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Method for constructing Caco-2/HUVEC cell co-culture system

A co-cultivation system and co-cultivation technology, applied in artificial cell constructs, cell culture active agents, animal cells, etc.

Inactive Publication Date: 2017-08-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

There is no report on the in vitro cell model that fully reflects the bioavailability of drugs by combining the Caco-2 cell model for the study of absorption with the HUVEC cell model for the study of pharmacological functions

Method used

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  • Method for constructing Caco-2/HUVEC cell co-culture system

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Embodiment 1

[0021] Use DMEM medium (containing 10% fetal bovine serum, 1% non-essential amino acid, 1% glutamine, 100U / mL penicillin, 100μg / mL streptomycin, pH7.4) for Caco-2 cells of passage 25-40 At 37°C, 5% CO 2 The environment was cultivated, the medium was changed every other day, the cells were digested with 0.25% trypsin / 0.53mmol / L EDTA solution every 3 days, and the cells were passaged at a ratio of 1:3. Cells in the logarithmic growth phase were divided into 1×10 5 / mL was inoculated on the upper layer of 24-well Transwell plate, cultured for 21 days, and the resistance value of Caco-2 cell layer measured by potentiometer TEER was greater than 550Ω·cm 2 , the cells are fully differentiated. HUVEC cells of passages 2 to 6 were incubated with DMEM medium (containing 10% fetal bovine serum, 100 μg / mL vascular endothelial growth factor, 2% penicillin-streptomycin double antibody solution, pH 7.4) at 37°C, 5% CO 2 The environment was cultivated, the medium was changed every other ...

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Abstract

The invention discloses a method for constructing a Caco-2 and HUVEC cell co-culture system. The method comprises inoculating an upper small cell of a Transwell plate with 25 to 40 passages of Caco-2 cells in a logarithmic phase, carrying out culture until the cells are completely differentiated, inoculating a lower small cell of the Transwell plate with 2 to 6 passages of HUVEC cells in a logarithmic phase, carrying out culture until 80-90% of the lower small cell is filled with the cells, inserting the completely differentiated Caco-2 cell Transwell upper small cell into the cultured HUVEC cell Transwell lower small cell, fine adjusting a DMEM medium and carrying out non-contact co-culture at 37 DEG C in 5% of CO2. The Caco-2 / HUVEC co-culture system realizes simultaneous research of absorption, metabolism and physiological activity of drugs, simulates the pharmacological action of the drug in the circulatory system after absorption and metabolism in the intestinal tract and provides a cell model for showing the bioavailability of the drug.

Description

[0001] 1. Technical field [0002] The invention relates to a method for constructing a co-culture system of Caco-2 and HUVEC cells, belonging to the field of biotechnology. [0003] 2. Background technology [0004] Human colon adenocarcinoma cell lines (Caco-2 cells for short) have the same microvilli structure and tight junctions as small intestinal epithelial cells, and contain metabolic enzymes, sugars, amino acids, P450s related to small intestinal brush border epithelium Isozymes and various active transport carriers are classic models for studying intestinal absorption and transport mechanisms. [0005] Human umbilical vein endothelial cells (HUVEC for short) are venous vascular endothelial cells isolated from normal human umbilical cords, which contain a large number of cytoplasmic inclusions, smooth muscle actomyosin, and ABG antigens. Function, can produce and secrete many biologically active substances, play an important role in maintaining vasomotor, anticoagulant...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071
CPCC12N5/069C12N5/0693C12N2500/32C12N2501/165C12N2502/28C12N2502/30
Inventor 黄午阳张钟元闫征吴寒李大婧刘春泉
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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