01N33/5008C12N2506/45C12N2506/02C12N2501/999
Inventor 周琪李伟胡宝洋郝捷王昱凯梁灵敏冯琳孙云王柳
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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