Culture medium for single cell culture, and preparation method and application thereof

Abstract

The invention discloses a culture medium for single cell culture, and a preparation method and application thereof. The culture medium for single cell culture comprises an ordinary culture medium andan additive, wherein the additive is an immortalized-cell secretion extracted from a culture product of immortalized cells, and the immortalized cells are identical to the cells of a unicellular species which is to be cultured with the culture medium for single cell culture. Since the immortalized-cell secretion is added into the culture medium, primary cells can realize normal division and proliferation under the condition of low density, and the problem that low density of fibroblasts cannot meet the requirements of monoclonal cell selection is overcome. With the culture medium in the invention, a marker gene is not needed, so the risk of damage to the functions of endogenous genes caused by the marker gene is avoided, and a foundation is laid for commercial application of genetically edited cells. Since drug resistance screening is not needed, reagent cost is reduced, screening time is shortened, damage of drugs to cells is avoided, and cells are allowed to maintain the form of wildtypes and proliferation and division capabilities.

Description

technical field [0001] The present application relates to the field of cell culture, in particular to a culture medium for single cell culture and its preparation method and application. Background technique [0002] Gene editing refers to modifying the genome DNA sequence of a specific organism according to human wishes with the help of precise nuclease gene cutting technology. Commonly used technologies include zinc finger nuclease (ZFN), transcription activator-like effector nuclease ( TALENs) and Regularly Interspersed Short Palindromic Repeats (CRISPR / cas9) system. Among them, CRISPR / cas9 has been rapidly and widely used in various fields, such as agriculture, medicine, and environmental protection, because of its simple construction and high DNA shearing efficiency, and has great potential for scientific research and commercial application. [0003] CRISPR / Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be us...

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Problems solved by technology

Moreover, for fibroblasts, if the drug resistance gene is not introduced, if the cell density is not enough when dividing into 10cm cell culture dishes, it will easily lead to poor growth ability of single cell clones, and the cell density that meets the selected clones cannot be obtained; When the cell density is too high, the single cell clones are easy to connect to form non-single clones, and finally obtain cell clones with impure genotypes, which cannot be used for later scientific research and production

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