Method for performing unicellular sorting through Namocell unicellular isolation instrument
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A single-cell sorting technology, applied to cell dissociation methods, animal cells, reproductive tract cells, etc., can solve the problems of cell damage, great impact on recovery rate, expensive consumables, etc., to reduce time, improve efficiency, The effect of improving efficiency
Inactive Publication Date: 2019-04-02
SHANGHAI WUXI BIOLOGIC TECH CO LTD
View PDF9 Cites 5 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
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
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0026] A specific implementation method is as follows:
[0027] 1. Preparation of fluorescent staining reagent: Calcein-AM is a fluorescent staining reagent with excitation wavelength and emission wavelength of 495nm and 515nm respectively, which can stain living cells. Calcein-AM (ThermoFisher, C3100MP) was prepared into a solution with a concentration of 100 μM (10,000×) using sterile dimethyl sulfoxide (DMSO), ready for use.
[0028] 2. Sample preparation: Take out 3-5 mL of CHO-K1 cells to be sorted, filter them with a 40 μm cell strainer, and count them with Vi-cell. Use subculture medium to dilute to a viable cell density of 1E5cells / mL, add 100 μM Calcein-AM at a ratio of 1:10,000, so that the final concentration of Calcein-AM is 10 nM, and stain the cells for 5 minutes. After staining, the cells were diluted to 5,000cells / mL (the expected sorting speed was 1.6cells / s) using the subculture medium for single cell sorting.
[0029] 3. Start the instrument: turn on the s...
Embodiment 2
[0036] Evaluation of recovery rate of clones sorted by amocell single cell separator based on the method of Example 1:
[0037]Add 100 μL of medium to each well of a 96-well plate in advance, and use a Namocell single cell separator to sort single cells, and sort 1 cell per well. After sorting, the cells were cultured statically in a carbon dioxide incubator (36.5°C, 6% CO2), and photographed using Cell Metric after 2 hours, 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 1.
[0038] Table 1 Clonal recovery rate of single cell sorting by single cell printer
[0039] Number of 96-well plates
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
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...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more