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Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method

A culture method and organoid technology, which is applied in the field of pig liver and spleen ductal stem cell isolation and three-dimensional organoid culture, can solve the problems of organoid activity and organoid proliferation ability, lack of ductal stem cell isolation, and obvious batch effect, etc. Achieve the effect of high organoid formation rate, high specificity advantage, and good cell activity

Active Publication Date: 2021-03-23
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

However, for domestic animals such as pigs, there is no separation technology and special medium for ductal stem cells based on the physiological, biochemical and developmental characteristics of pig liver and spleen
Although some people try to use mouse liver organoid medium for porcine liver organoid culture, the organoid formation rate, organoid activity and organoid proliferation ability cannot meet the basic nutritional and drug screening test requirements, and the batch effect is obvious

Method used

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  • Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method
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  • Pig liver and spleen duct stem cell separation and three-dimensional organoid culture method

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Embodiment 1

[0028] Isolation of pig liver and spleen ductal stem cells and three-dimensional organoid culture method:

[0029] A. Two-step dissociation of porcine liver and spleen tissue to obtain ductal stem cells

[0030] See Table 1 for the components and proportions of the pig liver and spleen tissue dissociation solution:

[0031] Table 1

[0032]

[0033] (1) Preparation before the experiment: a. According to Table 1, prepare 50 mL of tissue dissociation solution, and store it at 4°C for later use. Before use, the dissociation solution should be preheated to room temperature (15-25°C). b. Use 5 mL of anti-adherence rinsing solution (Anti-Adherence Riinsing Solution, STEMCELL) to completely cover and rinse the inside of a 15 mL sterile enzyme-free centrifuge tube (4 in total), and then discard the rinsing solution; use 15 mL of Advanced DMEM / F12 to completely wash Rinse, then discard all the liquid; in the same way, take 20mL of anti-adhesion rinsing solution and 20mL of Advance...

Embodiment 2

[0053] Weaning pig spleen organoid culture method:

[0054] (1) Take a portion of the spleen tissue of fresh 21-day-old weaned piglets in cold (4°C) DPBS, and isolate spleen duct cells based on the two-step method proposed by the present invention;

[0055] (2) Preheat the 24-well cell culture plate in the incubator before the experiment, and pre-cool the 200 μL pipette tip in the refrigerator at 4°C for at least 30 minutes; thaw the Matrigel on ice in advance;

[0056] (3) Use a pre-cooled 200 μL pipette tip to pipette 30 μL of thawed Matrigel to the isolated ductal cells described in Part A, slowly blow up and down to mix 5 to 8 times, and mix well to avoid air bubbles;

[0057] (4) Set the pipette to 50 μL, transfer all the suspension in step (2) to a small well of a 24-well plate and form a dome structure, and slowly drop to the center of the small well to avoid disturbance or formation of air bubbles; repeat step, the remaining ductal cells are processed;

[0058] (5) P...

Embodiment 3

[0062] Culture method of weaned piglet liver organoids:

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Abstract

The invention discloses a pig liver and spleen duct stem cell separation and three-dimensional organoid culture method. The method comprises two parts of dissociating pig liver and spleen tissue witha two-step method to obtain duct stem cells and carrying out three-dimensional culture on pig liver and spleen organoids. According to the three-dimensional organoid culture method based on pig liverand spleen duct stem cell separation, the technical problem of pig liver and spleen organoid establishment is solved, and the application of the organoid technology in animal nutrition research is promoted.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for separating pig liver and spleen ductal stem cells and culturing three-dimensional organoids. Background technique [0002] The liver and spleen are the important hubs of nutritional metabolism and immune regulation in animals, and have a strong self-repair ability, which is mainly driven by (lgr5-labeled) ductal stem cells. Based on the totipotency of ductal stem cells, mouse and human liver organoid culture techniques have emerged in recent years. Compared with mouse or human liver and spleen cell lines, organoids can better reproduce biological phenotypes at the tissue level, and have very broad prospects in pathology research and drug screening. For the field of animal nutrition research, due to the lack of mature liver and spleen cell lines from livestock for a long time, many nutrition and immune research cannot be carried out, and are in a tec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/071
CPCC12N5/0672C12N5/0671C12N5/0648C12N2509/00C12N2509/10C12N2513/00C12N2500/90C12N2501/405C12N2500/32C12N2500/38C12N2500/46C12N2501/345C12N2501/115C12N2501/119C12N2501/15C12N2501/415C12N2501/11C12N2533/90C12N2501/998
Inventor 周健熊霞万丹邵宜锐吴宇梁印遇龙
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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