Single cell clone culture method

A single-cell cloning and cell technology, which is applied in general culture methods, cell culture supports/coatings, tissue cell/virus culture devices, etc. Wait for the question

Active Publication Date: 2020-10-27
WEST CHINA HOSPITAL SICHUAN UNIV +1
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  • Description
  • Claims
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Problems solved by technology

This method can obtain relatively pure monoclonal cells, but requires the use of expensive micromanipulation systems, requires high operator experience, and cannot overcome the problem that individual cells cannot grow normally
[0006] The methods of the prior art also generally have the problem of low throughput, that is, few single clones are obtained in a single culture.

Method used

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Embodiment 1

[0030] Embodiment 1 monoclonal culture method

[0031] The present invention specifically comprises the following steps ( figure 1 is a schematic diagram of the main steps):

[0032] (1) Use a laser-etched silicon wafer with a specific pattern as a template, and obtain a polydimethylsiloxane (polydimethylsiloxane, PDMS) stamp by inverting the mold, and prepare a convex rectangular microarray (a single rectangular area of ​​225 μm 2 ) PDMS seal.

[0033] (2) 200 μl of fibronectin (fibronectin, FN) solution with a concentration of 100 μg / ml is coated on the surface of the stamp. In this embodiment, an additional green fluorescein Flourescein isothiocyanate isomer I is added to the surface of the stamp to indicate the stamp, at room temperature After 20 minutes of incubation, excess solution was washed off.

[0034] (3) Dry in a 37°C incubator for 8-15 minutes, then place a 50ml centrifuge tube containing 20-30ml of water upside down and press it on the stamp, and apply a cert...

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Abstract

The invention discloses a single cell clone culture container. The single cell clone culture container is a cell culture container with a fibronectin imprinting block array on the bottom surface, an area, that is not covered with fibronectin, on the bottom surface is sealed by a sealing agent, and an area of a single imprinting block is 225-625 [mu]m<2>. The invention also discloses a single cellclone culture method, and the method is to inoculate cells into the aforementioned culture container for culture. The single cell clone culture method of the invention has high throughput, can culturethousands of single cell clone groups at a time, and can also overcome the problem that the single cell clone is impure and cannot grow normally caused by the traditional limiting dilution method. Inaddition, the method can be applied to many fields such as gene editing, tumor clone culture, and single cell sequencing sample preparation, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of single cell clone culture method. Background technique [0002] A single cell clone is a cell group formed by culturing one cell and making it divide. Since this group of cells originates from common ancestor cells, they have the same genotype and high consistency in physiological state, so they are widely used in biological and medical research, such as: gene editing cell screening, tumor cloning, single-cell sequencing sample preparation, hybridoma preparation etc. [0003] The patent application document with publication number CN 108130314 A discloses a monoclonal culture method, which mainly includes: (1) first adding thrombin into a pre-cooled 96-well plate; (2) diluting the target cells with culture medium, Add fibrinogen, T7 buffer and medium to obtain a mixed system; (3) spread the mixed system of step (2) evenly over the 96-well plate of step (1), ensuring that each well is a single cell; ( 4) Cultivat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12N5/02C12N5/10C12N15/90C12N5/09C12N5/20
CPCC12M23/34C12M25/14C12N5/0068C12N15/907C12N5/069C12N5/0693C12N5/163C12N2533/52C12N2511/00C12N2510/00
Inventor 包骥步宏李顺张芸琳
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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