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Preparation of a three-dimensional cell scaffold for in vitro toxicological evaluation of tobacco products and method for cell culture using it

A three-dimensional cell, tobacco product technology, applied in biochemical equipment and methods, epidermal cells/skin cells, cell culture supports/coatings, etc. objective results

Active Publication Date: 2019-07-12
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • 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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  • Preparation of a three-dimensional cell scaffold for in vitro toxicological evaluation of tobacco products and method for cell culture using it
  • Preparation of a three-dimensional cell scaffold for in vitro toxicological evaluation of tobacco products and method for cell culture using it

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Experimental program
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Effect test

Embodiment 1

[0037] Prepare the oil phase first, the composition is 4ml styrene, 0.5ml divinylbenzene, 2.4g SPAN 80, and then prepare the water phase, the composition is 64ml water, 0.5g calcium chloride and 0.65g potassium persulfate. Under high-speed stirring at 500rpm, 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 36mm and a height of 2mm, and sealed after 10 minutes of nitrogen gas. It was then placed in an oven at 60° C. for 24 hours to polymerize. The samples were extracted by Soxhlet extraction with ethanol and water as solvents for 24 hours, and then dried in vacuum. Add L-polylysine with a molecular weight of 70,000 to 150,000 and a concentration of 0.1 mg / ml to the vacuum-dried materials, and soak for 24 hours. Then sterilized with a high-pressure steam pot, the sterilized samples were placed in a 6-well plate, 2ml of 1640 medium containing 10% fetal bovine serum and 1% doubl...

Embodiment 2

[0039] Prepare the oil phase first, the composition is 4ml styrene, 0.5ml divinylbenzene, 2.4g SPAN 80, and then prepare the water phase, the composition is 64ml water, 0.5g calcium chloride and 0.65g potassium persulfate. Under high-speed stirring at 500rpm, 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 36mm and a height of 2mm, and sealed after 10 minutes of nitrogen gas. It was then placed in an oven at 60° C. for 24 hours to polymerize. The samples were extracted by Soxhlet extraction with ethanol and water as solvents for 24 hours, and then dried in vacuum. Add L-polylysine with a molecular weight of 30,000 to 70,000 and a concentration of 0.1 mg / ml to the vacuum-dried materials, and soak for 24 hours. Then sterilized with a high-pressure steam pot, the sterilized samples were placed in a 6-well plate, 2ml of 1640 medium containing 10% fetal bovine serum and 1% double...

Embodiment 3

[0041] Prepare the oil phase first, the composition is 4ml styrene, 0.5ml divinylbenzene, 2.4g SPAN 80, and then prepare the water phase, the composition is 64ml water, 0.5g calcium chloride and 0.65g potassium persulfate. Under high-speed stirring at 500rpm, 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 36mm and a height of 2mm, and sealed after 10 minutes of nitrogen gas. It was then placed in an oven at 60° C. for 24 hours to polymerize. The samples were extracted by Soxhlet extraction with ethanol and water as solvents for 24 hours, and then dried in vacuum. Add D-type polylysine with a molecular weight of 30,000 to 70,000 and a concentration of 0.1 mg / ml to the vacuum-dried materials, and soak for 24 hours. Then sterilized with a high-pressure steam pot, the sterilized samples were placed in a 6-well plate, 2ml of 1640 medium containing 10% fetal bovine serum and 1% d...

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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 three-dimensional cell scaffold preparation, in particular to a method for preparing a three-dimensional cell scaffold for in vitro toxicological evaluation of tobacco products and using it for cell culture. Background technique [0002] At present, the monolayer cell culture method is generally used in the in vitro toxicity evaluation of tobacco products. It has the advantages of simple culture, easy operation, low cost, and large-scale application. It is widely used as an experimental model for in vitro toxicology research of tobacco products. 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 to two dimensions and cannot generate extracellular matrix. Unable to differentiate, thus losing the three-dimensional shape of the original body. Therefore, the biological characteristics reflected by monola...

Claims

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Application Information

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Patent Type & Authority Patents(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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