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Brain organoid model and preparation method and application thereof

A technology of organoids and models, applied in the field of brain organoid models and their preparation, can solve the problems of low throughput, large differences, easy pollution or fusion, etc., and achieve the effect of simplifying the cultivation steps and good compatibility

Pending Publication Date: 2022-06-24
合肥燃音生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of organoid transfer and suspension culture, organoids are affected by external stimuli of human manipulation and location uncertainty, and are easily contaminated or fused, and difficult to locate and observe.
The above limitations lead to complex, highly variable, low-throughput, and difficult real-time monitoring of the organoid culture process.

Method used

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  • Brain organoid model and preparation method and application thereof
  • Brain organoid model and preparation method and application thereof
  • Brain organoid model and preparation method and application thereof

Examples

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preparation example Construction

[0039] According to a typical embodiment of the present invention, a method for preparing a brain organoid model is provided, the method comprising:

[0040] Step S1, obtaining monolayer adherent growth pluripotent stem cells with boundary constraints;

[0041] The method for obtaining border confinement in a monolayer adherent-grown pluripotent stem cell with border confinement includes one of molecular stamping method, photolithography method, perforated film method and differential adhesion method.

[0042] As a specific embodiment, the perforated film method is adopted, and the specific operations are as follows:

[0043] Step S101, obtaining a film with multiple perforations;

[0044] Wherein, the step S101 specifically includes:

[0045] Step S1011 , obtaining a positive mold having a micro-pillar array; the height of the micro-pillar array of the positive film is 30 μm˜100 μm. If the height is too low, the film will be too thin and difficult to operate; if the height...

Embodiment 1

[0073] Embodiment 1 A kind of culture method of brain organoid model

[0074] 1. Preparation of Boundary-Restricted Pluripotent Stem Cells

[0075] The SU-8 template is prepared by soft lithography, and this template is a high-throughput micro-pillar array, the diameter of the micro-pillars is 500 microns, the height is 50 microns, and the distance between the micro-pillars is 1000 microns;

[0076] like figure 1 As shown in the figure, the PDMS prepolymer and the curing agent were mixed at a ratio of 10:1 and poured onto the SU-8 template. After vacuuming to remove air bubbles, a layer of 0.2 mm PMMA plate was covered on it and clamped with two coverslips. Hold and fix it with a bench vise, place it in an oven at 80 °C for polymerization for 120 min, take it out and return to room temperature, peel off the perforated PDMS film from the SU-8 template, use a 10 mm diameter punch to remove the film of a suitable size, and place it in the 48-well hole. Add 1 mL of 70% ethanol t...

Embodiment 2

[0091] In this example, the bounding shape of the pluripotent stem cells is a circle; the growth area of ​​the pluripotent stem cells is 0.008 mm 2 The growth area spacing of described pluripotent stem cells is 1mm; The growth area arrangement of described pluripotent stem cells is a square apex arrangement; The culture system of described brain organoids is a 48-well plate; Others are all the same as in Example 1.

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Abstract

The invention discloses a brain organoid model and a preparation method and application thereof, and the method comprises the following steps: obtaining pluripotent stem cells with boundary limitation and single-layer adherent growth, adding into an EB formation culture medium, and maintaining culture for 4-6 days; culturing for 2-6 days by adopting a nerve induction culture medium; and then absorbing and discarding the culture medium, coating with precooled Matrigel, curing, and sequentially replacing a neural differentiation culture medium for culturing for 3-5 days and inducing a mature culture medium for culturing for 10-30 days to obtain the brain organ-like model. According to the method, the brain organoid model is generated from single-layer cell culture for the first time, the organoid with a uniform form can be constructed in a high-throughput and one-step method, and the whole growth and development process of the organoid can be observed in situ.

Description

technical field [0001] The present invention relates to the technical field of stem cells, organoids, organoid chips and tissue engineering, in particular to a brain organoid model and a preparation method and application thereof. Background technique [0002] The brain is the most complex organ in the human body in structure and function, and understanding human brain development and disease is one of the greatest challenges in life sciences. However, the difficulty of obtaining human brain tissue has seriously hindered our progress in deciphering the secrets of the human brain. Researchers have long used cell culture models and animal models to study adult human brain development and disease. These studies provide the foundation for our current understanding of brain development and function. Nonetheless, our understanding of the human brain is limited to simple cellular interactions and features shared by humans and vertebrates. Therefore, in order to further study the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0735C12N5/10C12Q1/02
CPCC12N5/0618C12N5/0606C12N5/0696G01N33/5088C12N2506/02C12N2506/45C12N2503/02C12N2535/00G01N2500/10C12N2513/00
Inventor 陈璞李彬赵稳
Owner 合肥燃音生物科技有限公司
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