Collagen coated microcarrier for animal cell culture and preparation method thereof

A collagen and microcarrier technology, applied in animal cells, biochemical equipment and methods, tissue culture, etc., can solve the problems of weak protein connection, biocompatibility and unsatisfactory cell culture effect, and achieve optimal cell culture. effect, reduction in coating amount, cost reduction effect

Active Publication Date: 2015-08-19
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

), providing a method for preparing a collagen-coated microcarrier with agarose as a matrix, this method is to coat the polyelectrolyte formed by gum arabic and gelatin as a raw material, and the coating molecules are physically Adsorbed on agarose microspheres, the protein connection is not strong, and the biocompatibility and cell culture effect are not ideal

Method used

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  • Collagen coated microcarrier for animal cell culture and preparation method thereof
  • Collagen coated microcarrier for animal cell culture and preparation method thereof
  • Collagen coated microcarrier for animal cell culture and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The preparation of embodiment 1 collagen coating microcarrier

[0064] Select konjac glucomannan microspheres with a particle size of 50 μm-100 μm, wash and dry them with a sand core funnel, weigh 20 g, put them in a three-necked flask, add 20 mmol of activating reagent 1,4-butanediol diglycidyl ether, and hydroborate Sodium 40mg, 0.1M NaOH solution 40mL, react at 300rpm at 25°C for 18h. After the reaction, the microspheres were washed and dried, and 10 mL of NaHCO with pH=8 was added 3 -Na 2 CO 3 5 g of fish gelatin with an average molecular weight of 5,000 was added to the buffer, and reacted at 200 rpm at 15°C for 24 hours to couple the protein. Then, after mixing the reacted system, add 100mL of water at 10°C, and rinse twice with PBS with pH=7. Put the treated microspheres into the flask, then add 20mmol of cross-linking reagent malondialdehyde and 40mL of PBS buffer, stir and react at 65°C for 0.5h, clean the microspheres with a sand core funnel after the reac...

Embodiment 2

[0065] The preparation of embodiment 2 collagen coating microcarriers

[0066] Select konjac glucomannan microspheres with a particle size of 100 μm-160 μm, wash and extract 20 g with a sand core funnel, place it in a three-necked flask, add 0.1 moL of activating reagent diepoxidized butadiene, 1 g of sodium borohydride, and 100 mL of 0.5M NaOH solution, React at 50 rpm at 65 °C for 0.5 h. After the reaction, the microspheres were washed and dried, and 80 mL of NaHCO with pH=10 was added 3 -Na 2 CO 3Add 5 g of bovine gelatin with an average molecular weight of 5,000 to the buffer solution, react at 300 rpm at 40° C. for 5 h, and couple the protein. Then, after mixing the reacted system, add 500 mL of deionized water at 5°C, and rinse twice with PBS with pH=7.2. The treated microspheres were put into the flask, then added the cross-linking reagent glutaraldehyde 0.2mol and PBS buffer 100mL, stirred and reacted at 15°C for 24h, cleaned the microspheres with a sand core funne...

Embodiment 3

[0067] The preparation of embodiment 3 collagen coated microcarriers

[0068] Select konjac glucomannan microspheres with a particle size of 160 μm-250 μm, wash and extract 20 g with a sand core funnel, put it in a three-necked flask, add 0.2 mol of activating reagent n-butyl glycidyl ether, 10 mg of sodium borohydride, and 20 mL of 1.5M KOH solution , reacted at 150rpm at 25°C for 16h. After the reaction, wash and dry the microspheres, add 100mL NaHCO with pH=12 3 -Na 2 CO 3 Add 1 g of porcine gelatin with an average molecular weight of 10,000 to the buffer solution, react at 50 rpm at 65° C. for 0.5 h, and couple the protein. Then the reacted system was mixed evenly, added dropwise into 1 L of deionized water at 0°C, and rinsed twice with PBS of pH=7.4. The treated microspheres were added to the flask, and then 50 mmol of cross-linking reagent succinaldehyde and 150 mL of PBS buffer were added, and the reaction was stirred at 50°C for 5 h. After the reaction, the microsp...

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Abstract

The invention discloses a collagen-coated micro-carrier used in animal cell culture, and a preparation method thereof. According to the micro-carrier provided by the invention, konjac glucomannan microspheres are adopted as a substrate, and are coated by using collagen. The micro-carrier preparation method comprises the steps that: an activating reagent and a catalyst are added into the substrate microspheres, and an activation reaction is carried out in an alkaline environment; the prepared microspheres are cleaned and are vacuum-dried; the microspheres are added into a buffering solution, and collagen is added, such that a coupling reaction is carried out; the system after reaction is well mixed, and is added into water, such that collagen is coated and agglomerated on the surfaces of the microspheres; and the microspheres are washed; the microspheres are added into a reaction vessel; a crosslinking reagent and a PBS buffering solution are added, and the mixture is stirred, such that a reaction is carried out; after reaction, the product is washed, such that the micro-carrier product is obtained. The micro-carrier has uniform collagen coating on the surface, and the collagen is firmly connected, such that cell rapid adhesion and high-density growth can be facilitated. Also, the prepared micro-carrier has the advantages of low cost, short period, and suitability for industrialized productions.

Description

technical field [0001] The method relates to a microcarrier for animal cell culture and a preparation method thereof, in particular to a collagen-coated microcarrier for animal cell culture and a preparation method thereof, belonging to the field of biochemical engineering. Background technique [0002] Microcarriers are a type of non-toxic, non-rigid, uniform density, and usually transparent small particles used in cell culture, which can enable anchorage-dependent cells to grow in a single layer on the particle surface during suspension culture, thereby increasing the number of cells. The attached growth area is conducive to the large-scale cultivation and collection of cells. Microcarrier culture is currently the main technology used for large-scale culture of animal cells. Large-scale cultivation of anchorage-dependent animal cells using microcarrier systems is the main production method adopted by biopharmaceutical companies at home and abroad, and is used for the prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/02C08J9/36C12N5/07
Inventor 马光辉周炜清康跻耀张贵峰苏志国孙李靖李娟
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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