Method for single cell cloning and isolation

Abstract

The invention discloses a method for single cell cloning and isolation and particularly discloses a method for single-cell cloning and isolation of anchorage-dependent growth animal cells. In single-cell cloning and isolation, cells are dyed in short time through a neutral red solution so that small cell blocks are fast dyed and are visible and the accuracy of the operation is improved. In cell cluster picking, a low-concentration pancreatin solution is used so that cells can fall off from the wall of a vessel, cell damage is reduced and cell survival and proliferation are promoted. The method utilizes a manual pipette to suck a small amount of pancreatin and a small cell cluster local digestion method. Compared with the traditional cloning method, the method simplifies processes and is free of a special material-cloning ring. Compared with the limited dilution method, the method is conducive to cell growth and prevents individual growth of many cells in the independent environment. Compared with the method with the microscope, the method is easy to operate and greatly reduces the probability of pollution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new method for separating single-cell clones from adherent-growing animal cells. Background technique [0002] Single-cell cloning is the division and proliferation of a single cell to form a cell population with the same genetic traits. Since they are derived from the same progenitor cell, the expanded cell population also has very similar morphological characteristics and basically the same physiological and biochemical characteristics. [0003] Single cell cloning is an important link in the establishment of stable cell lines, so it is widely used in the fields of modern biotechnology and pharmaceutical research and development. Common methods for constructing stable cell lines include plasmid transfection and virus infection. Both methods require antibiotic selection and single-cell clone isolation to obtain cell populations with stable phenotype and consistent gen...

AI Technical Summary

Problems solved by technology

[0005] These three methods have their own limitations: the limiting dilution method is more suitable for suspension cells, but in practice, the demand for adherent cells accounts for the vast majority, and many cells grow very slowly or even cannot grow at very low densities, so it is impossible to carry out Single-cell cloning; the cloning ring method not only requires the purchase of cloning rings, but also because it is difficult to distinguish with the naked eye when the number of cells is small, the separation must wait until the number of cells has expanded to several hundred, so it takes a long time to wait, and there are many steps, The difficulty is high, and the experimenter usually needs to have rich experience; the micromanipulation method needs to use an expensive special microscope or move the conventional microscope to a safety cabinet for use, which makes the operation more difficult, and it is easy to cause cell contamination and waste all previous efforts