A monoclonal cell strain screening method based on fluorescence intensity

Abstract

The application discloses a monoclonal cell strain screening method based on fluorescence intensity, and the method comprises the following steps: determining a cell line, and stably transfecting cells of the cell line with fluorescent proteins; selecting a microwell plate and setting a reference hole, and separating the cells stably transfected with the fluorescent proteins into the microwell plate, wherein the reference hole is filled with multiple cells, and the remaining holes are filled with only one cell; screening single-cell holes with only one cell in the remaining holes except the reference hole; screening clone group holes capable of growing clone groups; transferring the cells in the clone group holes to new holes for culture after being respectively digested; screening fluorescence screening holes; screening division and proliferation holes; and determining the cells in the division and proliferation holes as screened monoclonal cell strains. The monoclonal cell strain screening method provided by the application can screen monoclonal cell strains with strong fluorescence intensity and normal proliferation speed, is convenient and fast compared with fluorescence microscope observation and imaging, and also reduces errors possibly caused by human eye comparison of fluorescence intensity.

Description

Technical Field

[0001] This invention belongs to the field of cell screening technology, specifically a method for screening monoclonal cell lines based on fluorescence intensity. Background Technology

[0002] In medical research, fluorescent proteins are often used to label cells. The most common method is stable transfection with GFP (Green fluorescent protein). However, even with stable transfection, there may be a decrease in fluorescent protein expression and large differences in fluorescence signals between cells. The best method is to isolate single cells to obtain monoclonal cell lines and then select monoclonal cell lines with strong fluorescence intensity. The fluorescence intensity of the cells obtained in this way is significantly better than that of the original cells. After single cells are inserted into wells, in order to select monoclonal cell lines with strong fluorescence intensity, it is necessary to select wells that meet the following conditions from all the wells: (1) a single cell is inserted into the well; (2) the cell can divide and proliferate normally; (3) the fluorescence intensity of the cells obtained from the division of a single cell is relatively strong. There are instruments on the market designed to observe and record the formation of single cell clones, such as the CloneSelectImager cell growth analysis system. However, due to the limited use of this function in scientific research, such instruments are not common in universities.

[0003] In the absence of the above instruments, other methods are required. The most common method is to observe and screen using a fluorescence microscope: (1) Screen out wells with exactly one cell. Commonly used single-cell separation methods, such as flow cytometry single-cell sorting or limiting dilution, cannot guarantee 100% that each well has exactly one cell. There is a certain possibility that there are two or more cells in one well. Therefore, it is not possible to wait several days after the cells are put into the well before observing. At this time, most wells already have multiple cells, and it is impossible to determine whether the single cell has divided or multiple cells were put into the well on the same day as the single cell separation. Therefore, after the cells are put into the well, place them in an incubator for 1 to 2 hours. After the cells settle to the bottom (not adherent), immediately use a microscope to confirm the number of cells in each well. It is necessary to carefully observe every area in each well to ensure that no cells are missed. Finally, select wells with exactly one cell. This is a tedious and time-consuming task, and it is difficult to avoid errors. (2) Further screen out wells where single cells can divide and proliferate and have strong fluorescence intensity. The tedious part is that: ① It is necessary to use a microscope to observe the cell division and proliferation status one well at a time; The fluorescence intensity of each well is compared, and the well with the strongest fluorescence intensity is selected. However, the difference between the wells is not significant and it is difficult to distinguish them with the naked eye. It is best to use software to analyze the fluorescence intensity. If imaging and analyzing the average fluorescence intensity of cells in each well during the cloning stage, the software cannot accurately identify each cell because the cells are crowded together, resulting in inaccurate data. If the cells are digested, broken up, and evenly distributed before imaging and analysis, multiple images can only be taken in different areas using a microscope. It is not possible to take an image of the entire well at once, so the software naturally cannot analyze the average fluorescence intensity of cells in the entire well.

[0004] Microplate imaging systems or similar instruments (such as high-content cell imaging analysis systems, live cell workstations, etc.) have been widely used in medical and life science research in recent years, and similar instruments are common in universities. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art by providing a method for screening monoclonal cell lines based on fluorescence intensity using a microplate imaging system commonly found in universities.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for screening monoclonal cell lines based on fluorescence intensity, comprising the following steps:

[0008] S1. Identify a cell line that is stably transfected with fluorescent proteins;

[0009] S2. Select a microplate for single-cell isolation (hereinafter referred to as "single-cell isolation microplate"). In step S1, cells stably transfected with fluorescent protein are introduced into the microplate, with multiple cells in the reference well and only one cell in each of the other wells.

[0010] On the same day that the cells stably transfected with fluorescent protein in step S2 are isolated into microplates, a microplate imaging system is used to screen out single-cell wells with only one cell in each well except the reference well.

[0011] S4. After culturing cells in single-cell wells for several days, a microplate imaging system is used to screen out the wells in which clonal clusters can grow.

[0012] S5. After digestion, the cells in each clone cluster well are transferred to the corresponding new wells on a new microplate for culture.

[0013] S6. After cells are transferred to new wells, the top 30% to 50% of the average fluorescence intensity of cells are selected as fluorescence screening wells using a microplate imaging system.

[0014] S7. After culturing the cells in the fluorescent screening wells for several days, the microplate imaging system is used to screen out the fluorescent screening wells in which the cells can proliferate at a normal rate to 4 to 8 times the number of cells in the fluorescent screening wells in step S6 as division and proliferation wells.

[0015] S8. Confirm that the cells in the cell division and proliferation wells are selected monoclonal cell lines.

[0016] The microporous plate used in step S2 is a microporous plate with a black wall and a transparent bottom.

[0017] The number of cells in the reference well in step S2 is 50 to 200. The reference well is used to set the imaging parameters of the microplate imaging system, mainly the focal length and exposure time.

[0018] The microplate imaging system in steps S3, S4, S6, and S7 needs to meet the following conditions: it should have bright-field and fluorescence imaging capabilities, be able to automatically present whole-well imaging of a single well, be able to complete whole-well imaging of all wells on the entire microplate in one automatic imaging session, and be able to analyze and display the number of cells in each well and the average fluorescence intensity of the cells in each well.

[0019] The microplate imaging systems in steps S3, S4, S6, and S7 include, but are not limited to, Tecan's SPARK CYTO, Zeiss's Celldiscoverer7, and Agilent's CYTATION5.

[0020] In step S3, the screening of single-cell wells is carried out by using the cell number analysis results of the microplate imaging system and / or by using the cell number to be determined by observing fluorescence plus bright field imaging with the human eye, and wells with exactly one cell are selected as single-cell wells.

[0021] The cloning cluster in step S4 contains at least 20 cells.

[0022] The normal rate in step S7 is the proliferation rate based on the proliferation rate of the original stable transfected cells.

[0023] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0024] S91. Digest the cells of the selected monoclonal cell line and proceed to step S92.

[0025] S92. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture (generally from 96-well plate → 24-well plate → 6-well plate → 6 cm culture dish → 10 cm culture dish), and proceed to step S93.

[0026] S93. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S92 have multiplied by 2 to 4 times or more, proceed to step S94.

[0027] S94. Determine whether the number of cells obtained in step S93 is sufficient for flow cytometry detection (generally, the number of cells collected by the flow cytometer should be greater than 10,000). If yes, proceed to step S96; otherwise, proceed to step S95.

[0028] S95. Digest the cells obtained in step S93 and return to step S92;

[0029] S96. After digesting the cells of the monoclonal cell line determined in step S94 that are sufficient for flow cytometry detection and the originally stable transfected cells cultured under the same culture conditions, the fluorescence intensity of the monoclonal cell line and the originally stable transfected cells is identified by flow cytometry.

[0030] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0031] S101. Digest the cells of the selected monoclonal cell line and proceed to step S102.

[0032] S102. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture (generally from 96-well plate → 24-well plate → 6-well plate → 6 cm culture dish → 10 cm culture dish), and proceed to step S103.

[0033] S103. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S102 have multiplied by 2 to 4 times or more, proceed to step S104.

[0034] S104. Determine whether the cells obtained in step S103 can cover 70% to 80% of the area of ​​a 10 cm culture dish. If yes, proceed to step S106; otherwise, proceed to step S105.

[0035] S105. Digest the cells obtained in step S103 and return to step S102.

[0036] S106. After digesting the monoclonal cell line that covers 70%–80% of the 10 cm culture dish area as determined in step S104 and the original stable transfected cells cultured under the same conditions, respectively, absorb the number of cells that can cover 40%–60% of the area of ​​a single well on the microplate, and seed them into the wells of the new microplate. Then, use a microplate imaging system to compare the difference in fluorescence intensity between the selected monoclonal cell line and the original stable transfected cells.

[0037] The selection criteria for the new microplate pores or new culture dishes in steps S92 and S102 are as follows: when the number of cells obtained from digestion can cover 70% to 80% of the area of ​​the current pores in the current microplate or the current culture dish, a new microplate with a larger pore diameter or a new culture dish with a larger diameter than the current culture dish is used.

[0038] Cells are classified into adherent cells and suspension cells based on whether they adhere to the culture medium. Since suspension cells are suspended in the culture medium and may be located at different levels, the microplate imaging system may not be able to clearly image all cells in the entire well. Therefore, this invention is determined to be applicable to adherent cells, but it is uncertain whether it is applicable to suspension cells.

[0039] The present invention has the following advantages over the prior art:

[0040] The monoclonal cell line screening method provided by this invention uses a microplate imaging system commonly used in universities to complete the imaging of all wells in one go, and analyzes indicators such as the number of cells in the well and the average fluorescence intensity of the cells. By using the imaging and analysis functions of the microplate imaging system several times, cell lines that are single-celled, have the strongest average fluorescence intensity, and can proliferate at a normal rate can be screened out as early as possible. This can greatly reduce the workload and also reduce the errors or even mistakes that may be caused by observation under a microscope or comparison of fluorescence intensity by human eyes. Attached Figure Description

[0041] Appendix Figure 1 A flowchart of a method for screening monoclonal cell lines based on fluorescence intensity provided by the present invention;

[0042] Appendix Figure 2 Green fluorescence imaging of well B4 during single-cell screening, as provided in the embodiments of the present invention;

[0043] Appendix Figure 3 Bright-field imaging of well B4 during single-cell screening, as provided in the embodiments of the present invention;

[0044] Appendix Figure 4 The cell count analysis results obtained by software analysis when screening single-cell wells in the embodiments provided by the present invention;

[0045] Appendix Figure 5 Green fluorescence imaging of well B4 during screening of clonal clusters in an embodiment of the present invention;

[0046] Appendix Figure 6 Green fluorescence imaging of well A1 (whose cells correspond to well B4 of a single-cell separation microplate) during the screening of fluorescence screening wells provided in the embodiments of the present invention.

[0047] Appendix Figure 7The average fluorescence intensity analysis results of cells in each well when screening fluorescence screening wells in the embodiments provided by the present invention;

[0048] Appendix Figure 8 Green fluorescence imaging of well A1 (whose cells correspond to well B4 of a single-cell separation microplate) during screening of cell division and proliferation wells provided in the embodiments of the present invention;

[0049] Appendix Figure 9 The green fluorescence intensity of the originally stably transfected cells as detected by flow cytometry is shown in Example 1 provided for verification of the present invention.

[0050] Appendix Figure 10 Example 1 provided for verification of the present invention shows the green fluorescence intensity of a monoclonal cell line derived from well B4 of a single-cell separation microplate as detected by flow cytometry.

[0051] Appendix Figure 11 Example 2 provided for verification of the present invention shows the confluence of originally stable transfected cells after seeding into a 96-well plate;

[0052] Appendix Figure 12 Example 2 provided for verification of the present invention shows the confluence of a monoclonal cell line derived from the B4 well of a single-cell isolation microplate after being seeded into a 96-well plate.

[0053] Appendix Figure 13 The green fluorescence image of the originally stably transfected cells after being seeded into a 96-well plate, as shown in Example 2 of the present invention;

[0054] Appendix Figure 14 The second verification example provided for this invention shows a green fluorescence image of a monoclonal cell line derived from well B4 of a single-cell isolation microplate after being seeded into a 96-well plate. Detailed Implementation

[0055] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided to make the technical solutions of the invention more comprehensive and complete, and to fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and therefore their detailed descriptions will be omitted.

[0056] The terms “a,” “one,” “the,” and “the” are used to indicate the existence of one or more elements / components / etc.; the terms “including” and “having” are used to indicate an open-ended meaning of inclusion, and to mean that there may be other elements / components / etc. in addition to the listed elements / components / etc.

[0057] A method for screening monoclonal cell lines based on fluorescence intensity, the flowchart of which is shown below. Figure 1 As shown, the steps of this monoclonal cell line screening method are as follows:

[0058] S1. Identify a cell line that is stably transfected with fluorescent proteins;

[0059] S2. Select a microplate for single-cell isolation. In step S1, cells stably transfected with fluorescent protein are introduced into the microplate, with multiple cells in the reference well and only one cell in each of the other wells.

[0060] On the same day that the cells stably transfected with fluorescent protein in step S2 are isolated into microplates, a microplate imaging system is used to screen out single-cell wells with only one cell in each well except the reference well.

[0061] S4. After culturing cells in single-cell wells for several days, a microplate imaging system is used to screen out the wells in which clonal clusters can grow.

[0062] S5. After digestion, the cells in each clone cluster well are transferred to the corresponding new wells on a new microplate for culture.

[0063] S6. After cells are transferred to new wells, the top 30% to 50% of the average fluorescence intensity of cells are selected as fluorescence screening wells using a microplate imaging system.

[0064] S7. After culturing the cells in the fluorescent screening wells for several days, use a microplate imaging system to screen out the fluorescent screening wells in which the cells can proliferate at a normal rate to 4 to 8 times the number of cells in the fluorescent screening wells in step S6. These fluorescent screening wells are used as division and proliferation wells. The normal rate is the proliferation rate based on the proliferation rate of the original stable transfected cells.

[0065] S8. Confirm that the cells in the cell division and proliferation wells are selected monoclonal cell lines.

[0066] The microplate used in step S2 is a black-walled, transparent-bottomed microplate; the number of cells in the reference well in step S2 is 50 to 200. The reference well is used to set the imaging parameters of the microplate imaging system, mainly the focal length and exposure time.

[0067] The microplate imaging system used in steps S3, S4, S6, and S7 must meet the following conditions: it must possess bright-field and fluorescence imaging capabilities; it must be able to automatically display whole-well imaging of a single well; it must be able to complete whole-well imaging of all wells on the entire microplate in a single automatic imaging session; and it must be able to analyze and display the number of cells in each well and the average fluorescence intensity of the cells in each well. The microplate imaging systems used in this invention include, but are not limited to, Tecumseh's SPARK CYTO, Zeiss's Celldiscoverer7, and Agilent's CYTATION5.

[0068] In step S3, the screening of single-cell wells is carried out by using the cell number analysis results of the microplate imaging system and / or by using the cell number to be determined by observing fluorescence plus bright field imaging with the human eye, and wells with exactly one cell are selected as single-cell wells.

[0069] The cloning cluster in step S4 contains at least 20 cells.

[0070] To illustrate the performance of the fluorescence intensity-based monoclonal cell line screening method provided by this invention, two verification methods are presented for validation.

[0071] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0072] S91. Digest the cells of the selected monoclonal cell line and proceed to step S92.

[0073] S92. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture, and proceed to step S93.

[0074] S93. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S92 have multiplied by 2 to 4 times or more, proceed to step S94.

[0075] S94. Determine whether the number of cells obtained in step S93 is sufficient for flow cytometry detection. If yes, proceed to step S96; otherwise, proceed to step S95.

[0076] S95. Digest the cells obtained in step S93 and return to step S92;

[0077] S96. After digesting the cells of the monoclonal cell line determined in step S94 that are sufficient for flow cytometry detection and the originally stable transfected cells cultured under the same culture conditions, the fluorescence intensity of the monoclonal cell line and the originally stable transfected cells is identified by flow cytometry.

[0078] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0079] S101. Digest the cells of the selected monoclonal cell line and proceed to step S102.

[0080] S102. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture, and proceed to step S103.

[0081] S103. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S102 have multiplied by 2 to 4 times or more, proceed to step S104.

[0082] S104. Determine whether the cells obtained in step S103 can cover 70% to 80% of the area of ​​a 10 cm culture dish. If yes, proceed to step S106; otherwise, proceed to step S105.

[0083] S105. Digest the cells obtained in step S103 and return to step S102.

[0084] S106. After digesting the monoclonal cell line that covers 70%–80% of the 10 cm culture dish area as determined in step S104 and the original stable transfected cells cultured under the same conditions, respectively, absorb the number of cells that can cover 40%–60% of the area of ​​a single well on the microplate, and seed them into the wells of the new microplate. Then, use a microplate imaging system to compare the difference in fluorescence intensity between the selected monoclonal cell line and the original stable transfected cells.

[0085] Example

[0086] The Tecsen SPARK CYTO is a multifunctional microplate reader with real-time imaging and analysis capabilities. It features bright-field and fluorescence imaging and can analyze fluorescence signals within the wells, making it suitable for fluorescent labeling experiments. The instrument's imaging and analysis software is relatively simple to operate and meets the imaging and analysis requirements of this experiment. Single-cell separation can be achieved using flow cytometry single-cell sorting or limiting dilution. While limiting dilution is instrument-independent, its single-cell entry rate is lower than flow cytometry single-cell sorting, and it cannot select single cells with strong fluorescence intensity. Flow cytometry, on the other hand, can separate single cells with strong fluorescence and has a high single-cell entry rate; therefore, flow cytometry single-cell sorting was used to separate single cells for microplate entry in this study.

[0087] This embodiment illustrates the process of using SPARK CYTO instead of instruments designed for observing and recording the formation of single-cell clones, such as the CloneSelect Imager cell growth analysis system or fluorescence microscopy, to screen for single-clonal cell lines with strong fluorescence intensity after flow cytometry sorting of adherent cells stably transfected with GFP (green fluorescent protein, the most commonly used of all fluorescent proteins).

[0088] This embodiment provides a method for screening monoclonal cell lines based on fluorescence intensity, the flowchart of which is shown below. Figure 1 As shown, the steps of this monoclonal cell line screening method are as follows:

[0089] S1. Identify a cell line that is stably transfected with fluorescent proteins;

[0090] HBE cells (bronchial epithelial cells, adherent cells) were identified and stably transfected with lentivirus, followed by selection with puromycin to ensure stable expression of GFP.

[0091] S2. Select a microplate for single-cell isolation. In step S1, cells stably transfected with fluorescent protein are introduced into the microplate, with multiple cells in the reference well and only one cell in each of the other wells.

[0092] After cell digestion, cells were resuspended in PBS and sorted using flow cytometry in single-cell sorting mode to insert cells with high fluorescence intensity into microplates. 96-well plates are the most commonly used microplates for single-cell sorting. Because 96-well plates with black walls and transparent bottoms provide better imaging than fully transparent 96-well plates, cells were sorted into 96-well plates with black walls and transparent bottoms. Well D7 was designated as the reference well, with 50 cells sorted into it (for rapid instrument acquisition of parameters such as focal length and optimal exposure time). One cell was sorted into each of the remaining 95 wells.

[0093] On the same day that the cells stably transfected with fluorescent protein in step S2 are isolated into the microplate, after 1-2 hours of cell separation and after the cells settle to the bottom, the microplate imaging system is used to screen out the single-cell wells with only one cell in the remaining wells except the reference well.

[0094] Specifically:

[0095] (1) Imaging: Open the imaging software, place the microplate into the instrument, select the corresponding brand and type of microplate, select the wells to be tested (all 96 wells), double-click the Fluorescence imaging function, select User defined for Application, select 4x objective (since there is no need to observe the internal structure of cells, 4x objective is sufficient for this embodiment. Although the instrument is equipped with higher magnification, using higher magnification would prolong the imaging time and increase the data file size, so it is not necessary to use high magnification), select Whole well for Pattern, keep the default "0μm" for Border offset, and check Brightfield and Green for Channel; click Live viewer, click Green to view green fluorescence imaging, click D7 well (D7 well is the reference well, set 50 cells to enter the well), select the area with cells in D7 well, click Acquisitionsettings, click Auto The exposure function automatically obtains parameters such as focal length and exposure time. If necessary, the exposure parameters (mainly the exposure time, which determines the intensity of green fluorescence in the image) are adjusted according to the imaging effect of the single cell well. Click Apply, then click Close to close the current interface. Data analysis retains the default settings. Click Start to start shooting. Image files and data files are automatically saved.

[0096] (2) Imaging Viewing: Open the analysis software and the automatically generated data file. The interface displays a microplate with 96 wells. Simply click on any well to view the whole-well fluorescence imaging and bright-field imaging of that well, thereby determining the number of cells in that well. If you need the imaging image file for each well, you can open the automatically saved image folder to view and copy it. In this embodiment, the flow cytometry ultimately verified that the homogeneity (percentage of cells with strong fluorescence) of the monoclonal cell line from well B4 of the single-cell separation microplate was the best among the three cell lines finally screened. Therefore, each subsequent step used the imaging of cells from well B4 of the single-cell separation microplate as the demonstration. The green fluorescence imaging of well B4 is shown below. Figure 2As shown, the image background is clean and the cell imaging is clear. Generally, when single cells expressing high levels of GFP are sorted into wells by flow cytometry, provided the flow cytometer is functioning well, the sorted cells will be highly fluorescent. Cell count can be determined using fluorescence imaging. If other methods are used to separate single cells into wells, or if there is a possibility of single cells with weak fluorescence, or if interference from autofluorescent impurities is ruled out, bright-field imaging can also be used to determine cell count. Bright-field imaging of well B4 is shown below. Figure 3 As shown, while bright-field imaging is not as immediately clear as fluorescence imaging, it still distinguishes cells from impurities quite well; cells that have not yet adhered are round and bright. If bright-field imaging cannot determine whether it is a cell or an impurity, it can be observed again after 3-5 days to see if it has divided. Cells that have not divided are either impurities or cells that cannot divide.

[0097] (3) Viewing Analysis Data: An Excel spreadsheet in the automatically generated folder displays multiple analysis results. If you are not satisfied with the analysis data obtained by the software's automatic parameter settings, you can reset the parameters and analyze again; the spreadsheet displays the cell count analysis results for each well. Figure 4 Due to the possibility of impurities in the wells, or two cells being too close together, or slight abnormalities in cell morphology, the cell count analysis of some wells may be inaccurate. Therefore, it is not recommended to directly adopt the cell count analysis results. Instead, the cell count as determined by human visual observation of fluorescence and bright-field imaging should be used to screen out wells containing exactly one cell. (The number of wells that can be screened out with a single cell depends on the flow cytometry single-cell sorting technology. If the flow cytometry single-cell sorting effect is good, at least half of the wells can be screened out here.) In this sorting, except for D7 (the reference well), the other 95 wells were set as single-cell wells. This step screened out 89 wells, excluding the 6 wells A2, A5, E7, F1, G8, and G9, as single-cell wells.

[0098] S4. After culturing cells in single-cell wells for 14 days, a microplate imaging system was used to screen out the wells in which clonal clusters could grow.

[0099] (1) Taking pictures: Same as step S3.

[0100] (2) Imaging examination: Among the single-cell wells, wells capable of growing clonal clusters (clonal clusters containing at least 20 cells) were further screened out. In this screening, 19 wells were selected as clonal cluster wells: B4, B7, B8, B10, C6, C11, D3, D9, D10, E4, E9, E10, F2, F3, F8, F10, G5, G11, and H7. Figure 5 Green fluorescence imaging of well B4 is shown.

[0101] S5. After digestion, the cells in each clone cluster well are transferred to the corresponding new wells on a new microplate for culture.

[0102] Since most cells in a clonal cluster are crowded together, which is not conducive to their division and proliferation, for wells that are confirmed to be single cells and capable of growing clonal clusters (well 19 in step S4), the cells in each well are digested and transferred to the corresponding new wells in a new 96-well plate for culture to promote their division and proliferation.

[0103] S6. After cells are transferred to new wells, the top 6 wells with the highest average fluorescence intensity of cells are selected as fluorescence screening wells using a microplate imaging system.

[0104] (1) Taking pictures: Same as step S3.

[0105] (2) Imaging observation: Green fluorescence imaging of cells from well B4 of the single-cell isolation microplate after digestion and transfer to new wells, as shown in the figure. Figure 6 As shown.

[0106] (3) Analyze and view the data: Figure 7 The provided table shows the average fluorescence intensity analysis results of cells in each new well. Each data point represents the average green fluorescence value (unit: relative fluorescence unit) in the corresponding new well. Cell lines with strong fluorescence intensity are screened out based on their average fluorescence intensity. The reason for analyzing the average fluorescence intensity after the first digestion, rather than before, is that before digestion, the cells form a clonal cluster, crowded together, leading to significant errors in cell identification by the software, and consequently, large errors in the calculation of average fluorescence intensity. After the first digestion, the cells are dispersed and seeded into new wells, with relatively clear spacing between cells, resulting in smaller errors in cell identification by the software. Therefore, the calculated average fluorescence intensity is more reliable, and the data from the software analysis of the average fluorescence intensity of each well can quickly screen out wells with strong average fluorescence intensity. In this study, wells A1, A5, B1, B2, B5, and B6 (corresponding to wells B4, C6, F2, F3, G5, and G11 from the single-cell isolation microplate) were selected from the 19 wells above as fluorescence screening wells.

[0107] S7. Nine days after digestion, based on the proliferation rate of the original stable transfected cells, a microplate imaging system was used to screen out fluorescent screening wells that could proliferate at a normal rate to 4 to 8 times the number of cells in the fluorescent screening wells in step S6 as division and proliferation wells.

[0108] (1) Taking pictures: Same as step S3.

[0109] (2) Imaging and observation: Cell proliferation capacity was inferred by the change in the number of cells in the same well. Based on the proliferation rate of the originally stably transfected cells, the microplate imaging system was used to screen out the fluorescent screening wells that could proliferate at a normal rate to 4-8 times the number of cells in the fluorescent screening wells in step S6 as proliferation wells. In this study, wells A1, B2, and B5 of the current microplate (whose cells correspond to wells B4, F3, and G5 of the single-cell separation microplate) were selected as proliferation wells. The green fluorescence imaging of well A1 of the current microplate (whose cells correspond to well B4 of the single-cell separation microplate) is shown below. Figure 8 As shown.

[0110] S8. Determine that the cells in wells A1, B2, and B5 of the current microplate (the cells in which are derived from wells B4, F3, and G5 of the single-cell isolation microplate) are the three selected single-clonal cell lines.

[0111] The number of days referred to in this embodiment is the number of days suitable for the cells used in this embodiment, which was explored through practice. The number of days may be different for different cells and can be adjusted according to the characteristics of the cells. If the cells divide and proliferate quickly, the number of days can be shortened accordingly, and if the cells divide and proliferate slowly, the number of days can be extended accordingly. The number of days referred to in the following verification examples is also only applicable to the verification examples.

[0112] Verification Example 1

[0113] Taking the monoclonal cell line derived from the B4 well of a single-cell isolation microplate as an example, the monoclonal cell line screened by the monoclonal cell line screening method provided in this invention is validated.

[0114] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0115] S91. Digest the cells of the selected monoclonal cell line and proceed to step S92.

[0116] S92. Based on the number of cells obtained from digestion, transfer the cells from the 96-well plate to the wells of a new microplate or a new culture dish for culture, and proceed to step S93.

[0117] S93. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S92 have multiplied by 2 to 4 times or more, proceed to step S94.

[0118] S94. Determine whether the number of cells obtained in step S93 is sufficient for flow cytometry detection. If yes, proceed to step S96; otherwise, proceed to step S95.

[0119] S95. Digest the cells obtained in step S93 and return to step S92;

[0120] S96. After digesting the cells of the monoclonal cell line determined in step S94 that are sufficient for flow cytometry detection and the originally stable transfected cells cultured under the same culture conditions, the fluorescence intensity of the monoclonal cell line and the originally stable transfected cells is identified by flow cytometry.

[0121] Steps S94, S95, and S96 are as follows: After sorting, the selected monoclonal cell lines and the previously stably transfected cells are continuously cultured under the same conditions. In the embodiment provided by this invention, 48 days after sorting, the selected monoclonal cell lines are proliferated to a sufficient number for flow cytometry detection. The monoclonal cell lines and the previously stably transfected cells are digested separately, and a portion of the cells is used for flow cytometry to identify fluorescence intensity, while the other portion is further cultured. Figure 9 , Figure 10 As can be seen, under identical analytical conditions and gating settings, the mean GFP (green fluorescence) value of the originally stably transfected cells was 1597, with cells highly expressing green fluorescent protein accounting for 15.7% of the total. In contrast, the mean GFP value of the monoclonal cell line from well B4 of the single-cell separation microplate was 16214, with cells highly expressing green fluorescent protein accounting for 99.8% of the total. In summary, the monoclonal cell line from well B4 of the single-cell separation microplate screened by this invention exhibits generally stronger fluorescence intensity. Both the average fluorescence intensity and the percentage of cells with strong fluorescence are significantly higher than those of the originally stably transfected cells. Therefore, the fluorescence intensity-based monoclonal cell line screening provided by this invention is highly successful.

[0122] Verification Example 2

[0123] Taking the monoclonal cell line from well B4 of the single-cell separation microplate as an example, the monoclonal cell line screened by the monoclonal cell line screening method provided in this invention is verified.

[0124] A method for validating monoclonal cell lines screened using a fluorescence intensity-based monoclonal cell line screening method, comprising the following steps:

[0125] S101. Digest the cells of the selected monoclonal cell line and proceed to step S102.

[0126] S102. Based on the number of cells obtained from digestion, transfer the cells to the wells of a new microplate or a new culture dish for culture, and proceed to step S103.

[0127] S103. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S102 have multiplied by 2 to 4 times or more, proceed to step S104.

[0128] S104. Determine whether the cells obtained in step S103 can cover 70% to 80% of the area of ​​a 10 cm culture dish. If yes, proceed to step S106; otherwise, proceed to step S105.

[0129] S105. Digest the cells obtained in step S103 and return to step S102.

[0130] S106. After digesting the monoclonal cell line that covers 70%–80% of the 10 cm culture dish area as determined in step S104 and the original stable transfected cells cultured under the same conditions, respectively, absorb the number of cells that can cover 40%–60% of the area of ​​a single well on the microplate, and seed them into the wells of the new microplate. Then, use a microplate imaging system to compare the difference in fluorescence intensity between the selected monoclonal cell line and the original stable transfected cells.

[0131] The specific details of steps S104, S105, and S106 are as follows: After sorting, the selected monoclonal cell lines and the previously stable transfected cells are continuously cultured under the same conditions. In the embodiment provided by this invention, 57 days after sorting, the selected monoclonal cell lines proliferate to cover 80% of the area of ​​a 10 cm culture dish (some cells were lost during this period due to the use of some cells from the monoclonal cell lines for verification in Example 1). After digesting the monoclonal cell lines and the previously stable transfected cells, a number of cells sufficient to cover 40% of the area of ​​each well in a 96-well plate are aspirated and seeded into new wells. Both wells are then simultaneously photographed and imaged for observation.

[0132] (1) Taking pictures: Same as step S3.

[0133] (2) Imaging and Viewing: Since it's best to compare the fluorescence intensity of different cells using fluorescence imaging while ensuring a similar total cell count, but bright-field imaging doesn't clearly show the cell number, the software automatically calculates the cell confluence level. The confluence value represents the area of ​​the pores covered by cells, expressed as a percentage of the total value. The confluence value has a good positive correlation with the cell number. The software automatically generates an image displaying the confluence level, displayed as a yellow overlay for visual confirmation by the user. Figure 11 , Figure 12 It is evident that the confluence of the previously stably transfected cells and the monoclonal cell lines derived from well B4 of the single-cell isolation microplate was essentially the same in the current well; Figure 13 , Figure 14 It is evident that the fluorescence intensity of the monoclonal cell line derived from the B4 well of the single-cell isolation microplate is significantly stronger than that of the original stable transfected cells. In other words, the fluorescence intensity of the monoclonal cell line screened by the monoclonal cell line screening method provided by this invention is significantly enhanced compared with the original cells.

[0134] (3) Data analysis: The confluence of the original stable transfected cells and the monoclonal cell line from well B4 of the single-cell isolation microplate in the current well is 43% and 42%, respectively, which further confirms that the confluence of the two is basically the same.

[0135] The embodiments provided by this invention were verified using two methods: flow cytometry and Spark Cyto Imaging from Teco. The verification results showed that the selected cell lines had excellent fluorescence intensity.

[0136] The single-clonal cell line screening method based on fluorescence intensity proposed in this invention has the following advantages compared with screening using fluorescence microscopy observation and imaging:

[0137] (1) One-time imaging: One automatic imaging can complete the imaging of all the holes of a whole microplate.

[0138] (2) Convenient image observation: The analysis software opens the original file and you can easily observe the bright field and fluorescence imaging of each well by simply clicking on any well without making any mistakes.

[0139] (3) Software analysis of data: The software analyzes the number of cells and the average fluorescence intensity of cells in each well. Especially after the first digestion after the formation of the clonal cluster, the cells are broken up. The software analysis of the number of cells in each well and the average fluorescence intensity of cells is relatively accurate. Based on this, wells with stronger average fluorescence intensity of cells can be selected and wells with weaker average fluorescence intensity of cells can be discarded. This step is very important and can reduce a considerable amount of workload.

[0140] In the description of the embodiments of the present invention, it should be understood that the terms "upper" and "lower" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or unit referred to must have a specific orientation or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of the present invention.

[0141] In the description of this specification, the terms "an embodiment," "a preferred embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0142] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solutions based on the technical concept proposed in this invention shall fall within the scope of protection of this invention. Technologies not covered in this invention can be implemented using existing technologies.

Claims

1. A method for screening monoclonal cell lines based on fluorescence intensity, characterized in that: The steps of this method are as follows: S1. Identify a cell line that is stably transfected with fluorescent proteins; S2. Select a microplate for single-cell isolation and set a reference well. Cells stably transfected with fluorescent protein in step S1 are introduced into the microplate, with multiple cells introduced into the reference well and only one cell introduced into the other wells. On the same day that the cells stably transfected with fluorescent protein in step S2 are isolated into microplates, a microplate imaging system is used to screen out single-cell wells with only one cell in each well except the reference well. S4. After culturing cells in single-cell wells for several days, a microplate imaging system is used to screen out the wells in which clonal clusters can grow. S5. After digestion, the cells in each clone cluster well are transferred to the corresponding new wells on a new microplate for culture. S6. After cells are transferred to new wells, the top 30% to 50% of the average fluorescence intensity of cells are selected as fluorescence screening wells using a microplate imaging system. S7. After culturing the cells in the fluorescent screening wells for several days, the microplate imaging system is used to screen out the fluorescent screening wells in which the cells can proliferate at a normal rate to 4 to 8 times the number of cells in the fluorescent screening wells in step S6 as division and proliferation wells. S8. Confirm that the cells in the cell division and proliferation wells are selected monoclonal cell lines.

2. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 1, characterized in that: The microporous plate used in step S2 is a microporous plate with a black wall and a transparent bottom.

3. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 1, characterized in that: The number of cells in the reference well in step S2 is 50 to 200, and the reference well is used to set the imaging parameters of the microplate imaging system.

4. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 1, characterized in that: The microplate imaging system in steps S3, S4, S6, and S7 needs to meet the following conditions: it should have bright-field and fluorescence imaging capabilities, be able to automatically present whole-well imaging of a single well, be able to complete whole-well imaging of all wells on the entire microplate in one automatic imaging session, and be able to analyze and display the number of cells in each well and the average fluorescence intensity of the cells in each well.

5. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 4, characterized in that: The microplate imaging systems in steps S3, S4, S6, and S7 include, but are not limited to, Tecan's SPARK CYTO, Zeiss's Celldiscoverer7, and Agilent's CYTATION5.

6. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 1, characterized in that: In step S3, the screening of single-cell wells is carried out by using the cell number analysis results of the microplate imaging system and / or by using the cell number to be determined by observing fluorescence plus bright field imaging with the human eye, and wells with exactly one cell are selected as single-cell wells.

7. The method for screening monoclonal cell lines based on fluorescence intensity according to claim 1, characterized in that: The cloning cluster in step S4 contains at least 20 cells.

8. A method for verifying monoclonal cell lines screened by the fluorescence intensity-based monoclonal cell line screening method as described in any one of claims 1-7, characterized in that: The steps of this verification method are as follows: S91. Digest the cells of the selected monoclonal cell line and proceed to step S92. S92. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture, and proceed to step S93. S93. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S92 have multiplied by 2 to 4 times or more, proceed to step S94. S94. Determine whether the number of cells obtained in step S93 is sufficient for flow cytometry detection. If yes, proceed to step S96; otherwise, proceed to step S95. S95. Digest the cells obtained in step S93 and return to step S92; S96. After digesting the cells of the monoclonal cell line determined in step S94 that are sufficient for flow cytometry detection and the originally stable transfected cells cultured under the same culture conditions, the fluorescence intensity of the monoclonal cell line and the originally stable transfected cells is identified by flow cytometry.

9. A method for verifying monoclonal cell lines screened by the fluorescence intensity-based monoclonal cell line screening method as described in any one of claims 1-7, characterized in that: The steps of this verification method are as follows: S101. Digest the cells of the selected monoclonal cell line and proceed to step S102. S102. Based on the number of cells obtained from digestion, transfer the digested cells to the wells of a new microplate or a new culture dish for culture, and proceed to step S103. S103. After several days of culture, when the cells in the wells of the new microplate or the new culture dish from step S102 have multiplied by 2 to 4 times or more, proceed to step S104. S104. Determine whether the cells obtained in step S103 can cover 70% to 80% of the area of ​​a 10 cm culture dish. If yes, proceed to step S106; otherwise, proceed to step S105. S105. Digest the cells obtained in step S103 and return to step S102. S106. After digesting the monoclonal cell line that covers 70%–80% of the 10 cm culture dish area as determined in step S104 and the original stable transfected cells cultured under the same conditions, respectively, absorb the number of cells that can cover 40%–60% of the area of ​​a single well on the microplate, and seed them into the wells of the new microplate. Then, use a microplate imaging system to compare the difference in fluorescence intensity between the selected monoclonal cell line and the original stable transfected cells.

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