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Single cell cloning culture method

A single-cell clone and culture method technology, applied in the field of single-cell clone culture, can solve the problems of difficult cell screening, difficult culture, low transfection efficiency, etc., and achieve the effect of high-efficiency screening, ensuring unity, and ensuring non-diffusion of concentration.

Inactive Publication Date: 2014-07-16
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to propose a single-cell clone culture method to solve the problems that the purity of single-cell clones cannot be guaranteed and difficult cultivation and low transfection efficiency cell selection are difficult

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0024] The method of culturing transgenic fibroblasts by single cell clone culture method comprises the following steps:

[0025] 1. Use DMEM (dulbecco's modified eagle medium) culture solution containing 20% ​​fetal bovine serum (FBS) to make a 5 μl droplet in a petri dish; the droplet is made in two steps, first

[0026] Add 2.5 μl of culture medium one by one at intervals of the petri dish to form droplets; then cover the droplets with paraffin oil, and then add 2.5 μl of culture medium to each droplet, so that the total volume of the droplets is 5 μl , to form micro-droplets of culture solution; put it into the incubator to balance, and wait for the cells to be put in;

[0027] 2. Prepare the micromanipulator: use a needle puller (Narishige company, model PN-30) to make a cell manipulation needle with an inner diameter of 20 μm and a blunt front end, and install it on the micromanipulator (Eppendorf company, model NK2);

[0028] 3. Screening monoclonal cells: blot dry the...

Embodiment 2

[0033] Screening and culturing myoblasts by single cell clone culture method, comprising the following steps:

[0034]1. Use DMEM (dulbecco's modified eagle medium) culture solution containing 20% ​​fetal bovine serum (FBS) to make a 5 μl droplet in a petri dish; the droplet is made in two steps, first

[0035] Add 2.5 μl of culture medium one by one at intervals of the petri dish to form droplets; then cover the droplets with paraffin oil, and then add 2.5 μl of culture medium to each droplet, so that the total volume of the droplets is 5 μl , to form micro-droplets of culture solution; put it into the incubator to balance, and wait for the cells to be put in;

[0036] 2. Prepare the micromanipulator: use a needle puller (Narishige company, model PN-30) to make a cell manipulation needle with an inner diameter of 12 μm and a blunt front end, and install it on the micromanipulator (Eppendorf company, model NK2);

[0037] 3. Screen monoclonal cells: drain the muscle cell cultu...

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Abstract

The invention discloses a single cell cloning culture method. The single cell cloning culture method comprises the steps: step one, preparing culture solution microdrops; step two, preparing a micromanipulation system; step three, screening monoclonal cells; step four, putting target cells in an operation needle into the culture solution microdrops obtained in the step one in such a manner of carrying one cell in each drop by using the micromanipulation system prepared in the second step, and then carrying out cell culture; step five, replacing the culture solution by a half amount for the cells cultured in the step four two days later after culturing; sixth step six, carrying out enlarge culturing to realize single cell cloning. The method is characterized in that a paraffin oil between the culture solution microdrops isolates the contact of the microdrops with each other, and thus the unicity of cell cloning is guaranteed; meanwhile, as the volume of the culture solution microdrops is less than 5 microliters, biological factors secreted by the cultured cells are not diluted excessively so as to provide a good microenvironment for the growth of cells.

Description

technical field [0001] The invention belongs to the technical field of cell culture, in particular to a single cell clone culture method. Background technique [0002] The single cell cloning culture method currently used is to infinitely dilute the cells and culture them in a culture dish or in a 96-well plate after reaching a certain dilution concentration, so as to realize the independent growth of a single cell into a clone. When cultured in a petri dish, the cells cultured in the petri dish are in a floating state before attaching to the wall, and it is easy to contact with another cell or attach to the wall at the same place to form a clone. With the increase in the number of cell proliferation, the cells in a dividing state Due to poor adherence, cells will also move to other cell clones, and the single source of clones cannot be fully guaranteed; on the other hand, due to the initial culture in order to prevent the cell density from being too large, multiple cells co...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 王立民周平甘尚权唐红郭延华张译元
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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