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Method for preparing three-dimensional cell scaffold for in-vitro toxicological evaluation of tobacco products and method for cell culture by use of three-dimensional cell scaffold

A three-dimensional cell, tobacco product technology, applied in epidermal cells/skin cells, biochemical equipment and methods, cell culture supports/coatings, etc., to improve adhesion and proliferation, convenient toxicology evaluation experiments, and evaluation objective results

Active Publication Date: 2018-02-09
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, there is no report on the preparation of three-dimensional cell scaffolds for in vitro toxicological evaluation of tobacco products in cell culture dishes through high internal phase emulsions.

Method used

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  • Method for preparing three-dimensional cell scaffold for in-vitro toxicological evaluation of tobacco products and method for cell culture by use of three-dimensional cell scaffold

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Embodiment 1

[0037] First, the oil phase was prepared to consist of 4ml of styrene, 0.5ml of divinylbenzene, and 2.4g of SPAN 80, and then the water phase was prepared to consist of 64ml of water, 0.5g of calcium chloride and 0.65g of potassium persulfate. Under high-speed stirring at 500 rpm, the water phase was slowly added to the oil phase to form a high internal phase emulsion, which was poured into a cell culture dish with a diameter of 36 mm, a height of 2 mm, and sealed after 10 min of nitrogen. It was then polymerized in an oven at 60°C for 24 hours. The samples were extracted by Soxhlet method with ethanol and water as solvent for 24 h and then dried in vacuum. L-type polylysine with a molecular weight of 70,000 to 150,000 and a concentration of 0.1 mg / ml was added to the vacuum-dried materials, and soaked for 24 hours. Then sterilized in a high-pressure steam cooker. The sterilized samples were placed in a 6-well plate, and 2 ml of 1640 medium containing 10% fetal bovine serum a...

Embodiment 2

[0039] First, the oil phase was prepared to consist of 4ml of styrene, 0.5ml of divinylbenzene, and 2.4g of SPAN 80, and then the water phase was prepared to consist of 64ml of water, 0.5g of calcium chloride and 0.65g of potassium persulfate. Under high-speed stirring at 500 rpm, the water phase was slowly added to the oil phase to form a high internal phase emulsion, which was poured into a cell culture dish with a diameter of 36 mm, a height of 2 mm, and sealed after 10 min of nitrogen. It was then polymerized in an oven at 60°C for 24 hours. The samples were extracted by Soxhlet method with ethanol and water as solvent for 24 h and then dried in vacuum. L-type polylysine with a molecular weight of 30,000 to 70,000 and a concentration of 0.1 mg / ml was added to the vacuum-dried materials, and soaked for 24 hours. Then sterilized in a high-pressure steam cooker. The sterilized samples were placed in a 6-well plate, and 2 ml of 1640 medium containing 10% fetal bovine serum an...

Embodiment 3

[0041] First, the oil phase was prepared to consist of 4ml of styrene, 0.5ml of divinylbenzene, and 2.4g of SPAN 80, and then the water phase was prepared to consist of 64ml of water, 0.5g of calcium chloride and 0.65g of potassium persulfate. Under high-speed stirring at 500 rpm, the water phase was slowly added to the oil phase to form a high internal phase emulsion, which was poured into a cell culture dish with a diameter of 36 mm, a height of 2 mm, and sealed after 10 min of nitrogen. It was then polymerized in an oven at 60°C for 24 hours. The samples were extracted by Soxhlet method with ethanol and water as solvent for 24 h and then dried in vacuum. D-type polylysine with a molecular weight of 30,000 to 70,000 and a concentration of 0.1 mg / ml was added to the vacuum-dried materials, and soaked for 24 hours. Then sterilized in a high-pressure steam cooker. The sterilized samples were placed in a 6-well plate, and 2 ml of 1640 medium containing 10% fetal bovine serum an...

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Abstract

A method for preparing a three-dimensional cell scaffold for in-vitro toxicological evaluation of tobacco products is characterized in that an interconnected and porous material provided with a polymer is obtained through high-internal-phase emulsion polymerization in a cell culture dish, the material has large holes of the size of cells for in-vitro toxicological evaluation of tobacco products, the holes are communicated with one another, thereby facilitating input of oxygen and nutrients required for cell growth and output of cell metabolism waste, and the material surface is modified, so that the material has good cell affinity. Compared with existing panel type monolayer cell culture methods commonly adopted for toxicological evaluation, the method has the advantages that cells can realize three-dimensional growth on the scaffold and have more closer three-dimensional shape and biochemical and functional properties as compared with that originally in vivo. Therefore, when the cellscultured by use of the three-dimensional cell scaffold are used for in-vitro toxicological evaluation of the tobacco products, the evaluation result is more objective and real. The cell scaffold is prepared in the culture dish and is more convenient for following experiments.

Description

technical field [0001] The invention belongs to the technical field of preparation of three-dimensional cell scaffolds, in particular to a preparation of three-dimensional cell scaffolds for in vitro toxicology evaluation of tobacco products and a method for using the three-dimensional cell scaffolds for cell culture. Background technique [0002] At present, the in vitro toxicity evaluation of tobacco products generally adopts the monolayer cell culture method, which has the advantages of simple culture, easy operation, low cost, and can be widely used. However, in monolayer cell culture, cells grow on the plane of the attached substrate. Due to the lack of three-dimensional scaffolds, they can only develop in two dimensions and cannot generate extracellular matrix. Moreover, due to the lack of specific growth factors and differentiation factors in vivo, cells can only develop in two dimensions. Can not differentiate, thus losing the three-dimensional shape of the original ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F212/08C08F212/36C08F2/30C08F220/18C08F220/06C08F222/14C08F220/14C08F220/32C08J9/42C08J9/26C12N5/071C12N5/09
CPCC08F2/30C08F212/08C08F220/14C08F220/32C08F220/325C08J9/26C08J9/42C08J2201/0444C08J2325/08C08J2325/14C08J2333/12C08J2333/14C12N5/0625C12N5/0693C12N2513/00C12N2533/30C08F222/103C08F220/1808C08F220/06C08F222/102
Inventor 杨松李茹洋孙培健孙学辉贾云祯王宜鹏秦亚琼聂聪
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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