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A method for constructing a three-dimensional cell culture microenvironment and its application

A three-dimensional culture and construction method technology, which is applied in the application of organ chips and the construction of three-dimensional cell culture microenvironment, can solve the problems of large damage to the active components of the extracellular matrix, long time to remove DNA, and complicated 3D forming methods. Achieve the effects of shortening the preparation time, low price, and retaining active ingredients

Active Publication Date: 2020-07-14
江苏艾玮得生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem is that it takes a long time to remove DNA by acid and alkali treatment, and the damage to the active components of the extracellular matrix is ​​large; the organic matter treatment also greatly damages the active components of the extracellular matrix
The biomaterial used in this method has good biocompatibility and universal applicability, but it cannot provide suitable support for specific cells, and the 3D molding method is complicated and requires special equipment
[0012] However, there are no reports on the use of specific extracellular matrix and modified gelatin to prepare hydrogels and use them as a three-dimensional culture microenvironment for cells, especially for organ chips.

Method used

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  • A method for constructing a three-dimensional cell culture microenvironment and its application
  • A method for constructing a three-dimensional cell culture microenvironment and its application
  • A method for constructing a three-dimensional cell culture microenvironment and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0059] (1) Select fresh pig heart, wash to remove fat, blood and other appendages, freeze at -20°C, cut into thin slices of about 500 μm with a scalpel on ice, stir and wash the thin slices with 0.01M PBS buffer solution for 15 minutes, repeat 3 times; after draining the flakes, stir them with 0.1% trypsin for 2 hours, treat them with 3% Triton X-100 for 2 hours, treat them with 1% SDS for 4 hours, and wash them with 0.01M PBS buffer solution for 15 minutes every 2 hours. Stir the 0.01MPBS mixed solution of 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1 hour, after the decellularization treatment is completed, stir and wash with deionized water, replace the deionized water every 15min, repeat 3 times, and detect that the conductivity is less than 10μs / cm Drain the matrix, freeze-dry it, and pulverize it into powder with a grinder. Take the powder and prepare 10mg / mL matrix mother solution with 0.1M hydrochloric acid solution containing 5mg / mL pepsin, shake at room temp...

Embodiment 2

[0061] (1) Select fresh pig lungs, wash and remove fat, blood and other appendages, freeze at -20°C, cut into slices of about 500 μm with a scalpel on ice, stir and wash the slices with 0.01M PBS buffer solution for 15 minutes, repeat 3 times; after draining the flakes, they were treated with 1% CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt) for 2h, 2% sodium deoxycholate for 4h, each treatment was 2h, Stir and wash with 0.01M PBS buffer solution for 15min, stir and treat with 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1h, after the decellularization treatment is completed, stir and wash with deionized water, replace it every 15min Ionized water, repeated 3 times, no surfactant in the washing was detected by high performance liquid chromatography; the matrix was drained of water, freeze-dried, and pulverized into powder with a grinder. Take the powder and prepare 10mg / mL matrix mother solution with 0.01...

Embodiment 3

[0063] (1) Select fresh pig lungs, wash and remove fat, blood and other appendages, freeze at -20°C, cut into slices of about 500 μm with a scalpel on ice, stir and wash the slices with 0.01M PBS buffer solution for 15 minutes, repeat 3 times; drain the flakes and treat them with 1% SDS for 4 hours, and wash them with 0.01M PBS buffer solution for 15 minutes every 2 hours, and then treat them with 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease 1h, the deionized water treatment is over, stir and wash with deionized water, replace the deionized water every 15min, repeat 3 times, and detect that the conductivity is less than 10μs / cm; drain the matrix, freeze-dry it, and pulverize it with a grinder powder. Take the powder and prepare 10mg / mL matrix mother solution with 0.01M hydrochloric acid solution containing 5mg / mL pepsin, shake at room temperature for 48h, centrifuge at 10000rpm / min, and take the supernatant; prepare 50mg / mL methacrylic a...

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Abstract

The invention discloses a microenvironment construction method for three dimensional culture of cells based on a specificity extracellular matrix and an application. A specificity extracellular matrix extraction method, the specificity extracellular matrix and a photocuring material are combined to prepare hydrogel, and the invention relates to an application of the hydrogel to in vitro three dimensional culture and particularly an organ chip technique. According to the technical scheme, in accordance with tissue or organs of different density, surfactants having different decellularization capacity are selected, through combination of the manner that enzyme treatment is selected and an ammonia solution is used as a solvent, particularly-compact tissue or organs are treated, and the preferred combination of the enzyme and surfactants comprises Trypsin and a Triton X-100 solution, and the solvent is a 0.05%-0.5% ammonia solution. The extracellular matrix and a light-initiating colloid-forming material are compounded for preparing the hydrogel so as to construct cell culture microenvironment. The method has the advantages of being high in colloid-forming speed, simple in colloid-forming condition, controllable in hydrogel mechanical property and the like.

Description

technical field [0001] The invention relates to the fields of biological materials and tissue engineering, in particular to the extraction of specific extracellular matrix and a method for constructing a three-dimensional cell culture microenvironment, and in particular to the application in organ chips. Background technique [0002] Tissue engineering is a discipline that is emerging in recent years and belongs to the category of high biotechnology. In 1993, Langer and Vacanti conceptually proposed the definition of tissue engineering: applying the principles and technologies of life science and engineering to develop biological substitutes for repairing, maintaining, and promoting tissue functions. It mainly involves three aspects: seed cells, tissue engineering scaffolds and tissue regeneration. [0003] Chinese invention patent 201410145791.7 discloses an acellular matrix repair gel and a new method for its preparation. Animal tissues and organs are inactivated with per...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/071C12N5/077C12N5/09
CPCC12N5/0062C12N5/0656C12N5/0657C12N5/069C12N5/0693C12N2513/00C12N2533/54C12N2533/72C12N2533/80C12N2533/90
Inventor 顾忠泽张静陈早早王菲孙晓玮葛健军
Owner 江苏艾玮得生物科技有限公司
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