Intestinal cancer organoid culture medium, culture reagent combination and culture method

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: 2022-05-27
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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  • Intestinal cancer organoid culture medium, culture reagent combination and culture method
  • Intestinal cancer organoid culture medium, culture reagent combination and culture method
  • Intestinal cancer organoid culture medium, culture reagent combination and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Intestinal cancer organoid culture medium

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

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

[0109] Table 1

[0110]

[0111] The colorectal cancer organoid culture medium of the present Example 1 can be mixed...

Embodiment 2

[0125] The intestinal cancer organoid culture solution of this 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 and 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% and Noggin conditioned medium 20%, Advanced DMEM / F12 50%. The rest of Advanced DMEM / F12 and growth factors were the same.

[0126] Similarly, according to the same type and content of the growth factors in Example 1, 5 kinds of intestinal cancer organoid culture medium were also provided, which were recorded as culture medium 2-I, culture medium 2-II, culture medium 2-III, culture medium 2-IV and culture medium 2-V.

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

Embodiment 3

[0128] Example 3 Intestinal cancer organoid culture reagent combination

[0129] A colorectal cancer organoid culture reagent combination, comprising an enzymatic hydrolysis solution and the colorectal cancer organoid culture solution of Example 1 or Example 2. Wherein, 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 was 0.2 to 2 mg / mL. Intestinal cancer organoid culture solution and enzymatic hydrolysis solution are packaged separately.

[0130] In Example 3, the basal medium in the enzymatic hydrolysis solution was Advanced DMEM / F12.

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

[0132] table 3

[0...

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Abstract

The application relates to the technical field of biological tissue engineering, and discloses a colon cancer organoid culture solution, a culture reagent combination and a culture method. The colon cancer organoid culture solution includes a conditioned medium, a basal medium, compound antibiotics and growth factors. Among them, compound antibiotics include Primocin, penicillin-streptomycin, and metronidazole; growth factors include epidermal growth factor, N2, B27, acetylcysteine, nicotinamide, Gastrin, Prostaglandin E2, Y-27632, HGF, IL ‑6, SDF1. The synergistic effect between the components of the culture medium accelerates the formation and growth of intestinal cancer organoid cells, increases the number of harvested cells and also improves the culture speed. It is suitable for the culture of human-derived intestinal cancer organoids, such as small intestinal cancer, rectal cancer, colon cancer, etc.

Description

technical field [0001] The present application relates to the technical field of biological tissue engineering, for example, to a colorectal cancer organoid culture solution, a combination of culture reagents 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 colorectal cancer is not completely clear, and it is believed that it is mainly the result of the combined effect of environmental factors and genetic factors. Bowel cancer organoids, eg, colorectal cancer organoids, are an advantageous model for studying bowel cancer. Although there are some research reports on the culture medium of intestinal cancer organoids, the growth of the culture process is slow, which increases the time investment cost. [0003] During the research and development process, it was found that there are at least the following problems in the related tech...

Claims

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Application Information

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Patent Type & Authority Patents(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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