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Culture solution, culture reagent combination and culture method for intestinal cancer organs

An organoid and culture medium technology, applied in the field of colorectal cancer organoid culture medium, can solve the problems of long time, low cell volume, slow growth during the culture process, etc. Effect

Active Publication Date: 2022-04-12
BEIJING DAXIANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are some research reports on intestinal cancer organoid culture medium at present, the culture process grows slowly, which increases the time input cost
[0003] During the research and development process, at least the following problems were found in related technologies: In the culture process of existing intestinal cancer organoids, each passage needs to be cultured for at least 7 days, or even more days, to meet the passage requirements
Moreover, the amount of cells obtained is also very small. In order to harvest more cells and conduct downstream experiments, it takes longer, which makes the construction process of the entire organ slow, resulting in high time costs for bowel cancer organoid research.

Method used

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  • Culture solution, culture reagent combination and culture method for intestinal cancer organs
  • Culture solution, culture reagent combination and culture method for intestinal cancer organs
  • Culture solution, culture reagent combination and culture method for intestinal cancer organs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Colon cancer organoid culture medium

[0107] A intestinal cancer organoid culture medium, including conditional medium, basic medium, compound antibiotics and growth factors. Wherein, the volume ratio of conditioned medium and basal medium is 7:3; in conditioned medium, by volume percentage, including Wnt-3a conditioned medium 20%, R-spondin1, R-spondin3 conditioned medium 50% and Noggin The conditioned medium is 30%, and the volume ratio of the three is 0.5:1.5:1. Basal media consisted of Advanced DMEM / F12, HEPES 10 mM and Glutamax 1x. Compound antibiotics include Primocin 100 μg / mL, penicillin-streptomycin 1x and metronidazole 4 μg / mL.

[0108] In Example 1, the types and contents of the growth factors are shown in Table 1 below, so as to obtain different colon cancer organoid culture fluids correspondingly.

[0109] Table 1

[0110]

[0111] For the intestinal cancer organoid culture medium of Example 1, the ingredients can be mixed in proportion. ...

Embodiment 2

[0125] The colorectal cancer organoid culture solution of Example 2 is different from Example 1 in that the conditioned medium includes R-spondin1, R-spondin3 conditioned medium and Noggin conditioned medium; and the R-spondin1, R-spondin3 The volume ratio of the conditioned medium to the Noggin conditioned medium was 1.5:1. That is, by volume ratio, R-spondin1, R-spondin3 conditioned medium 30%, Noggin conditioned medium 20%, Advanced DMEM / F12 50%. The rest of the Advanced DMEM / F12 and growth factors were the same.

[0126] Similarly, according to the same types and contents of the growth factors in Example 1, five kinds of intestinal cancer organoid culture fluids were also provided, which were denoted as culture fluid 2-I, culture fluid 2-II, culture fluid 2-III, and culture fluid 2-IV and medium 2-V.

[0127] In this Example 2, R-spondin1, R-spondin3 conditioned medium and Noggin conditioned medium were obtained by the preparation method from Step S101 to Step S105 in Ex...

Embodiment 3

[0128] Example 3 Colon cancer organoid culture reagent combination

[0129] A colon cancer organoid culture reagent combination, comprising enzymatic hydrolysis solution and the colon cancer organoid culture solution of embodiment 1 or embodiment 2. Among them, the enzymatic hydrolysis solution includes basal medium, type I collagenase, type III collagenase and Primocin; wherein, the concentration of type I collagenase is 0.1-2 mg / mL, and the concentration of type III collagenase is 0.1-1 mg / mL and the concentration of Primocin is 0.2-2mg / mL. Intestinal cancer organoid culture medium and enzymatic hydrolysis solution are packaged separately.

[0130] In Example 3, the basal medium in the enzymolysis solution is Advanced DMEM / F12.

[0131] According to the different concentrations of type Ⅰ collagenase, type Ⅲ collagenase and Primocin, five kinds of enzymatic hydrolysis solutions as shown in Table 3 below were respectively obtained.

[0132] table 3

[0133]

[0134] In...

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Abstract

The invention relates to the technical field of biological tissue engineering, and discloses an intestinal cancer organ culture solution, a culture reagent combination and a culture method, and the intestinal cancer organ culture solution comprises a conditioned culture medium, a basal culture medium, a compound antibiotic and a growth factor. Wherein the compound antibiotic is prepared from Primocin, penicillin-streptomycin and metronidazole; the growth factors comprise an epidermal growth factor, N2, B27, acetylcysteine, nicotinamide, Gastrin, Prostaglandin E2, Y-27632, HGF (Hepatitis Growth Factor), IL-6 (Interleukin-6) and SDF1 (Sodium Dodecyl Factor 1). The components of the culture solution have a synergistic effect, so that the formation and growth of intestinal cancer organ cells are accelerated, and the culture speed is increased while the cell harvesting quantity is increased. The method is suitable for culturing human intestinal cancer organs, such as small intestinal cancer, rectal cancer, colon cancer and the like.

Description

technical field [0001] The present application relates to the technical field of biological tissue engineering, for example, to a colon cancer organoid culture medium, a culture reagent combination and a culture method. Background technique [0002] Intestinal cancer, including colon cancer and rectal cancer, is one of the top ten malignant tumors in my country. At present, the etiology of colon cancer is not yet fully understood, and it is currently considered to be the result of the combined effects of environmental factors and genetic factors. Bowel cancer organoids, for example, colorectal cancer organoids, are an advantageous model for studying bowel cancer. Although there are some research reports on intestinal cancer organoid culture medium, the culture process grows slowly, which increases time investment and cost. [0003] During the research and development process, at least the following problems were found in related technologies: in the culture process of exis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/071
Inventor 肖荣荣李珮文刘建闯孙艳廷张晓会周宇
Owner BEIJING DAXIANG BIOTECH CO LTD
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