Standardized culture medium and culture method for three-dimensional culture of lung and lung cancer tissue organoids

A three-dimensional culture and culture medium technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve the problems of lack of standardized culture medium, achieve the maintenance of tissue cell specificity, appropriate nutritional components, and meet the needs of scientific research Effect

Pending Publication Date: 2021-03-16
HAIXI NEW TEXTILE MATERIAL OF JINJIANG IND TECH ACAD +1
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  • Application Information

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Problems solved by technology

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

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  • Standardized culture medium and culture method for three-dimensional culture of lung and lung cancer tissue organoids
  • Standardized culture medium and culture method for three-dimensional culture of lung and lung cancer tissue organoids
  • Standardized culture medium and culture method for three-dimensional culture of lung and lung cancer tissue organoids

Examples

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Comparison scheme
Effect test

Embodiment 1

[0076] A standardized culture medium for three-dimensional culture of lung and lung cancer tissue organoids, including at least one or more of Reagent A and Reagent B;

[0077] Reagent A contains one or more of R-spondin1, Noggin, N-acetylcysteine ​​(N-acetylcysteine), Nicotinamide (Nicotinamide), Y-27632, A8301, SB202190, FGF-7 and FGF-10 ;

[0078] Reagent B comprises reagent B1, reagent B2 and reagent B3;

[0079] Reagent B1 contains Bovine Pitituary Extract (BPE), Y-27632, DAPT, Hydrocortisone, Insulin, Gentamicin, Amphotericin ), Retinoic Acid, Transferrin, Triiodothyronine, Epinephrine and human epidermal growth factor (hEGF);

[0080] Reagent B2 contains heparin (Heparin);

[0081] Reagent B3 contains one or more of DMEM / F12, L-ascorbic acid-2-phosphate magnesium and sodium selenium.

[0082] Preferably, in reagent A, based on the final volume of the standardized medium, the concentration of R-spondin1 is 10%, the concentration of Noggin is 10%, and the concentratio...

Embodiment 2

[0139] This example provides a standardized medium for three-dimensional culture of lung and lung cancer tissue organoids. The standardized medium is used to establish human lung organoids. The formula of the standardized medium is as follows:

[0140] Basal medium:

[0141] Use Advanced F12 / DMEM as solvent and contain the following ingredients:

[0142] N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid 10~1000mM (specifically, 100mM)

[0143] L-alanyl-L-glutamine 20~2000mM (specifically 200mM)

[0144] Penicillin 10000U / ml

[0145] Streptomycin 10000ug / ml;

[0146] Reagent A:

[0147] Calculated based on the final volume of standardized medium containing the following components:

[0148] R-spondin1 10%

[0149] Noggin 10%

[0150] N-acetylcysteine ​​0.125~12.5mM (specifically, 1.25mM)

[0151] Niacinamide 1~100mM (for example, 10mM)

[0152] Y-2763 2.5~250uM (specifically 25uM)

[0153] A8301 50~5000nM (for example, 500nM)

[0154] SB202190 0.1~10uM (for examp...

Embodiment 3

[0186] This example provides a standardized medium for three-dimensional culture of lung and lung cancer tissue organoids. The standardized medium is used to expand human lung organoids. The formula of the standardized medium is as follows:

[0187] Basal medium:

[0188] Use Advanced F12 / DMEM as solvent and contain the following ingredients:

[0189] N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid 10~1000mM (specifically, 100mM)

[0190] L-alanyl-L-glutamine 20~2000mM (specifically 200mM)

[0191] Penicillin 10000U / ml

[0192] Streptomycin 10000ug / ml;

[0193] Reagent A:

[0194] Calculated based on the final volume of standardized medium containing the following components:

[0195] R-spondin1 10%

[0196] Noggin 10%

[0197] N-acetylcysteine ​​0.125~12.5mM (specifically, 1.25mM)

[0198] Niacinamide 1~100mM (for example, 10mM)

[0199] Y-2763 2.5~250uM (specifically 25uM)

[0200] A8301 50~5000nM (for example, 500nM)

[0201] SB202190 0.1~10uM (for example,...

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Abstract

The invention discloses a standardized culture medium and a culture method for three-dimensional culture of lung and lung cancer tissue organoids. The standardized culture medium comprises at least one or more of a reagent A and a reagent B, wherein the reagent A comprises one or more of R-spondin1, Noggin, N-acetylcysteine, nicotinamide, Y-27632, A8301, SB202190, FGF-7 and FGF-10; the reagent B comprises a reagent B1, a reagent B2 and a reagent B3; the reagent B1 comprises one or more of bovine pituitary protein extract, Y-27632, DAPT, hydrocortisone, insulin, gentamicin, amphotericin, tretinoin, transferrin, iodomethacin, epinephrine and human epidermal growth factors; the reagent B2 comprises heparin; and the reagent B3 comprises one or more of DMEM / F12, L-ascorboic acid-2-phosphate magnesium and sodium selenium. According to the standardized culture medium and the culture method, aiming at the growth characteristics of adult stem cells in lung tissues, multiple growth factor components are selected for blending, and by optimizing the proportion of growth factors in the culture medium, normal lung tissues and lung cancer cells can effectively form lung and lung cancer organoidsin 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-dimensional culture of lung and lung 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 for the rese...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0688C12N5/0693C12N2501/11C12N2500/25C12N2500/34C12N2500/38C12N2501/71C12N2500/46C12N2500/36C12N2500/32C12N2501/119C12N2501/81C12N2501/91C12N2500/05C12N2500/12C12N2500/30
Inventor 郑小佳刘晓娟王佳佳
Owner HAIXI NEW TEXTILE MATERIAL OF JINJIANG IND TECH ACAD
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