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Primary intestinal cell based in-vitro intestinal model and construction method and application thereof

A technology of intestinal cells and construction methods, which is applied in the field of in vitro intestinal model and its construction based on primary intestinal cells, can solve the problem of inconvenient absorption, transport, metabolism and bioavailability of drugs and nutrients. Good simulation of intestinal interface absorption, difficulty in stem cell separation, etc., to achieve ideal stability and repeatability, strong proliferation and differentiation ability, and accurate evaluation results

Active Publication Date: 2019-10-25
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the separation of stem cells is difficult and costly
More importantly, the Lgr5+ intestinal epithelial stem cells are cultured in vitro to obtain spherical organoids, and the intestinal epithelial layer is distributed in the inner cavity of the formed closed spherical organoids, which is useful for subsequent evaluation of the absorption of drugs and nutrients at the intestinal interface , transportation, metabolism, and bioavailability have brought great inconvenience. The absorption experiment needs to be completed by microneedle injection under a microscope. The organoids formed by the stem cells cannot well simulate the intestinal interface. absorb

Method used

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  • Primary intestinal cell based in-vitro intestinal model and construction method and application thereof
  • Primary intestinal cell based in-vitro intestinal model and construction method and application thereof
  • Primary intestinal cell based in-vitro intestinal model and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Using electrospun cellulose acetate nanofiber membrane as the carrier material of primary intestinal cells to construct an in vitro intestinal model

[0047] In vitro intestinal models such as figure 1 As shown, including an outer container 1 and an inner container 3, the electrospun cellulose acetate nanofiber membrane material 5 with an average fiber diameter of 180 nm is fixed in the middle of the upper and lower chambers formed by the outer container 1 and the inner container 3 as the upper and lower chambers. Compartment material. The unpatterned morphology of electrospun cellulose acetate nanofibrous membrane materials such as image 3 As shown in (a), the patterned pit structure with a diameter of about 250 μm is as follows image 3 (b) shown.

[0048] The separation of small intestinal crypt cells is as follows: take the primary intestinal segment, wash it, cut it into pieces of about 1mm, transfer it into a 15ml centrifuge tube, add 10mL of EDTA (...

Embodiment 2

[0051] Example 2. Constructing an In vitro Intestinal Model Using Electrospun Chitosan / Collagen Nanofiber Membrane as the Carrier Material of Primary Intestinal Cells

[0052] In vitro intestinal models such as figure 1 As shown, it includes an outer container 1 and an inner container 3, and an electrospun chitosan / collagen nanofiber membrane material 5 with an average fiber diameter of 700 nm is fixed in the middle of the upper and lower chambers formed by the outer container 1 and the inner container 3 as the upper and lower chambers As for the interlayer material of the chamber, the electrospun chitosan / collagen nanofiber membrane material has pits of about 50 μm.

[0053] The separation of large intestine crypt cells is as follows: take the primary intestinal segment, clean it, cut it into pieces of about 1mm, transfer it into a 15ml centrifuge tube, add 10mL of EDTA (5mM) dissociation solution, and place it on a shaker for 20min (during Change fresh dissociation solution...

Embodiment 3

[0056] Example 3: Using electrospun polylactic acid / zein nanofiber membrane as primary intestinal cell carrier material to construct an in vitro intestinal model

[0057] In vitro intestinal models such as figure 1 As shown, including an outer container 1 and an inner container 3, the average fiber diameter of 1200 nm electrospun polylactic acid / zein nanofiber membrane material 5 is fixed in the middle of the upper and lower chambers formed by the outer container 1 and the inner container 3 as the upper and lower chambers compartment material.

[0058] The separation of small intestinal crypt cells is as follows: take the primary intestinal segment, wash it, cut it into pieces of about 1mm, transfer it into a 15ml centrifuge tube, add 10mL of EDTA (5mM) dissociation solution, and place it on a shaker for 50min (during Change fresh dissociation solution twice); wash 3 times with PBS, start to collect crypts while shaking (first collect with 100 μm filter membrane, then filter ...

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Abstract

The invention discloses a primary intestinal cell based in-vitro intestinal model and a construction method and application thereof. The method includes: taking an electrospun nanofiber film as a carrier material of intestinal cells, performing inoculation of separated intestinal cells to the surface of the carrier material, and culturing until a complete intestinal cell layer is formed and intestinal villi are differentiated, so that the in-vitro intestinal model with perfect bioactivity and physiological functions is obtained. On the basis of a three-dimension structure, similar to the structure of a natural extracellular matrix, of the electrospun nanofiber film, bionic simulation of the structure of the natural extracellular matrix is realized, and adhering and growing performances ofprimary intestinal cells on the surface of the carrier material are remarkably improved. The constructed model is capable of simulating the real intestinal tract and can be extensively used for analyzing absorption and transferring of nutrient substances and pharmaceutical components to quickly and accurately evaluate nutrient substance and medicine absorptivity and bioavailability, and the modelcan also be used for assessment of colonization or interaction of intestinal microorganisms and can be applied to tissue engineering.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro intestinal model based on primary intestinal cells and its construction method and application. Background technique [0002] The in vitro intestinal absorption model is widely used to evaluate the absorption of drugs and nutrients, and its absorbability data are used for rapid in vitro analysis and screening of early drug development and nutrient development. In the past ten years, cell models have been widely used at home and abroad as the intestinal absorption model of drugs and nutrients. Among them, the Caco-2 intestinal cell model is derived from human colon cancer cells. It is a famous intestinal absorption model and has been widely adopted by drug research institutions or development companies in developed countries. It is currently the most classic in vitro intestinal model. As an attachment-dependent cell, the most common culture model for Caco-2 ce...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0679C12N2533/00C12N2533/50C12N2533/70C12N2535/10G01N33/5005
Inventor 胡梦欣牛红梅
Owner ZHEJIANG GONGSHANG UNIVERSITY
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