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Standardized culture medium and culture method for three-dimensionally culturing intestinal and intestinal cancer tissue organs

A three-dimensional culture and culture method technology, applied in biochemical equipment and methods, tissue culture, cell culture active agents, etc., can solve the problem of lack of standardized medium, and achieve the effect of meeting the needs of scientific research and suitable nutrients

Pending Publication Date: 2021-03-12
HAIXI NEW TEXTILE MATERIAL OF JINJIANG IND TECH ACAD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of lack of standardized culture medium in the process of intestinal organoid culture in vitro, the purpose of the present invention is to provide a standardized culture medium and culture method for three-dimensional culture of intestinal and intestinal cancer tissue organoids. The culture medium of the present invention is aimed at intestinal For the growth characteristics of adult stem cells in tissues, a variety of growth factor components are selected for reconciliation. After optimizing the ratio of growth factors in the medium, intestinal adult stem cells and intestinal cancer cells can effectively form intestinal cells in a three-dimensional culture environment. Organoids and bowel cancer organoids

Method used

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  • Standardized culture medium and culture method for three-dimensionally culturing intestinal and intestinal cancer tissue organs
  • Standardized culture medium and culture method for three-dimensionally culturing intestinal and intestinal cancer tissue organs
  • Standardized culture medium and culture method for three-dimensionally culturing intestinal and intestinal cancer tissue organs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] A standardized culture medium for three-dimensional culture of intestinal and intestinal cancer tissue organoids, including at least one or more of Reagent A, Reagent B and Reagent C;

[0063] Reagent A contains one or more of N-acetylcysteine ​​(N-acetylcysteine), Y-27632, A8301, recombinant human epidermal growth factor (EGF) and gastrin (Gastrin);

[0064] Reagent B contains one or more of R-spondin1, Noggin, Nicotinamide and SB202190;

[0065] Reagent C comprises Wnt-3A.

[0066] Preferably, in reagent A, based on the final volume of the standardized medium, the concentration of N-acetylcysteine ​​is 0.1~10mM (such as 1mM), and the concentration of Y-27632 is 0.5~50uM (such as 5uM ), the concentration of A8301 is 50~5000nM (such as 500nM), the concentration of recombinant human epidermal growth factor is 5~500ng / ml (such as 50ng / ml), and the concentration of gastrin is 1~100nM (such as 10nM).

[0067] Preferably, in reagent B, based on the final volume of the stan...

Embodiment 2

[0112] This example provides a standardized medium for three-dimensional culture of intestinal and intestinal cancer tissue organoids. The standardized medium is used to establish and expand human intestinal organoids. The formula of the standardized medium is as follows:

[0113] Reagent A:

[0114] Calculated based on the final volume of standardized medium containing the following components:

[0115] N-acetylcysteine ​​0.1~10mM (specifically 1mM)

[0116] Y-27632 0.5~50uM (specifically 5uM)

[0117] A8301 50~5000nM (for example, 500nM)

[0118] Recombinant human epidermal growth factor 5~500ng / ml (specifically 50ng / ml)

[0119] Gastrin 1~100nM (for example, 10nM);

[0120] Reagent B:

[0121] Calculated based on the final volume of standardized medium containing the following components:

[0122] R-spondin1 10%

[0123] Noggin 10%

[0124]Niacinamide 1~100mM (for example, 10mM)

[0125] SB202190 1~100uM (for example, 10uM);

[0126] Reagent C:

[0127] Wnt-3A 5%...

Embodiment 3

[0163] This example provides a standardized medium for three-dimensional culture of intestinal and intestinal cancer tissue organoids. The standardized medium is used for differentiating and culturing human intestinal organoids. The formula of the standardized medium is as follows:

[0164] Reagent A:

[0165] Calculated based on the final volume of standardized medium containing the following components:

[0166] N-acetylcysteine ​​0.1~10mM (specifically 1mM)

[0167] Y-27632 0.5~50uM (specifically 5uM)

[0168] A8301 50~5000nM (for example, 500nM)

[0169] Recombinant human epidermal growth factor 5~500ng / ml (specifically 50ng / ml)

[0170] Gastrin 1~100nM (for example, 10nM);

[0171] Basal medium:

[0172] Use Advanced F12 / DMEM as the solvent, and calculate the final volume of the basal medium to include the following components:

[0173] N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid 10~1000mM (specifically, 100mM)

[0174] L-alanyl-L-glutamine 20~2000mM (spe...

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Abstract

The invention discloses a standardized culture medium for three-dimensional culture of intestinal and intestinal cancer tissue organs and a culture method. The standardized culture medium comprises atleast one or more of a reagent A, a reagent B and a reagent C; wherein the reagent A comprises one or more of Nacetylcysteine, Y-27632, A8301, a recombinant human epidermal growth factor and gastrin;and the reagent B is prepared from one or more of R-spodin1, Noggin, nicotinamide and SB202190, and the reagent C comprises Wnt-3A. According to the culture medium disclosed by the invention, aimingat the growth characteristics of adult stem cells in intestinal tissues, various growth factor components are selected for blending, and by optimizing the proportion of growth factors in the culture medium, the adult stem cells of intestines and cells of intestinal cancer can effectively form organoids of intestines and organoids of intestinal cancer respectively in a three-dimensional culture environment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a standardized culture medium and a culture method for three-dimensionally culturing intestinal and intestinal cancer tissue organoids. Background technique [0002] Organoid technology is a major breakthrough in the field of biology in recent years. Through organoid culture technology, scientists have established a new in vitro culture model for culturing normal or diseased cancer tissues. Normal organoids can mimic the morphology, structure and functional characteristics of normal tissues; cancerous organoids exhibit the morphological characteristics, gene mutation spectrum and phenotypic characteristics of cancer in vivo. Normal and cancerous organoids can be stably cultured in vitro for a long time, so organoid culture technology can carry out most of the biological processes in vivo in petri dishes. This revolutionary technological advancement establishes a platform...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0679C12N5/0693C12N2501/11C12N2501/20
Inventor 李军李存郑云波
Owner HAIXI NEW TEXTILE MATERIAL OF JINJIANG IND TECH ACAD
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