Method for detecting activation of microencapsulated cells

A technology of cell activity and detection method, which is applied in the detection field of characterizing the activity of microencapsulated cells by energy charge calculation, can solve the problems of inapplicability, the detection result is lower than the actual value, and the stability of adenylate is affected, and the detection method can be achieved. Stable and reliable results

Inactive Publication Date: 2011-05-11
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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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 a 50°C water bath.

[0023] B: collect APA microencapsulated cultured Hepg2 cells, filter out the culture medium, wash with pre-cooled PBS until the wash solution is colorless, and then filter and dry the liquid. A 1ml syringe was ground to aspirate to break the capsule. After microcapsule membranes were completely damaged under the microscope, add preheated lysate, incubate in a 50°C water bath for 30 minutes, and shake at intervals of 5 minutes. Transfer the reaction system to an 80°C water bath to continue the reaction for 15 minutes, and collect the supernatant by filtration;

[0024] 2) Separation and quantification of adenylic acid:

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

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 a 50°C water bath.

[0031] B: collect monomethoxypolyethylene glycol polylactic acid (PELA) microencapsulated cultured 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 lysate, Incubate in a water bath at 50°C for 30 minutes, shaking at intervals of 5 minutes. Transfer the reaction system to an 80°C water bath to continue the reaction for 15 minutes, and collect the supernatant by filtration;

[0032] 2) Separation and quantification of adenylic acid:

[0033] The content of adenylic acid in the extracted samples was separated by high performance liquid chromatography. Determination conditions are UV detector, wavelength 260nm, C18 reverse 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 has been widely used in the fields of monoclonal antibody production, cell transplantation and cell expansion because of its special microcapsule environment that can make cells grow three-dimensionally. [0003] The activity of microencapsulated cells directly affects the downstream yield and application effect during the culture process. At present, the activity of microencapsulated cells is detected by thiazolium 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 su...

Claims

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Application Information

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Patent Type & Authority Applications(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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