Monoclonal cell selection method and application
The method addresses the inefficiencies of traditional monoclonal cell selection by using trypsin digestion and a pressure glass syringe to transfer monoclonal cells efficiently and accurately, reducing cross-contamination and time.
Patent Information
- Application Number
- CN202310683490.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-09
- Publication Date
- 2024-12-10
AI Technical Summary
Traditional methods for selecting monoclonal cells are prone to leakage, cause cross-contamination, are cumbersome, and take a long time, failing to accurately select cell monoclones.
A method involving trypsin digestion of cells in a culture dish, followed by washing and transferring monoclonal cell colonies using a pressure glass syringe with liquid columns to a cell culture plate, ensuring accurate and efficient selection without a cloning ring.
This method reduces the risk of cross-contamination and shortens the time required for monoclonal cell selection, allowing for rapid and precise transfer to culture plates.
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of cell cloning, and in particular relates to a method for selecting monoclonal cells. Background Art
[0002] With the development of modern biotechnology, the selection of monoclonal cells is an important process for obtaining cells of the target genotype. Monoclonal cells are cell colonies formed by the proliferation of a single cell. The traditional method of selecting monoclonal cells is to use a cloning ring, mark the cells that meet the conditions after observing them under a microscope, place the cloning ring on the cell monoclone, digest the cells and culture them separately. This method is prone to leakage, cannot accurately select cell monoclones, causes cell cross-contamination, is cumbersome to operate, takes a long time, and has a large workload. Summary of the invention
[0003] In view of the problems raised by the background technology, the purpose of the present invention is to propose a method and application for selecting monoclonal cells, which can directly digest cells without using a cloning ring, and can transfer monoclonal cells to a cell culture plate for culture in batches, quickly and accurately, thereby ensuring the normal progress of subsequent experiments, and solving the problems of cumbersome monoclonal cell selection steps and easy cross contamination.
[0004] The present invention provides a method for selecting monoclonal cells, comprising:
[0005] 1) Wash the adherent cells and digest them with trypsin. After digestion is terminated, the cells are evenly inoculated in a culture dish and cultured until a monoclonal cell colony grows;
[0006] 2) Wash the monoclonal cell colony and digest it with trypsin until the cells become round and translucent, then terminate the digestion;
[0007] 3) Select monoclonal cell colonies with full and transparent morphology, and gently draw the monoclonal cell colonies into the cell culture plate using a pressure glass syringe containing 2-4 sections of liquid column, mix well, and culture.
[0008] Wherein, the washing steps in step 1) and step 2) are washing with 1×PBS buffer solution; the washing times are at least twice.
[0009] Wherein, the concentration of pancreatic enzyme in step 1) is 0.05% or 0.25%; and the added amount is 0.5-2 mL.
[0010] Wherein, the density of the inoculated cells in step 1) is 20 cells / mL-200 cells / mL, preferably 50-100 cells / mL; the inoculation volume is 2-6 mL, preferably 3 mL.
[0011] Wherein, the method for terminating digestion in step 1) and step 2) is to add a culture medium containing 10% fetal bovine serum in an amount of 1-5 mL.
[0012] The digestion time in step 2) is 2-10 min, preferably 5-8 min; the monoclonal cell colony is a colony of more than 10 cells formed by the division of a single cell.
[0013] Wherein, the selection step in step 3) is performed under a microscope in a clean bench.
[0014] Among them, the 2-4 sections of liquid columns in step 3) are cell culture media separated by air, preferably 3 sections; there is a section of air between two adjacent sections of cell culture media; the lengths of the liquid columns and the air are both 2 cm.
[0015] Among them, the pressure glass needle tube in the step 3) is a mouth pipette or a rubber-tipped tube, and one end of the needle tube is a smooth round mouth with an inner diameter of 0.2mm-1mm, preferably 0.5-0.8mm; the cell culture plate is a 24-, 48- or 96-well plate, preferably a 48-well plate; the culture dish in the step 1) is a 35, 60, or 100mm cell culture dish, preferably a 60mm dish.
[0016] The application of the above-mentioned monoclonal cell selection method in monoclonal cell passage should also be within the protection scope of the present invention.
[0017] The above technical scheme has the following beneficial effects: 0.05% concentration or 0.25% concentration of pancreatic enzyme can be selected according to different cell types to digest cells at a suitable concentration. The inoculation cell density of 20 / mL-200 / mL can ensure that single cells grow into colonies and avoid aggregation and adhesion of multiple cells. Digest the cells at room temperature for 2-10 minutes, and select the appropriate time according to different cell types to ensure that the cells are digested into a transparent round shape to avoid over-digestion. Use a 2-4 segment liquid column pressure glass syringe to absorb cells. You can choose a mouth pipette or a rubber-headed tube. The speed can be effectively controlled to accurately absorb monoclonal cell colonies. The smooth round mouth with an inner diameter of 200um-1mm at one end of the syringe can avoid damaging cells. The appropriate inner diameter can be selected according to the size of the monoclonal cell clumps. The present invention shortens the growth time of monoclonal cells and improves the efficiency of obtaining target monoclonal cells. DETAILED DESCRIPTION
[0018] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0019] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0020] The reagents involved in the following examples are as follows: 1×PBS (Biosharp, BL302A), 0.05% or 0.25% trypsin (Gibco, 25200072, 25300062), 10% fetal bovine serum (Gibco, 10099-141C), DMEM culture medium (Gibco, C11995500BT). The materials involved in the following examples are as follows: 35, 60, 100 mm cell culture dishes, 96, 48, 24-well cell culture plates, clean bench, microscope, glass syringe, cell culture box.
[0021] Example 1
[0022] A method for selecting monoclonal cells comprises the following steps:
[0023] Step (1) CRISPR / Cas9 gene-edited pig kidney cells (PK-15) with a density of 90% in a T25 cell culture flask were mixed and washed twice with 5 mL 1×PBS, and then digested with 0.25% trypsin (the amount of trypsin added was 10 6 1 mL of trypsin was added to each cell), and digestion was terminated with 3 mL of DMEM medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were resuspended and mixed with DMEM medium containing 10% fetal bovine serum. The cells were counted and the cell density was adjusted to 50 cells / mL. 3 mL of the cell suspension was added to a 60 mm cell culture dish, pipetted evenly, and gently moved to a 37°C incubator containing 5% CO 2 cultured in a cell culture incubator.
[0024] Step (2), when the single cells in the dish of step (1) proliferate to a cluster of about 50 cells, wash twice with 3 mL of 1×PBS, add 1 mL of 0.25% trypsin and digest at room temperature for 3-6 minutes, observe the cell morphology under a microscope, and when the cells become transparent, round and do not fall off, add 2 mL of DMEM culture medium containing 10% fetal bovine serum to terminate the digestion.
[0025] Step (3), use an alcohol burner to draw a glass tube with an inner diameter of 2mm and an outer diameter of 3mm into a glass tube with an inner diameter of 0.5mm, cut the thin end with a grinding wheel, and burn it under a flame to form a smooth round mouth. Connect the drawn glass needle with a mouth pipette, draw a 2cm cell culture medium liquid column, and then draw 2cm of air to make three consecutive liquid columns. Place the cells digested in step (2) under a microscope in a clean bench for observation, and select monoclonal cell colonies with full and translucent morphology. Use a mouth pipette to gently draw the monoclonal cell colony and transfer it to a 48-well cell culture plate containing complete culture medium. The movement of the liquid column can be used to determine whether the cells have been removed. Mix well with a 200uL pipette and gently transfer to a 37°C medium containing 5% CO 2 cultured in a cell culture incubator.
[0026] The method of Example 1 was used to pick monoclonal cells, which could effectively avoid cross contamination of cells of different genotypes and pick monoclonal cells with better morphology, with a success rate of 100%.
[0027] Example 2
[0028] This embodiment provides a method for selecting monoclonal cells, which differs from Embodiment 1 in that: in step (1), the cells are primary porcine fibroblasts, the trypsin concentration is 0.05%, and the cell density is 100 cells / mL; in step (2), the cells proliferate to form a mass of about 80 cells; in step (3), the inner diameter of the thin end of the glass tube is 0.8 mm, and the cell culture plate is a 96-well plate, and the operations in other steps remain unchanged.
[0029] The method of Example 2 was used to pick monoclonal cells, which could effectively avoid cross contamination of primary cells of different genotypes, pick monoclonal cells with better morphology, and ensure that primary cells could grow quickly in the cell culture plate, with a success rate of 100%.
[0030] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.
Claims
1. A method for selecting monoclonal cells, characterized in that: include: 1) Wash the adherent cells and digest them with trypsin. After digestion is terminated, the cells are evenly inoculated in a culture dish and cultured until a monoclonal cell colony grows; 2) Wash the monoclonal cell colony and digest it with trypsin until the cells become round and translucent, then terminate the digestion; 3) Select monoclonal cell colonies with full and transparent morphology, and gently draw the monoclonal cell colonies into the cell culture plate using a pressure glass syringe containing 2-4 sections of liquid column, mix well, and culture.
2. The method for selecting monoclonal cells according to claim 1, characterized in that: The washing steps in step 1) and step 2) are washing with 1×PBS buffer solution; the washing times are at least twice.
3. The method for selecting monoclonal cells according to claim 1, characterized in that: The concentration of pancreatin in step 1) is 0.05% or 0.25%; the amount added is per 10 6 Add 0.5-2 mL of trypsin per cell.
4. The method for selecting monoclonal cells according to claim 1, characterized in that: The density of the inoculated cells in step 1) is 20 cells / mL-200 cells / mL; the inoculation volume is 2-6 mL.
5. The method for selecting monoclonal cells according to claim 1, characterized in that: The method for terminating digestion in step 1) and step 2) is to add a culture medium containing 10% fetal bovine serum in an amount of 1-5 mL.
6. The method for selecting monoclonal cells according to claim 1, characterized in that: The digestion time in step 2) is 2-10 min; the monoclonal cell colony is a colony of more than 10 cells formed by the division of a single cell.
7. The method for selecting monoclonal cells according to claim 1, characterized in that: The selection step in step 3) is performed under a microscope in a clean bench.
8. The method for selecting monoclonal cells according to claim 1, characterized in that: The 2-4 segments of liquid columns in step 3) are cell culture media separated by air; there is a segment of air between two adjacent segments of cell culture media; the lengths of the liquid columns and the air are both 2 cm.
9. The method for selecting monoclonal cells according to claim 1, characterized in that: The pressure glass needle tube in step 3) is a mouth pipette or a rubber-tipped tube, one end of the needle tube is a smooth round mouth with an inner diameter of 0.2mm-1mm, and the cell culture plate is a 24-, 48- or 96-well plate; the culture dish in step 1) is a 35-, 60-, or 100mm cell culture dish.
10. Use of the monoclonal cell selection method according to any one of claims 1 to 9 in monoclonal cell passaging.