Method for performing unicellular sorting through Namocell unicellular isolation instrument

Abstract

The invention discloses a method for performing unicellular sorting. The method comprises the following steps of (1) performing sample preparation: diluting CHO-K1 cells to be sorted with subculturingmediums until the density of living cells is 1E5cells / mL, adding a calcein reagent for dyeing, and after dyeing is completed, diluting the cells with the subculturing mediums to 5000cells / mL; (2) loading the samples: starting a Namocell unicellular isolation instrument, pouring the cells into a chip cavity, and then loading chips to be detected; (3) performing position calibration: adjusting a laser spot to a microfluid passage central position; (4) setting unicellular sorting parameters: setting fluorescence value that FITCH=500, FITCL=30, PEH=3, and PEL=0; (5) performing unicellular sorting: beginning to sort single cells, accurately capturing active cells through a high-speed electronic valve, finally forming 1[mu]L volume of liquid drops, and performing sorting into a hole plate, wherein one cell is sorted in each hole. Through the adoption of the method disclosed by the invention, the unicellular sorting efficiency and the monoclone recovery rate can be improved, and can be remarkably better than those of CHO-K1 cells under other sorting and culturing condition in the prior art.

Description

technical field [0001] The invention belongs to the fields of biopharmaceuticals and biotechnology, in particular to a method for single cell sorting using a Namocell single cell separator, which can be applied to the construction of cell lines for production. Background technique [0002] Monoclonal antibodies have the characteristics of strong targeting, high specificity and low toxicity and side effects, and have been successfully used in the treatment of various diseases such as immunity and cancer. Stable cell lines for the production of monoclonal antibodies are generally screened through mammalian expression systems, among which Chinese Hamster Ovary (CHO) cells are one of the most commonly used host cells. In order to ensure the safety of biopharmaceuticals, regulatory authorities have very strict requirements on the monoclonality of production cell lines. Therefore, cell monoclonalization is a very critical step in the entire production process of cell line construc...

AI Technical Summary

Problems solved by technology

The disadvantage is that the pressure of the sheath fluid can damage the cells to a certain extent, the consumables used are expensive, the cleaning process of the instrument is complicated and time-consuming, and a special person is required to be responsible for maintenance, which consumes economic, time and labor costs

However, the single cell sorting efficiency and monoclonal recovery rate of the Namocell single cell separator are greatly affected by cell sorting and culture conditions, and the sorting and culture conditions of different cells are significantly different. Therefore, targeting a specific cell The development of suitable sorting and culture conditions to improve the efficiency of single cell sorting and the recovery rate of single clones is a problem that those skilled in the art need to solve