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Construction method for human intestinal tract epithelial cell model

A technology of intestinal epithelial cells and construction methods, applied in the field of cell model construction, can solve problems such as long time required, difficulty in large-scale application, complicated cell inoculation process, etc.

Active Publication Date: 2019-12-31
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this model is that the cell inoculation process is complicated and takes a long time, and additional mechanical devices (peristaltic pumps, vacuum controllers) need to be added during the culture process, making it difficult to apply on a large scale

Method used

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  • Construction method for human intestinal tract epithelial cell model
  • Construction method for human intestinal tract epithelial cell model
  • Construction method for human intestinal tract epithelial cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] (1) Preparation of intestinal cell suspension: first prepare DMEM complete medium: 88% DMEM high glucose medium + 10% FBS + 1% double antibody + 1% non-essential amino acid. Take the purchased Caco-2 cells stored in liquid nitrogen, melt them in a 37°C water bath, add 10 times the complete DMEM medium dropwise, mix well, and centrifuge at 1200rpm for 5min; discard the supernatant, and transfer to a centrifuge tube Add DMEM complete medium to resuspend the cells, count and adjust the cell density to l×l0 6 cell / mL, then inoculated into culture flasks, and placed the culture flasks at 37°C, 95% humidity, 5% CO 2 Concentration in the incubator for static culture (replace the culture medium every two days). Until the Caco-2 cells were cultured until the cell confluence reached 80-90%, the intestinal cell suspension was collected, and the intestinal cell concentration in the intestinal cell suspension was adjusted to (2.0-3.0)×10 7 cell / mL is the obtained intestinal cell s...

Embodiment 2

[0093] (1) Preparation of intestinal cell suspension: first prepare DMEM complete medium: 88% DMEM high glucose medium + 10% FBS + 1% double antibody + 1% non-essential amino acid. Take the purchased Caco-2 cells stored in liquid nitrogen, melt them in a 37°C water bath, add 10 times the complete DMEM medium dropwise, mix well, and centrifuge at 1200rpm for 5min; discard the supernatant, and transfer to a centrifuge tube Add DMEM complete medium to resuspend the cells, count and adjust the cell density to l×l0 6 cell / mL, then inoculated into culture flasks, and placed the culture flasks at 37°C, 95% humidity, 5% CO 2 Concentration culture in the incubator (replace the culture medium every other day). Until the Caco-2 cells were cultured until the cell confluence reached 80-90%, the intestinal cell suspension was collected, and the intestinal cell concentration in the intestinal cell suspension was adjusted to (2.0-3.0)×10 7 cell / mL is the obtained intestinal cell suspension....

Embodiment 3

[0100] (1) Preparation of intestinal cell suspension: first prepare DMEM complete medium: 88% DMEM high glucose medium + 10% FBS + 1% double antibody + 1% non-essential amino acid. Take the purchased Caco-2 cells stored in liquid nitrogen, melt them in a 37°C water bath, add 10 times the complete DMEM medium dropwise, mix well, and centrifuge at 1200rpm for 5min; discard the supernatant, and transfer to a centrifuge tube Add DMEM complete medium to resuspend the cells, count and adjust the cell density to l×l0 6cell / mL, then inoculated into culture flasks, and placed the culture flasks at 37°C, 95% humidity, 5% CO 2 Concentration in the incubator for static culture (replace the culture medium every two days). Until the Caco-2 cells were cultured until the cell confluence reached 80-90%, the intestinal cell suspension was collected, and the intestinal cell concentration in the intestinal cell suspension was adjusted to (2.0-3.0)×10 7 cell / mL is the obtained intestinal cell su...

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Abstract

The invention discloses a construction method for a human intestinal tract epithelial cell model, and relates to the technical field of cell model construction. The human intestinal tract epithelial cell model is characterized in that a Caco-2 cell is taken as a model cell, and a DMEM (dulbecco's modified eagle medium) containing 20%FBS (fetal calf serum) is used for carrying out 3D culture on theCaco-2 cell in a micro-fluidic chip so as to construct the in vitro model of the human intestinal tract epithelial cell. On one hand, a human cell is adopted for culture, the problem that an experiment result is in accurate since an animal cell is adopted for culture is avoided, and on the other hand, the 3D cell culture enables intestinal tract cells to own a mechanical microenvironment which ismore similar to the human physiology, so that intestinal tract cells can be accelerated to differentiate. Therefore, the human intestinal tract epithelial cell model constructed by the construction method disclosed by the invention can more clearly and accurately reflect the characteristics of small intestine epithelial barriers.

Description

technical field [0001] The invention relates to the technical field of cell model construction, in particular to a method for constructing a human intestinal epithelial cell model. Background technique [0002] Currently, there are two methods for constructing human intestinal epithelial cell models, namely the Transwell model and the Gut-on-a-Chip intestinal organ model. [0003] The Transwell model was proposed by borchardt and workers in 1989. Obtained the certification of the US FDA. Caco-2 cells were isolated from human intestinal tumors, and intestinal absorption and metabolism were studied in vitro by culturing these cells in standard plastic dishes or Transwell inserts. Although these cultures formed an epithelial monolayer barrier for molecular transport, the cells exhibited a nonphysiological squamous epithelial morphology (rather than the columnar protrusions of the real physiological environment) and did not recapitulate most of the cellular differentiation of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N5/0679C12N2513/00
Inventor 李岩石万吴扬宫智勇吴永宁邓子逸姚丽云吴倩刘孙燕
Owner WUHAN CHOPPER BIOLOGY
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