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Non-trypsinization harvested cell culture method based on temperature sensitive hydrogel material

A temperature-sensitive hydrogel and cell culture technology, which is applied in the field of non-trypsinized harvested cell culture based on temperature-sensitive hydrogel materials, can solve the problems of cell function loss, cell membrane damage, weakening the application value of seed cells, etc., and achieve high viability Effect

Active Publication Date: 2019-07-26
HANGZHOU TUOAIMU MEDICAL INSTR CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such methods will cause damage to cell membrane proteins, resulting in damage to cell membranes, loss of cell surface markers, or partial loss of cell functions, which will weaken the application value of seed cells in the field of regenerative medicine

Method used

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  • Non-trypsinization harvested cell culture method based on temperature sensitive hydrogel material
  • Non-trypsinization harvested cell culture method based on temperature sensitive hydrogel material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1) Take 1.5g of chitosan powder, 1.75g ​​of N-isopropylacrylamide, 0.075g of ammonium persulfate with a viscosity-average molecular weight of 65w and a degree of deacetylation of 90%, and disperse them in 95 milliliters of deionized water, then, Add 0.2mL of hydrochloric acid, stir and dissolve evenly, react at 60°C for 8 hours, then use deionized water to dialyze the reaction product to neutrality, and vacuum freeze-dry to obtain poly-N-isopropylacrylamide modified chitosan powder ;

[0022] 2) Weigh 1.0 g of the poly-N-isopropylacrylamide modified chitosan powder obtained in step 1), disperse it in 25 milliliters of PBS solution, stir and dissolve to obtain poly-N-isopropylacrylamide modified chitosan powder with a mass concentration of 4%. Propylacrylamide modified chitosan aqueous solution;

[0023] 3) Mix 0.75mL of the poly-N-isopropylacrylamide-modified chitosan solution prepared in step 2) and 0.25mL of the cell suspension at room temperature, then add it to the...

Embodiment 2

[0027] 1) Take 1.5g of chitosan powder, 1.75g ​​of N-isopropylacrylamide, 0.075g of ammonium persulfate with a viscosity-average molecular weight of 65w and a degree of deacetylation of 90%, and disperse them in 95 milliliters of deionized water, then, Add 0.2mL of hydrochloric acid, stir and dissolve evenly, react at 60°C for 8 hours, then use deionized water to dialyze the reaction product to neutrality, and vacuum freeze-dry to obtain poly-N-isopropylacrylamide modified chitosan powder ;

[0028] 2) Weigh 1.0 g of the poly-N-isopropylacrylamide modified chitosan powder obtained in step 1), disperse it in 25 milliliters of PBS solution, stir and dissolve to obtain poly-N-isopropylacrylamide modified chitosan powder with a mass concentration of 4%. Propylacrylamide modified chitosan aqueous solution;

[0029] 3) Apply 0.75 mL of the poly-N-isopropylacrylamide-modified chitosan solution prepared in step 2) to the bottom of the culture dish at room temperature, and then place ...

Embodiment 3

[0031] 1) Take 1.5g of chitosan powder, 1.75g ​​of N-isopropylacrylamide, 0.075g of ammonium persulfate with a viscosity-average molecular weight of 65w and a degree of deacetylation of 90%, and disperse them in 95 milliliters of deionized water, then, Add 0.2mL of hydrochloric acid, stir and dissolve evenly, react at 60°C for 8 hours, then use deionized water to dialyze the reaction product to neutrality, and vacuum freeze-dry to obtain poly-N-isopropylacrylamide modified chitosan powder ;

[0032] 2) Weigh 1.5g of the poly-N-isopropylacrylamide modified chitosan powder obtained in step 1), disperse it in 30 milliliters of PBS solution, stir and dissolve to obtain poly-N-isopropylacrylamide modified chitosan powder with a mass concentration of 5%. Propylacrylamide modified chitosan aqueous solution;

[0033] 3) Mix 0.75mL of the poly-N-isopropylacrylamide-modified chitosan solution prepared in step 2) and 0.25mL of the cell suspension evenly at a lower temperature, then add ...

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Abstract

The invention discloses a non-trypsinization harvested cell culture method based on a temperature sensitive hydrogel material. The method includes first preparing a hydrogel solution by an emulsion polymerization method, wherein the hydrogel can maintain a solution state at the low temperature, and the hydrogel can be rapidly formed after approaching the physiological temperature of a human body.Based on the temperature sensitive hydrogel material, the method achieves non-enzymatic harvesting of cells by the following two schemes: 1) mixing a cell suspension and the hydrogel solution, placingthe mixture in a 37 DEG C cell incubator, adding a 37 DEG C medium for culture after the mixture forms the hydrogel rapidly and then releasing the cells by cooling; 2) first applying the hydrogel solution on the surface of a substrate, placing the hydrogel solution in the 37 o C cell incubator, adding the 37 DEG C cell suspension and the medium for culture after the hydrogel is formed and releasing the cells by cooling. According to the cell culture method, trypsinization is not required, a cell scraper is not required, cell surface proteins and markers can be well preserved to ensure high cell viability, and the method is a non-destructive cell culture method.

Description

technical field [0001] The invention relates to a cell harvesting method without trypsinization and a scraper, in particular to a method for harvesting cells without trypsinization based on a temperature-sensitive hydrogel material. Background technique [0002] Chitosan is an amino polysaccharide derived from nature. Its macromolecular chain is composed of acetylglucosamine and deacetylglucosamine through random or block distribution. It has good biocompatibility, blood compatibility, hemostatic performance, antibacterial Performance and natural drug activity can promote wound healing. It can be degraded by lysozyme in vivo, and the final degradation product is glucosamine, which is weakly alkaline, consistent with the pH value of body fluid, and can be absorbed by the body. and medical fields have great application potential. [0003] In the development and utilization of chitosan resources, hydrogel is an important form. Hydrogel materials have a variety of excellent pr...

Claims

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Application Information

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IPC IPC(8): C12N5/00C08J3/075C08F251/00C08F220/54C08L51/02
CPCC08F251/00C08J3/075C08J2351/02C12N5/00C12N2533/72C08F220/54
Inventor 胡巧玲董晓飞王征科乔丰慧姜质琦姚远
Owner HANGZHOU TUOAIMU MEDICAL INSTR CO LTD
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