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Thermosensitive microcarrier as well as preparation technology and application method thereof

A micro-carrier and temperature-sensitive technology, applied in biochemical equipment and methods, microorganisms, artificial cell constructs, etc., can solve the problems of cell surface protein damage, high cost, limited application, etc., to maintain cell activity, easy operation, The effect of less energy consumption

Inactive Publication Date: 2013-11-27
上海瀚正生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The existing cell culture microcarriers include commercial products such as Cytodex1 and Cytodex3, but the use of these traditional microcarriers has disadvantages such as high cost and damage to cell surface proteins when cells are digested, which greatly limits their application in the field of cell therapy

Method used

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  • Thermosensitive microcarrier as well as preparation technology and application method thereof
  • Thermosensitive microcarrier as well as preparation technology and application method thereof
  • Thermosensitive microcarrier as well as preparation technology and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. Preparation of thermosensitive microcarriers:

[0056] 1) Add 1.0 g of dextran microspheres (G50) and 10 mL of dimethyl sulfoxide (DMSO) to a 25 mL round bottom flask, add 1.0 g of 2-chloropropionyl chloride, add a magnet and stir for 1.5 h, wash with methanol After 3-5 times, chemically modified dextran microspheres containing a large number of chlorine atoms on the surface are obtained;

[0057] 2) N-isopropylacrylamide (NiPAAm) was grafted by free radical ATRP polymerization, the method is as follows: dextran microspheres containing a large number of chlorine atoms modified by chloromethylation on the surface were added to a 50mL round bottom flask containing magnetons, Add N isopropylacrylamide (NIPAAm) 2.0 g, copper chloride (CuCl 2 ) 0.34 g, tris-(N'N dimethylaminoethylamine) (Me6Tren) 0.46 g, 25 mL deionized water, under anaerobic environment, add ascorbic acid 0.13 g, react at room temperature for 24 h, then wash with water for 3- After drying for 5 times, ...

Embodiment 2

[0066] 1. Preparation of thermosensitive microcarriers:

[0067] 1) Add 1.0 g of sodium alginate microspheres, 10 mL of dimethyl sulfoxide (DMSO) and 3 g of 2-aminomethyl chloride into a 25 mL round bottom flask, add a magnet and stir for 1 h, wash with methanol for 3 After -5 times, the chemically modified sodium alginate microspheres containing a large amount of amino groups on the surface were obtained;

[0068] 2) N-isopropylacrylamide (NiPAAm) is grafted by free radical ATRP polymerization, the method is as follows: add the above-mentioned chemically modified sodium alginate microspheres containing a large number of amino groups on the surface into a 50mL round bottom flask containing magnetons, and add N isopropylacrylamide (NIPAAm) 2.0 g, copper bromide (CuBr 2 ) 0.52 g, tris-(N'N dimethylaminoethylamine) (Me6Tren) 0.46 g, 20 mL deionized water, in an oxygen-free environment, add ascorbic acid 0.13 g, react at room temperature for 24 h, and then wash with water for 3 ...

Embodiment 3

[0077] 1. Preparation of thermosensitive microcarriers:

[0078] 1) Add 5.0 g of chitosan microspheres, 30 mL of dimethyl sulfoxide (DMSO) and 5.5 g of 2-bromopropionyl bromide into a 100 mL round bottom flask, add a magnet and stir for 1 h, wash with methanol for 3- After 5 times, the chemically modified dextran microspheres containing a large amount of bromine atoms on the surface were obtained; the chemically modified dextran microspheres containing a large amount of bromine atoms on the surface were mixed with 3.0 g of potassium ethyl xanthate in 20 mL of dichloro In methane and 20 mL of methanol, react at room temperature for 24 h, wash with water three times and dry to obtain chitosan microspheres with a large amount of ethyl xanthate structure on the surface;

[0079] 2) N-isopropylacrylamide (NiPAAm) grafting reaction method by plasma treatment, the method is as follows: Add 3.0 g of chitosan microspheres with a large amount of ethyl xanthate structure on the surface a...

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Abstract

The invention discloses a thermosensitive microcarrier as well as a preparation technology and an application method thereof. The thermosensitive microcarrier can be used for amplifying, culturing and harvesting high-quality and high-activity cells used for clinical treatment, and the high-quality and high-activity cells are applied to the fields of cell therapy and tissue engineering. The preparation technology comprises the following steps of: grafting active groups by virtue of chemical surface treatment while a high polymer material cell culture microballoon sphere as a matrix, then carrying out reaction on the high polymer material cell culture microballoon sphere and thermosensitive polymer to graft the thermosensitive polymer, so as to obtain a thermosensitive microcarrier used for cell culture, and controlling the temperature during cell culture, so that cells grow and are amplified while being attached onto the surface of the thermosensitive microcarrier; then lowering temperature and haversting cells with complete surface protein. On one hand, a method for amplifying massive cells used for treatment in a short time, on the other hand, no pancreatin or traditional dissociative reagent is used, so that damage on surface protein and ion channel of cells is reduced, the original function of the cells is maintained, and the thermosensitive microcarrier has strong clinical application value and economic value.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to cell culture, in particular to a temperature-sensitive microcarrier used for amplifying culture and harvesting high-quality and high-activity cells for clinical treatment, as well as a preparation method and an application method thereof. Background technique [0002] Cell culture refers to the technology in which animal cells grow and proliferate in vitro and maintain their characteristics and functions under appropriate culture conditions. Most cells from solid animal tissues require adherent monolayer growth. As long as they are not converted to anchorage-independent, they must be attached and spread on a suitable solid medium before growth can begin. [0003] Traditional cell culture is to use cell plate for culture, the plate is processed for cell attachment and growth, a traditional plate provides 78.52cm 2 and can support 10 6 -10 7 attached growth of individual cells. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/071C12N5/0775C08J7/16C08J7/18
Inventor 齐念民冯见杜明明陈彦田
Owner 上海瀚正生物技术有限公司
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