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Culture method for improving stability of hERG-HEK293 cells in patch clamp test

A technology of herg-hek293 and culture method, applied in cell dissociation methods, embryonic cells, animal cells, etc., can solve problems such as inability to seal, prone to instability, and affecting drug screening efficiency

Pending Publication Date: 2020-10-30
成都华西海圻医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] During the patch clamp test, hERG-HEK293 cells are affected by the state of the cell membrane and are prone to instability. The specific manifestations are: the cell membrane is brittle, and the cell membrane ruptures when the electrode just contacts the cell, making it impossible to seal.
These instabilities can lead to test failures and affect drug screening efficiency
However, there is no report on the method of improving the stability of hERG-HEK293 cells in the patch clamp test.

Method used

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  • Culture method for improving stability of hERG-HEK293 cells in patch clamp test

Examples

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Embodiment 1

[0021] Example 1 Culture method for improving the stability of hERG-HEK293 cells in patch clamp test

[0022] 1. Cell culture

[0023] 1.1 Experimental group (method of the present invention)

[0024] (1) Take the DMEM high sugar medium containing 20% ​​v / v (or 10% v / v) FBS that has been preheated in a 37°C water bath, add it to the culture flask, and take hERG-HEK293 cells (purchased from BPS , And has been resuscitated and cultivated) to about 4×10 4 Pcs / cm 2 The density of inoculation to the culture medium;

[0025] (2) Passage is performed when the degree of fusion reaches about 80-90%. During passaging, add an effective amount (sufficient enough to make the cells break away from the adherent state to the free state. In this example, the dosage is 0.04 mL / cm 2 Cell adhesion area) TrypLE TM Express (1×), digest at room temperature for 20 seconds, discard the digestive enzymes, and then incubate for 2 minutes in a 37° incubator. Use 2 times the volume of digestive enzymes preheated...

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Abstract

The invention discloses a culture method for improving hERG-HEK293 cell stability in a patch clamp test, and belongs to the field of cell culture. According to the method, by increasing the content ofFBS in the culture medium to 20% v / v and optimizing the operation steps of passage digestion, the stability of hERG-HEK293 cells in the patch clamp test can be improved, and then the success rate ofthe patch clamp test is increased.

Description

Technical field [0001] The invention belongs to the field of cell culture. Background technique [0002] The patch-clamp test is a special technique for detecting ultra-weak pA-level current signals. This technique involves touching the tip of a microelectrode (usually with a diameter of <5μm) to the surface of the cell membrane through a micromanipulation device, and then passing the negative Press to gently suck up the cell membrane to form a relatively closed area (ie, seal). Since the material is mainly non-conductive silica, a resistance of 10 at the tip area will be generated. 9 High impedance above Ω, thus forming an electrical protective cover effect that makes Na + , K + , Ca 2+ Plasma cannot pass through other places, it can only enter and exit from the partial diaphragm area in the tube, so that Na can be recorded + , K + , Ca 2+ The ultra-weak current generated by ions that play an important role in maintaining cell physiology when they flow through the cell membran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0606C12N5/0686C12N2510/00C12N2509/00
Inventor 卜迁岑小波魏倩宋文博李耀
Owner 成都华西海圻医药科技有限公司
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