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Low-cost mouse intestinal organ culture medium and culture method

A culture method and culture medium technology, applied in the field of cell biology, can solve the problem of less mouse intestinal organoids, etc., and achieve the effects of low cost and convenient production

Pending Publication Date: 2020-11-10
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been some reports on organoid culture methods of various human-derived tissues, there are few studies and reports on the culture methods of mouse intestinal organoids.

Method used

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  • Low-cost mouse intestinal organ culture medium and culture method
  • Low-cost mouse intestinal organ culture medium and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] A mouse intestinal organoid medium comprising the following components:

[0056] DMEM / F12 medium: 90%;

[0057] 293T-HA-Rspo1-Fc cell supernatant: 5%;

[0058] Vitamin A-free B27 additive: 2%;

[0059] Acetyl cysteine ​​amide 0.2μmol / L;

[0060] Prostaglandin E2 0.1μmol / L;

[0061] Mouse epidermal growth factor 10ng / mL;

[0062] Selective small molecule inhibitor A83-01 1μmol / L;

[0063] ROCK inhibitor Y27632 5 μmol / L;

[0064] Gastrin I 5ng / mL;

[0065] Penicillin: 100U / mL;

[0066] Streptomycin: 100 μg / mL;

[0067] Amphotericin B 0.5 μg / mL.

[0068] A method for culturing mouse intestinal organoids is as follows:

[0069] (1) Take fresh mouse intestines for pretreatment to obtain a cell mass of 3 cells, centrifuge to remove the supernatant, and obtain a cell mass precipitate;

[0070] (2) Take an appropriate amount of the above medium and mix it with Matrigel, then use the mixture to resuspend the cell pellet obtained in step (1), and then use a pipette to dr...

Embodiment 2

[0075] A mouse intestinal organoid medium comprising the following components:

[0076] DMEM / F12 medium: 70%;

[0077] 293T-HA-Rspo1-Fc cell supernatant: 25%;

[0078] Vitamin A-free B27 additive: 4%;

[0079] Acetyl cysteine ​​amide 2μmol / L;

[0080] Prostaglandin E2 0.5μmol / L;

[0081] Mouse epidermal growth factor 60ng / mL;

[0082] Selective small molecule inhibitor A83-01 5 μmol / L;

[0083] ROCK inhibitor Y27632 25 μmol / L;

[0084] Gastrin I 25ng / mL;

[0085] Penicillin: 200U / mL;

[0086] Streptomycin: 100 μg / mL;

[0087] Amphotericin B 2.5 μg / mL.

[0088] A method for culturing mouse intestinal cells, comprising the following steps:

[0089] (1) Take fresh mouse intestines for pretreatment, after obtaining a cell mass with a cell number of 30 cells, centrifuge to remove the supernatant, and obtain a cell mass precipitate;

[0090] (2) Take an appropriate amount of the above medium and mix it with Matrigel, then use the mixture to resuspend the cell pellet obtai...

Embodiment 3

[0095] A mouse intestinal organoid medium comprising the following components:

[0096] DMEM / F12 medium: 85%;

[0097] 293T-HA-Rspo1-Fc cell supernatant: 5%;

[0098] Vitamin A-free B27 additive: 10%;

[0099] Acetyl cysteine ​​amide 5μmol / L;

[0100] Prostaglandin E2 1μmol / L;

[0101] Mouse epidermal growth factor 100ng / mL;

[0102] Selective small molecule inhibitor A83-01 10μmol / L;

[0103] ROCK inhibitor Y27632 50μmol / L;

[0104] Gastrin I 50ng / mL;

[0105] Penicillin: 500U / mL;

[0106] Streptomycin: 500 μg / mL;

[0107] Amphotericin B 5 μg / mL.

[0108] A method for culturing mouse intestinal cells, comprising the following steps:

[0109] (1) Take fresh mouse intestines for pretreatment, after obtaining a cell mass with a cell number of 50 cells, centrifuge to remove the supernatant, and obtain a cell mass precipitate;

[0110] (2) Take an appropriate amount of mouse intestinal cell culture medium and mix it with matrigel, then use the mixture to resuspend the c...

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Abstract

The invention relates to a low-cost mouse intestinal organ culture medium and a culture method, and belongs to the technical field of cell biology. The culture medium for the mouse intestinal organs mainly comprises a basal culture medium, a 293T-HA-Rspo1-Fc cell supernatant and an additive. The culture method of the mouse intestinal organs comprises the following steps: pretreating mouse intestines to prepare cell clusters; resuspending the cell clusters by using the mixed solution of the mouse intestinal organ culture medium and matrix gel; and inoculating the cell clusters into a culture medium for culturing. The cytokines added into the culture medium are all mouse sources and are closer to the normal growth state in vivo, so that growth of organoid in a morphological structure can bemaintained. Besides, the 293T-HA-Rspo1-Fc cell supernatant is used for replacing an expensive cell factor RSpondin 1, so that the cost is low, and large-scale production is facilitated.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a low-cost mouse intestinal organoid culture medium and a culture method. Background technique [0002] Humans have used mouse models for biological and medical research for more than 100 years. Mice are small in size, short in reproduction cycle, large in number of litters, easy to raise, and rich in genetic resources; the genetic similarity between mice and humans is as high as 95%, and they have a human-like immune system and are easy to carry out genetic engineering and cell engineering transformation. These advantages determine the unique advantages of the mouse model in the biological medical research center. However, even though the mouse animal model has many advantages, it still has disadvantages such as complicated modeling process, long modeling time, difficult operation, high hardware requirements and high modeling cost. [0003] Organoids are in vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0679C12N2500/32C12N2501/02C12N2501/11C12N2501/15C12N2501/405C12N2501/345C12N2513/00C12N2533/90
Inventor 黄敏徐丛李刚
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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