Organoid culture medium material and preparation method and application thereof

A technology for culturing substrate materials and organ culture, which is applied in the field of organoid culture substrate materials and its preparation, which can solve the problems of large batch-to-batch differences, complex components, and expensive prices, and achieve good dryness, simple preparation process, and dryness Effect

Active Publication Date: 2020-11-17
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Matrigel is a very complex system, which is reflected in its complex composition, large batch-to-batch variation, and high price. Many scientific research institutions and scholars in the world are working on the research of its substitute products in order to develop a quality-reliable product. Organoid culture substrate with excellent performance and favorable price

Method used

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  • Organoid culture medium material and preparation method and application thereof
  • Organoid culture medium material and preparation method and application thereof
  • Organoid culture medium material and preparation method and application thereof

Examples

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Embodiment 1

[0033] This embodiment provides a method for preparing an organoid culture matrix material, comprising the following steps:

[0034] (1) Dissolving collagen type 1, collagen type 4, laminin, hyaluronic acid, and heparin with 1% acetic acid solution to obtain a collagen solution, and storing it on an ice box;

[0035] (2) Add sodium dihydrogen phosphate, EGF, FGF, A83-01, and Y27632 into the high-sugar DMEM / reduced serum DMEM mixed nutrient solution, mix well, add cells to be cultured, and then mix well with the collagen solution , adjust the pH to 7.5 with 0.1M NaOH, wherein the volume ratio of high-sugar DMEM and reduced-serum DMEM in the high-sugar DMEM / reduced serum DMEM mixed nutrient solution is 5:1, and the final concentration of each component in the cell culture medium is: Type 1 collagen 12mg / ml, laminin 3.2mg / ml, type 4 collagen 0.1mg / ml, sodium dihydrogen phosphate 1%, hyaluronic acid 1mg / ml, heparin 0.9mg / ml, A83-01 0.9ng / ml, Y27632 15uM / ml, EGF 10ng / ml, FGF 100n...

Embodiment 2

[0037] This embodiment provides a method for preparing an organoid culture matrix material, comprising the following steps:

[0038] (1) Dissolving collagen type 1, collagen type 4, laminin, hyaluronic acid, and heparin with 1% acetic acid solution to obtain a collagen solution, and storing it on an ice box;

[0039] (2) Add sodium dihydrogen phosphate, EGF, FGF, A83-01, and Y27632 into the high-sugar DMEM / serum-reduced DMEM mixed nutrient solution, mix well and then mix with the collagen solution, adjust the pH to 7.5 Obtained, wherein the volume ratio of high-sugar DMEM and reduced-serum DMEM in the mixed nutrient solution of high-sugar DMEM / reduced serum DMEM is 5:1, and the final concentration of each component in the cell culture medium is: collagen type 18 mg / ml, Laminin 1.5mg / ml, type IV collagen 1mg / ml, sodium dihydrogen phosphate 0.1%, hyaluronic acid 5mg / ml, heparin 0.5mg / ml, A83-01 1.9ng / ml, Y27632 8uM / ml, EGF 100ng / ml, FGF 10ng / ml.

Embodiment 3

[0041] This embodiment provides a method for preparing an organoid culture matrix material, comprising the following steps:

[0042] (1) Dissolving collagen type 1, collagen type 4, laminin, hyaluronic acid, and heparin with 1% acetic acid solution to obtain a collagen solution, and storing it on an ice box;

[0043](2) Add sodium dihydrogen phosphate, EGF, FGF, A83-01, and Y27632 into the high-sugar DMEM / serum-reduced DMEM mixed nutrient solution, mix well and then mix well with the collagen solution, and adjust the pH to 7.5 Obtained, wherein the volume ratio of high-sugar DMEM and reduced-serum DMEM in the mixed nutrient solution of high-sugar DMEM / reduced serum DMEM is 5:1, and the final concentration of each component in the cell culture medium is: collagen type 15 mg / ml, Laminin 2mg / ml, type IV collagen 0.5mg / ml, sodium dihydrogen phosphate 0.5%, hyaluronic acid 3mg / ml, heparin 0.6mg / ml, A83-01 1.2ng / ml, Y27632 10uM / ml, EGF 50ng / ml, FGF 50ng / ml.

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Abstract

The invention provides an organoid culture medium material and a preparation method and application thereof. The preparation method of the organoid culture medium material comprises the following steps: dissolving type I collagen, type IV collagen, laminin, hyaluronic acid and heparin with an acetic acid solution to obtain a collagen solution; and adding sodium dihydrogen phosphate, growth factors, A83-01 and Y27632 into a high-glucose DMEM / serum-reduced DMEM mixed nutrient solution, uniformly mixing the substances, uniformly mixing the mixture with the collagen solution, and regulating the pHvalue to 7.2-8.3 by using NaOH, thereby obtaining the product. When the organoid culture medium material is used for culturing partial types of organoids, the cell proliferation rate is higher than the cell proliferation rate existing when Matrigel is used as the medium material for culturing, the dryness of the organoids can be better maintained in the organoid culture and passage process, and the price is far lower than that of a general organoid culture material Matrigel.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an organoid culture substrate material and a preparation method and application thereof. Background technique [0002] One of the key advances in stem cell research over the past decade has been the development of organoid systems. Organoids are three-dimensional (3D) cell cultures that contain some of the key properties of their representative organs. These in vitro culture systems include a population of self-renewing stem cells that can differentiate into multiple organ-specific cell types, possess similar spatial organization to, and reproduce some of the functions of, corresponding organs, thereby providing a highly physiologically relevant system . Organoids can be generated from tissue samples containing adult stem cells, from single adult stem cells, or through directed induced differentiation of pluripotent stem cells. Because some organoid model syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0671C12N5/0677C12N5/0679C12N5/0688C12N5/0693C12N2500/12C12N2501/11C12N2501/115C12N2501/15C12N2501/405C12N2513/00C12N2533/52C12N2533/54C12N2533/70C12N2533/80
Inventor 黄敏尹天武徐丛廖传荣
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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