Establishment of humanized three-dimensional intestinal mucosa model and application thereof

A technology of intestinal mucosa and humanization, applied in the field of medicine, can solve problems such as the undiscovered three-dimensional intestinal mucosa model

Active Publication Date: 2013-08-21
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after searching the literature, there is still no relevant report on the construction of a thr

Method used

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  • Establishment of humanized three-dimensional intestinal mucosa model and application thereof
  • Establishment of humanized three-dimensional intestinal mucosa model and application thereof
  • Establishment of humanized three-dimensional intestinal mucosa model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Constructing a three-dimensional intestinal mucosal model with polarity using the Transwell system

[0031] as follows figure 1 As shown, mesenchymal cells (such as fibroblasts) were seeded in extracellular matrix (such as collagen), and after incubation for 2 hours, the extracellular matrix was solidified, and a layer of mixed epithelial cells was planted on it, that is, a certain proportion of Caco- 2 cells and mucus-secreting cells (such as HT29-MTX cells), after two weeks of co-culture, a layer of immune cells (such as macrophages derived from THP-1 cells) was inoculated on the basal side of the Transwell. After the above four kinds of cells were co-cultured for one week, the model was completely differentiated, and the transmembrane electrical resistance value (TEER) was measured with a resistivity meter to determine the tightness and integrity of the epithelial cell layer, and then it could be used for the transmembrane transport experiment of drugs.

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Embodiment 2

[0034] Example 2 Using the three-dimensional model to study the membrane-permeability of passive paracellular transport marker compounds

[0035] Fluorescent yellow was used as a marker compound for paracellular transport, and the Caco-2 monolayer cell model was used as a control. The three-dimensional model was established according to the above method. Transmembrane electrical resistance (TEER) detects the tightness and integrity of the epithelial cell layer, and the TEER value reaches 250Ω·cm 2 The 3D model of can be used for transport experiments. Add 0.5ml of 20μg / ml fluorescent yellow liquid to the culture medium on the top side of the cell layer (villi side, side A), and add 1.5ml blank buffer solution to the bottom side (basal side, side B) at the same time to start the transport experiment, respectively At 30 min, 60 min, 90 min, and 120 min, 100 μl of the drug solution was taken from the bottom side, and 100 μl of blank buffer solution was supplemented at the same t...

Embodiment 3

[0036] Example 3 Using the three-dimensional model to study the membrane-permeability of marker compounds passively transported across cells

[0037] Propranolol was used as a marker compound for passive transcellular transport, and the Caco-2 monolayer cell model was used as a control. The three-dimensional model was established according to the above method. During the culture period, the medium was changed every other day until about 21 days after incubation, and the epithelial cells were completely differentiated. At the same time, the tightness and integrity of the epithelial cell layer were detected by measuring the transmembrane electrical resistance (TEER), and the TEER value reached 250Ω·cm 2 The 3D model of can be used for transport experiments. Add 0.5ml of 50μM propranolol drug solution to the culture medium on the top side of the cell layer (villi side, side A), and add 1.5ml blank buffer solution to the bottom side (basal side, side B) at the same time to start t...

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Abstract

The invention provides an establishment of humanized three-dimensional intestinal mucosa model and an application thereof. The model improves a traditional Caco-2 monolayer cell model mainly based on normal physiological structure characteristics of small intestinal epithelium, wherein on basis of adding extracellular matrix, the Caco-2 cells are co-cultured with mucus-secreting cells, interstitial cells and immune cells for a period of time to form a three-dimensional intestinal mucosa model being similar with structure and function of the small intestinal epithelium in vivo. By using the model, a membrane permeating capability at small intestine epithelium as well as a metabolism degree of oral drug molecules can be predicted. The model is mainly used for researching transmembrane transportation and absorption, and metabolism process of medicament molecules at the small intestine in vitro.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to the establishment and application of a humanized three-dimensional intestinal mucosa model. Background technique [0002] Oral administration, as a traditional way of administration, is widely accepted by people because of its relative convenience and safety. The absorption process is one of the key factors determining the bioavailability of orally administered drugs. The small intestine is the main site of oral drug absorption, so at present, various in vitro and in vivo intestinal absorption models are mainly used at home and abroad to study the transmembrane transport process of drugs in the small intestine, so as to predict their oral absorption. Among them, the Caco-2 monolayer cell model is one of the in vitro models widely used to study the biomembrane permeability and transmembrane transport mechanism of drug molecules. The composition of this model system i...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
Inventor 杨凌李娜王秀丽
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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