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Method for detecting activation of microencapsulated cells

A technology of cell activity and detection method, which is applied in the field of detection of microencapsulated cell activity through energy charge calculation, can solve the problems of inapplicability, reduced extraction efficiency, and detection results lower than the actual value, etc., to achieve reliable measurement results and determination The effect of method stability

Inactive Publication Date: 2013-09-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

But this extraction method is not suitable for the culture system of special microencapsulated cells. The reasons are as follows: 1. The pH change in the extraction process affects the stability of adenylic acid, causing some products to decompose and make the test result lower than the actual value; 2. Interaction of microcapsule material and acid, reducing extraction efficiency

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  • Method for detecting activation of microencapsulated cells
  • Method for detecting activation of microencapsulated cells

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Experimental program
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Embodiment 1

[0020] A detection method for characterizing the activity of microencapsulated cells by energy charge

[0021] 1) Sample preparation:

[0022] A: Lysis solution: 50mM HEPES, (PH7.4), 50mAU / ml proteinase k, preheated in 50°C water bath.

[0023] B: Collect APA microencapsulated and cultured Hepg2 cells, filter out the culture medium, wash with pre-cooled PBS until the washing liquid is colorless, and then filter the liquid. A 1ml syringe is ground and extracted to break the capsule. After the microcapsule membranes were all damaged under the microscope, preheated lysis solution was added, incubated in a water bath at 50°C for 30 minutes, and shaken at 5-minute intervals. The reaction system was transferred to a water bath at 80°C for 15 minutes, and the supernatant was collected by filtration;

[0024] 2) Separation and quantification of adenylate:

[0025] The content of adenylate in the extracted samples was separated by high performance liquid chromatography. The measur...

Embodiment 2

[0028] A detection method for characterizing the activity of microencapsulated cells by energy charge

[0029] 1) Sample preparation:

[0030] A: Lysis solution: 50mM HEPES, (PH7.4), 50mAU / ml proteinase k, preheated in 50°C water bath.

[0031] B: Collect monomethoxy polyethylene glycol polylactic acid (PELA) microencapsulated microbial cells S. cerevisiae, filter out the culture medium, wash with pre-cooled PBS until the washing liquid is colorless, filter the liquid and add the preheated lysis buffer, Incubate in a 50°C water bath for 30 minutes with shaking at 5-minute intervals. The reaction system was transferred to a water bath at 80°C for 15 minutes, and the supernatant was collected by filtration;

[0032] 2) Separation and quantification of adenylate:

[0033] The content of adenylate in the extracted samples was separated by high performance liquid chromatography. The measurement conditions are UV detector, wavelength 260nm, C18 reversed-phase column, mobile phas...

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Abstract

The invention relates to a method for detecting activation of microencapsulated cells. The detection comprises the steps of sample extraction and phosphagen separation and quantification. The activation of the cell in a representation microcapsule is calculated through energy charge. The invention designs an operation scheme for the phosphagen quantification of microencapsulated cells to such special microcapsule system, which can accurately quantify the phosphagen content in the microencapsulated cells, and avoids the defects in the prior art such as large sample loss during extraction, unstable test, bad repeatability, etc., and the test results can be regarded as the evaluation indexes for the activation of the microencapsulated cells.

Description

technical field [0001] The invention relates to a detection method for cell activity, in particular to a detection method for characterizing the activity of microencapsulated cells through energy charge calculation. Background technique [0002] As a large-scale cell culture technology, microencapsulated cell culture is widely used in the fields of monoclonal antibody production, cell transplantation and cell expansion because of its special microencapsulated environment that enables cells to grow in three dimensions. [0003] The viability of microencapsulated cells during culture directly affects downstream productivity and application effects. At present, the microencapsulated cell viability was detected by thiazolyl blue (MTT) method. The method is to use intracellular mitochondrial succinate dehydrogenase to reduce exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit it in the cell, and then dissolve the crystal with dimethyl sulfoxide (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N1/28G01N30/06C12Q1/00
Inventor 马小军肖静王为于炜婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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