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A kind of cell stationary phase based on microcarrier cells and preparation method thereof

A cell immobilization and microcarrier technology, applied in the field of cell membrane chromatography, can solve the problems that the medium has a certain influence and the cells are toxic, and achieve the effect of improving specificity and large specific surface area.

Active Publication Date: 2016-04-27
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The selection of microcarriers should be based on the principle of being conducive to cell adherence and non-toxicity. However, ion-exchange gels have many disadvantages as carriers, such as being toxic to cells to a certain extent, and also have a certain impact on the pH of the medium. Therefore, in recent years The carrier has been improved. There are three main types of carriers currently in use. They are macromolecules formed by cross-linking dextran with different groups. )

Method used

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  • A kind of cell stationary phase based on microcarrier cells and preparation method thereof
  • A kind of cell stationary phase based on microcarrier cells and preparation method thereof
  • A kind of cell stationary phase based on microcarrier cells and preparation method thereof

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preparation example Construction

[0030] The preparation method of the cell stationary phase based on microcarrier cells provided comprises the following operations:

[0031] 1) Place the microcarriers in a siliconized culture vessel, activate and sterilize, then add DMEM medium overnight, discard the original medium before use, and replace with warm DMEM medium at 37°C to make a microcarrier suspension ;

[0032] 2) Put the microcarrier suspension into the culture container, inoculate the cell suspension, and then add DMEM culture solution; place the culture container upright in CO 2 Cultivate in the incubator for 2 to 3 hours, remove the rotation and shake for 1 to 2 minutes; then shake once every 1 to 2 hours, and replace the medium every 12 to 24 hours;

[0033] 3) After the cells adhere to the wall and grow on the microcarrier, take the cell suspension in the logarithmic phase of the adherent growth on the microcarrier, add paraformaldehyde solution with a mass concentration of 4%, fix at room temperatur...

Embodiment 1

[0037] The preparation of the FGFR4 cell chromatographic column based on microcarrier cells comprises the following operations:

[0038] 1) Processing of microcarriers

[0039] Pretreatment of glassware: wash and dry all glassware used for pretreatment of microcarriers, soak in 5% simethicone-ethyl acetate solution for uniform siliconization for 4 hours, pick up and dry in the air, and use ultra- Wash 3 times with pure water, soak overnight, and dry for later use.

[0040] Microcarrier Cytodex treatment: Weigh 0.2g microcarrier Cytodex into a siliconized beaker, wash twice with 20mL calcium and magnesium-free PBS (PH=7.4), replace with fresh PBS and soak overnight, autoclave, discard PBS , replace with fresh DMEM medium (containing 0.3mg / l G418, 10% FBS) and soak overnight, discard the original medium before use, replace with a proper amount of warm medium at 37°C, and adjust the medium content in the bottle The content is 20mg / ml.

[0041] Treatment of the surface of the c...

Embodiment 2

[0055] The difference from Example 1 is that human umbilical vein endothelial cells (ECV304) were used instead of fibroblast growth receptor (FGFR4) cells, ECV304 cells were cultured in DMEM medium, and other operations were prepared according to Example 1 to prepare ECV304 cell chromatography column.

[0056] Further, use the prepared ECV304 chromatographic column to analyze the retention of emodin on the ECV304 cell chromatography, as follows:

[0057] Preparation of emodin reference solution: take an appropriate amount of emodin, add methanol to dissolve and set the volume to 10ml, and obtain a concentration of 0.5mg / ml.

[0058] Mobile phase: 0.8%NaH 2 PO 4 -0.974%Na 2 HPO 4 (20:80), wherein NaCl 0.432g is added per 100ml;

[0059] Chromatographic column: 10mm×1mm;

[0060] Flow rate: 0.2ml / min; detection wavelength: 254nm.

[0061] Test results such as image 3 As shown, wherein a is emodin (blank microcarrier), b is emodin (microcarrier with cells), and c is metha...

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Abstract

The invention provides a cell stationary phase based on microcarrier cells and a preparation method of the cell stationary phase. Cells are cultured on a microcarrier surface; after being immobilized by paraformaldehyde, cells cultured by a microcarrier and the microcarrier supporting the growth of the cells are used as the stationary phase; a cell chromatographic column is prepared by wet-process packing. The method is applied to prepare the stationary phase, operation is simple, reactions are gentle and amplification is easy. The stationary phase has the characteristics of cells and keeps original base characteristics of the cells; a drug acceptor also keeps a base space structure; the cell stationary phase prepared by applying the method can be applied to screening drugs and improving drug screening specificity.

Description

technical field [0001] The invention belongs to the technical field of cell membrane chromatography, and relates to a cell stationary phase based on microcarriers and a preparation method thereof. Background technique [0002] Cell membrane chromatography (CMC) is a bio-affinity chromatography method proposed by Professor He Langchong and his research group in 1996, which is suitable for the screening and separation of active ingredients in complex systems of traditional Chinese medicine. After mixing with silica gel, the chromatographic column is filled to prepare a cell membrane chromatographic column, combined with high performance liquid chromatography to screen the active ingredients in Chinese medicinal materials. The target of drug screening is the receptor on the cell membrane. Cell membrane chromatography can be directly extracted from the complex components of traditional Chinese medicine. Identifying active ingredients that can interact with drug targets is a fast...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12N11/04C12Q1/02
Inventor 王嗣岑贺浪冲黄静张涛张杰李西玲魏芬
Owner XI AN JIAOTONG UNIV
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