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In vitro 3D liver model, enterohepatic co-culture model, building methods of the models and applications of the models

A method and model building technology, applied in 3D culture, biochemical equipment and methods, tissue culture, etc., can solve problems such as lack of, lack of in vitro models, etc.

Pending Publication Date: 2019-03-05
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It can be seen that there is currently a lack of a liver model that simulates the actual physiological conditions of the liver in vivo, maintains liver function in vivo for a long time, and a stable combination of this model and an intestinal model, thus lacking an in vitro model that can accurately predict drug intestinal absorption and liver toxicity with high throughput. Models, especially multiple-dose and chronic-dose in vitro toxicity evaluation

Method used

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  • In vitro 3D liver model, enterohepatic co-culture model, building methods of the models and applications of the models
  • In vitro 3D liver model, enterohepatic co-culture model, building methods of the models and applications of the models
  • In vitro 3D liver model, enterohepatic co-culture model, building methods of the models and applications of the models

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The establishment of an in vitro 3D liver model comprises the following steps:

[0082] 1. Cell culture.

[0083] HepaRG cells (source: ATCC, purchased by Guangzhou Geneo Biological Co., Ltd.) and human hepatic stellate cells (Human Hepatic Stellate Cell, HHSC, source: ScienCell, purchased by Beijing Yuhengfeng Technology Co., Ltd.) The RPMI 1640 complete medium (containing 10% FBS) was used for cell culture, and the cells grew well and were used for future use.

[0084] Incubate the above cells at 37°C with 5% CO 2Culture in an incubator with a relative humidity of 90%, replace the medium every other day, and subculture after 2 to 3 days of adherent growth and cover 80% to 90% of the bottom of the bottle.

[0085] Second, cell induction.

[0086] exist T150 conventional culture bottle was inoculated with 2×10 6 HepaRG cells were incubated with 1640 complete medium containing 10% FBS at 37 °C in 5% CO 2 and cultured in an incubator with a relative humidity of 90...

Embodiment 2

[0120] The establishment of an in vitro intestinal-liver co-culture model, comprising the following steps:

[0121] 1. Establishment of intestinal absorption model.

[0122] 1. Model establishment.

[0123] 1.1. Prepare cell suspension: when MDCKII / Caco-2 cells grow to 80%-90% of the bottom of the bottle, digest with trypsin and stop digestion with culture medium, centrifuge at 1000rpm for 5min, and then use RPMI1640 containing 10% FBS Complete medium was prepared at a density of 2 × 10 6 cells / ml of cell suspension.

[0124] 1.2. Add fresh 1640 blank culture solution containing 10% FBS to each well on the side of the Transwell receiver pool (bottom well) (keep the pages of the receiver pool and the supply pool flush).

[0125] 1.3. Add the prepared cell suspension (equivalent to 1×10 6 cells / 1.12cm 2 ).

[0126] 1.4. Place at 37°C, 5% CO 2 Cultured in an incubator, the medium was replaced every other day.

[0127] 1.5. Measure the transmembrane resistance after 15 day...

experiment example 1

[0149] Albumin and urea secretion level experiments.

[0150] 1. Albumin secretion level.

[0151] At different time points of cell culture, the albumin expression levels in Example 1 and Comparative Example 1 were measured by albumin kit and high-content fluorescent labeling. The result is as Figure 10 shown.

[0152] The results show that after the hanging drop 3D liver model and scaffold 3D liver model prepared in Example 1 were cultured for 3 days, the secretion of albumin reached a higher level, and both were significantly higher than the 2D culture level (more than 2 times the same number of cells) . And this level has been maintained until 30 days after culture.

[0153] Second, the level of urea secretion.

[0154] At different time points of cell culture, the expression level of cells to albumin was measured with urea kit, and the results were as follows: Figure 11 shown.

[0155] The results showed that after 5 days of culture in the hanging drop 3D liver mo...

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Abstract

The invention relates to an in vitro 3D liver model, an enterohepatic co-culture model, building methods of the models and applications of the models, belonging to the technical field of drug evaluation. The building method of the liver model includes: a step of cell culture, namely a step of culturing HepaRG cells and human hepatic stellate cells respectively, and keeping the cells for later use;a step of cell induction, namely a step of inoculating the HepaRG cells into a culture disk to perform cell culture, after the amount of the HepaRG cells adherently grown reaches a predetermined amount, performing replacement with an induction medium to perform induction culture to obtain induced HepaRG cells, and keeping the induced HepaRG cells for later use; a step of model building, namely astep of preparing a mixed cell suspension from the induced HepaRG cells and the human hepatic stellate cells, inoculating the mixed cell suspension to a preset carrier, and performing culturing to obtain a micro-tissue having a hepatic globule three-dimensional structure, that is the 3D liver model. The liver model that is the micro-tissue having a hepatic globule three-dimensional structure can well simulate actual physiological condition of the liver in the body, thereby accurately predicting liver toxicity of a drug.

Description

technical field [0001] The invention relates to the technical field of drug evaluation, in particular to an in vitro 3D liver model and an intestinal-liver co-culture model and their establishment method and application. Background technique [0002] Drug-induced liver injury has been the main reason for acute liver injury and withdrawal of marketed drugs. However, due to differences in liver function between humans and animals, it is difficult to accurately predict drug metabolism and toxicity in preclinical animal experiments. In recent years, with the development of cell biology, many isolated human models have been applied to the study of drug hepatotoxicity, such as human liver microsomes, human hepatocytes, primary hepatocytes and so on. Human hepatocytes can be expanded and passaged, and have the functional characteristics of whole cells, and are widely used in drug toxicity screening. However, most immortalized hepatocytes lose some of the liver-specific metabolic f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
CPCG01N33/502G01N33/5044G01N33/5067C12N5/0671C12N5/0679C12N2502/23C12N2502/14C12N2513/00
Inventor 淡墨卢贤欢
Owner NAT INST FOR FOOD & DRUG CONTROL
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