Molecular culture medium for culturing small intestine organoids of pigs

A medium and organoid technology, which is applied in the field of molecular medium for culturing porcine intestinal organoids, can solve the problems of easy apoptosis, inability to differentiate, and prone to swelling, and achieves high specificity, stable repeat effect, and high plasticity. Effect

Active Publication Date: 2021-03-16
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method is to establish in vitro models based on porcine small intestinal epithelial cells IPEC~J2, IPEC~J1 and human colon cancer cells CACO~2 to investigate intestinal nutrient transport, metabolism and regulation, but the cell model has essential defects, mainly manifested as In three aspects: 1. Usually composed of single or multiple cell lines, it cannot truly reproduce the various cell types of intestinal villi, such as goblet cells, Paneth cells and endocrine cells, etc.
2. The specificity of intestinal tissue structure cannot be reproduced, and the unidirectional and complex nutrient transport at the tissue level cannot be simulated
3. Unable to reproduce intestinal crypt-villous axis nutrient pools and crypt stem cell microenvironment
However, there are still some problems in the use of mouse culture medium to cultivate porcine intestinal organoids: 1. The formation rate of organoids isolated from piglet intestinal crypt stem cells is low and they are prone to apoptosis
2. Cell composition and gene expression of organoids are different from porcine small intestine gene expression
3. Unable to repeatedly cryopreserve and resuscitate at high passages (more than 20 passages), prone to swelling and unable to differentiate

Method used

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  • Molecular culture medium for culturing small intestine organoids of pigs
  • Molecular culture medium for culturing small intestine organoids of pigs
  • Molecular culture medium for culturing small intestine organoids of pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Preparation of WNR-J2 Conditioned Medium

[0041] The components of JM medium and its selection medium are listed in Table 1.

[0042] Table 1

[0043]

[0044] The preparation steps of WNR-J2 conditioned medium are as follows:

[0045] (1) Preparation of JM medium: Add 100 mL of thawed pig serum (Porcine Serum, Gibco, 26250084) at about 4 °C to 395 mL of DMEM high-glucose medium (SIGMA, D6429), and then add 1% penicillin / streptomycin Double antibody (Penicillin~Streptomycin, Gibco, 15140122) is 5mL, mix thoroughly, and store at 4°C for later use;

[0046] (2) Take 10 mL of JM culture, preheat it in a 37°C water bath, and then transfer it to a T25 culture flask;

[0047] (3) Thaw WNR-J2 cells quickly in a water bath at 37°C, rotate the cryopreservation tube clockwise to melt, and stop the water bath when only soybean-sized ice cubes remain;

[0048] (4) Quickly transfer the cell solution to a T25 culture flask containing medium, at 37°C, 5% CO 2 Incubator, un...

Embodiment 2

[0073] Example 2 Isolation of crypts in different intestinal segments of weaned piglets and culture of 3D organoids

[0074] In this example, the basic molecular medium was used, prepared according to the proportion of 50% WNR-J2 conditioned medium and the recommended molecular concentration, without adding any inhibitors, and only adjusting the pH value to the recommended range for different intestinal segments.

[0075] (1) Thaw Matrigel on ice, the specific amount can be adjusted according to the actual situation. Preheat the TC 24-well culture plate in the cell culture incubator for at least 30 minutes;

[0076](2) Take 10 cm sections of duodenum, jejunum, and ileum of weaned piglets, cut them open and wash them with saline immediately, add 5 mL of cold (2-8°C) DPBS to a 10 cm round petri dish, and put them into the intestines. Use a glass slide to scrape off the fluff, then re-add 5 mL of cold DPBS, scrape off the crypt vigorously, and discard the smooth muscle layer. T...

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Abstract

The invention discloses a molecular culture medium for culturing small intestine organoids of pigs. The molecular culture medium consists of a WNR-J2 condition culture medium and a basal culture medium, and the volume percentage concentration of the WNR-J2 condition culture medium in the molecular culture medium is 50-60%, and the basic culture medium is composed of Advanced DMEM/F12, an HEPES buffer solution, a B27 serum substitute, gastrin I, N-acetyl-L-cysteine, niacinamide10M, L-glutamine, N2-MAX, ethanolamine and an EGF epidermal growth factor. The molecular culture medium is suitable forsmall intestine crypt separation and culture of suckling piglets, weaned piglets and fattening pigs, long-term passage and maintenance of the small intestine organs of the pigs and intestinal nutrition and disease modeling of the pigs.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a molecular culture medium for culturing porcine small intestine organoids. Background technique [0002] The development of intestinal epithelial cells and their nutritional regulation have always been the core issues in animal nutrition research, so much so that there is a saying in the industry that "raising pigs is raising intestines". The traditional method is to establish in vitro models based on porcine small intestinal epithelial cells IPEC~J2, IPEC~J1 and human colon cancer cells CACO~2 to investigate intestinal nutrient transport, metabolism and regulation. In three aspects: 1. It is usually composed of a single or multiple cell lines, and cannot truly reproduce the various cell types of intestinal villi, such as goblet cells, Paneth cells, and endocrine cells. 2. The specificity of intestinal tissue structure cannot be reproduced, and the unidirection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0679C12N2501/345C12N2500/32C12N2500/38C12N2500/46C12N2501/11C12N2501/15C12N2501/405
Inventor 熊霞周健邵宜锐吴宇梁印遇龙
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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