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Method for unicellular sorting by using unicellular printer

A single-cell, printer technology, applied in animal cells, reproductive tract cells, vertebrate cells, etc., can solve the problems of differentiation of sorting and culture conditions, and the impact of recovery rate, so as to reduce time, improve efficiency, and improve operation efficiency. Effect

Inactive Publication Date: 2019-04-02
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the single-cell sorting efficiency and monoclonal recovery rate of single-cell printers are greatly affected by cell sorting and culture conditions, and the sorting and culture conditions of different cells are significantly different. It is a problem that those skilled in the art need to solve in order to improve the efficiency of single cell sorting and the recovery rate of monoclonal

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A specific implementation method is as follows:

[0025] 1. Start the instrument: turn on the main power switch of the single-cell printer and start the instrument. Click the software to automatically perform ROI (Region of Interest, which defines the volume ejected from the chip nozzle when droplets are generated) detection, and wait for the software initialization to complete.

[0026] 2. Sample preparation: centrifuge the subcultured CHO-K1 cells at 300 g for 5 min, and resuspend them in sterile Dulbecco's phosphate buffered saline (D-PBS, Hyclone SH30028.02) or subculture medium to make the cell density 1E6 cells / mL. Then use a 40 μm filter to filter the resuspended cell suspension for use.

[0027] 3. Instrument preparation: first start the droplet quality control, press the "start quality control" button to start the droplet QC. Droplet QC results can be stored as images. Then perform droplet adjustment, set the trigger delay to 3ms, the depth to 10μm, and the ...

Embodiment 2

[0032] Based on the method of Example 1, single-cell printer sorting monoclonal rate evaluation:

[0033] Add 100 μL of ethanol-containing medium (90% subculture medium + 10% 75% ethanol) to each well of a 96-well plate in advance, and use a single-cell printer to perform single-cell sorting, and sort one cell per well. Ethanol is added to the medium to stop the cells from dividing. After the sorting was completed, the 96-well plate was left to stand for 2 hours to allow the cells to fully settle, and then Cell Metric (Solentim) was used for imaging to determine whether each well of the 96-well plate was monoclonal. In order to distinguish the impurities in the 96-well plate and the medium, the 96-well plate was pre-scanned with Cell Metric after adding the medium. After 24 hours of sorting, the 96-well plate was rescanned using Cell Metric to reconfirm the monoclonality of each well of the 96-well plate. The evaluation results of the sorted monoclonal rate are shown in Tabl...

Embodiment 3

[0037] Evaluation of the recovery rate of clones sorted by single-cell printer based on the method of Example 1:

[0038] Add 100 μL of medium to each well of a 96-well plate in advance, and use a single-cell printer to sort single cells, sorting 1 cell per well. After sorting, the cells were cultured statically in a carbon dioxide incubator (36.5 °C, 6% CO 2 ), use Cell Metric to take pictures after 2h, 1 day, 2 days, 5 days and 9 days respectively. After 9 days, cells were assessed for recovery. The recovery of the evaluated cells is shown in Table 2.

[0039] Table 2 Clonal recovery rate of single cell sorting by single cell printer

[0040] Number of 96-well plates

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Abstract

The invention discloses a method for unicellular sorting by using a unicellular printer. The method comprises the steps of (1) preparing samples: performing centrifugation on subcultured CHO-K1 cellsfor 5min, and using a bacteria-free Dulbecco's phosphate buffer solution or a subculturing substrate for resuspending, wherein the cell density is 1E6cells / mL; 2 ) setting liquid drop adjustment parameters: starting the liquid drop quality control of the unicellular printer, setting the trigger delay as 3ms, setting depth as 10[mu]m, and setting the speed as 120[mu]s / ms; (3) setting sorting parameters: setting the diameters of cells as 12[mu] m-18[mu]m, setting the cell roundness as 55-100%, and setting the cell detection radius as 60[mu]m; and (4) performing sorting: sorting the cell samplesin an ink box of the unicellular printer to hole plates, and sorting one cell for each hole. Through the adoption of the method, the unicellular sorting efficiency and the monoclonal recovery rate areincreased. Compared with CHO-K1 cells in the prior art, the other sorting and culturing conditions are notably improved /

Description

technical field [0001] The invention belongs to the fields of biopharmaceuticals and biotechnology, and in particular relates to a single-cell sorting method using a single-cell printer, which can be applied to the construction of cell lines for production. Background technique [0002] Biopharmaceuticals such as monoclonal antibodies have become more and more popular areas of research and development in recent years, and the requirements of relevant regulations have become more and more stringent. For the construction of stable cell lines for production, Chinese Hamster Ovary cells (Chinese Hamster Ovary, CHO) are one of the most commonly used host cells. In order to ensure the safety of biopharmaceuticals, regulatory authorities have very high requirements for the monoclonality of cell lines used in production. Therefore, cell monoclonization is a very critical step in the entire process of cell line construction. [0003] Currently, two commonly used methods for cell mon...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0682G01N33/5005
Inventor 李琴张肖肖张文康张朗董慧芳蔡洁行周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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