Detection kit for density and motility rate of feeder cells and detection method thereof

Abstract

The invention provides a feeder cell density and motility rate detection kit and a detection method thereof, and belongs to the technical field of cell detection. The invention specifically discloses a method for detecting cell density and / or motility rate, which comprises the following steps: (1) exposing feeder cells to a CFSE dye environment for dyeing; (2) removing the CFSE dye in the environment; (3) culturing the feeder cells under proper conditions; (4) adding absolute counting microspheres into the cultured feeder cells and exposing the feeder cells in a dead cell dye environment for dyeing; and (5) calculating the density or the motility rate of the living cells according to a dyeing result and absolute counting microspheres. The method has good specificity, accuracy, precision and durability, and has good popularization value and market application prospect.

Description

Technical Field

[0001] This invention patent belongs to the field of cell detection technology, specifically relating to a detection kit and detection method for feeder cell density and viability. Background Technology

[0002] Feeder cells are a "key support" for the in vitro culture of NK cells—they solve the bottleneck problems of difficult in vitro proliferation and low activity of NK cells through three core functions: signal stimulation, nutrient buffering, and activity maintenance. Whether it is to obtain sufficient NK cells for mechanism exploration in basic research or to prepare NK cell preparations that meet infusion standards in clinical applications, the addition of feeder cells is one of the core strategies to improve culture efficiency and cell quality.

[0003] Safety evaluation of feeder cells is an indispensable part of NK cell in vitro culture (especially in clinical applications). The core objective is to eliminate contamination of the final product (NK cells) by feeder cells and avoid immune rejection or potential carcinogenic risks to recipients. Tumorigenicity is a crucial indicator in feeder cell safety evaluation, therefore, the detection of feeder cell proliferation capacity is essential.

[0004] In the early stages of culture, feeder cells typically exhibit high viability and density, facilitating rapid and accurate cell counting using equipment such as cell counters. However, as culture time increases, feeder cells gradually initiate apoptosis, leading to a decrease in density and viability. When these levels fall below a certain threshold, the accuracy and reliability of traditional methods like cell counters significantly decline, rendering them unsuitable for monitoring cell status. Therefore, there is an urgent need to develop a stable and effective new method for assessing the density and viability of cultured feeder cells. Summary of the Invention

[0005] In view of this, and in response to the shortcomings of the existing technology, the purpose of this application is to provide a test kit and method for detecting the cell density and viability of feeder cells. By analyzing the changing trends of cell density and viability of K562 feeder cells, the proliferation status of feeder cells can be determined, thereby confirming whether the feeder cells meet the release testing standards. This method is applicable to the tumorigenicity and safety evaluation of feeder cells such as K562 feeder cells.

[0006] To address the aforementioned technical problems, in a first aspect, the present invention provides a kit for detecting cell density and / or viability, the kit comprising CFSE dye, dead cell dye, and absolute counting microspheres.

[0007] In a second aspect, the present invention provides a method for detecting cell density and / or viability, the method comprising: (1) Expose the feeder cells to the above-mentioned CFSE dye environment; the concentration of the CFSE dye is 0.5 µM-10 µM; (2) After step (1), remove CFSE dye from the environment; (3) After step (2), the feeder cells are cultured for 0-21 days under suitable conditions; (4) After step (3), the cultured feeder cells are added to the absolute counting microspheres as described in any of the above and exposed to the dead cell dye environment as described in any of the above.

[0008] Thirdly, this invention provides the application of the above-mentioned kit in detecting the cell density and viability of feeder cells.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: (1) After the feeder cells are revived, cell counting and viability testing are performed.

[0010] (2) Use a certain concentration of CFSE dye to stain and label feeder cells. After labeling, quantitatively transfer feeder cells into 96-well culture plates.

[0011] (3) Samples of feeder cells in the 96-well plate were taken and tested on the day of plate laying (i.e., D0), D7, D14 and D21 respectively.

[0012] (4) Add a certain amount of absolute counting magnetic beads and live / dead dye to the cell suspension in each well, mix thoroughly, and then use a flow cytometer for detection.

[0013] (5) The labeled feeder cells can be circled by FITC (CFSE), and then double-gating is performed by FITC (CFSE) and PC5.5 (7-AAD) to obtain the proportion of live cells in the labeled feeder cells. The density of the feeder cells can be calculated by absolute counting microspheres.

[0014] Beneficial effects: This invention employs flow cytometry absolute counting to accurately quantify the density and viability of feeder cells. Simultaneously, CFSE dye labeling of target cells effectively distinguishes feeder cells from impurities such as debris generated by apoptosis in the culture. Combined with 7-AAD live / dead dye, the viability of feeder cells can be directly analyzed and delineated using flow cytometry. The concentration of CFSE dye was optimized, showing good results at concentrations of 0.5 µM–10 µM, especially 2 µM. Further screening of culture time revealed that D0, D7, and D14 (or D21) can be used as detection time points for the method.

[0015] This method has undergone systematic methodological validation, and its key indicators such as specificity, accuracy, precision, and robustness strictly comply with the relevant pharmacopoeia regulations. It is applicable to cell therapy products in multiple stages such as quality research, release testing, stability and comparability studies, and has good promotional value and market application prospects. Attached Figure Description

[0016] Figure 1 This is a gate logic diagram, where Target cells are target cells, Lives cells are live cells, Dead cells are dead cells, and Beads are counting microspheres.

[0017] Figure 2 The diagram illustrates the use of the FSC-A and SSC-A channels to delineate the target cell population. The middle section shows the use of the CFSE (FITC or FL1) and 7-AAD (PC5.5 or FL3) channels to delineate the live cells and dead cells populations from the target cell population. The right section shows the use of the PB450 (or V1) and SSC-A channels to delineate the bead population.

[0018] Figure 3 The graph shows the number of events and percentage for each group; where Events represents the number of events recorded by each group, %Total represents the percentage of each group in the total number of events, and %Parent represents the percentage of each group in the total number of events; Targetcells represents the target cells, Lives cells represent live cells, Dead cells represent dead cells, and Beads represent the counting microspheres.

[0019] Figure 4 This is a trend graph of live cell density and cell viability; the red curve represents viability, and the blue curve represents live cell density.

[0020] Figure 5 CFSE spectra of Miltenyi (left) and Beckman (right) on a flow cytometer.

[0021] Figure 6 The graph shows the changes in cell density at different culture times; the blue curve represents the growth curve of feeder cells (K562 feeder cells), and the red curve represents the growth curve of positive control cells (K562).

[0022] Figure 7The reference gate diagram for identifying the proportion of CFSE+ populations in data analysis is as follows: the top left side shows the target cell population identified using the FSC-A and SSC-A channels; the top right side shows the bead population identified using the PB450 (or V1) and SSC-A channels; the middle left side shows the Live cells and Dead cells populations identified from the target cell population using the CFSE (FITC or FL1) and 7-AAD (PC5.5 or FL3) channels; the middle right side shows the peak plot analysis of CFSE (FITC or FL1) to identify the CFSE+ population and its proportion; and the bottom middle side shows the number of events and percentage of each population.

[0023] Figure 8 This is a graph showing the results of the specificity test; the dark blue curve represents the density change curve of the positive control reference cells (K562), the light blue curve represents the viability change curve of the positive control reference cells (K562), the dark red curve represents the density change curve of the feeder cells (K562), and the light red curve represents the viability change curve of the feeder cells (K562). Detailed Implementation

[0024] The specific embodiments of this application will be further described in detail below with reference to the accompanying drawings. These embodiments are only for illustrating this application and are not intended to limit the scope of the invention.

[0025] The terms “K562 Feeder cells”, “K562”, “K562 cells”, “K562 Feeder”, or “K56 cells” used in this application refer to the same cell.

[0026] The term "CFSE" or "CFSE dye" used in this application refers to carboxyfluorescein succinimidyl ester, a green fluorescent dye that can penetrate cell membranes and is used to label target cells for analysis. Specifically, it can be 5(6)-carboxyfluorescein succinimidyl ester, 6-carboxyfluorescein succinimidyl ester, or 5-carboxyfluorescein succinimidyl ester.

[0027] The “7-AAD” used in this application refers to 7-Aminoactinomycin D, CAS number 7240-37-1, which is a fluorescent dye that can bind to the DNA of cells with membrane damage and is used to mark dead cells in a cell population.

[0028] Both CFSE and 7-AAD cell dyes are excited by 488 nm excitation light and generate fluorescence reception signals in the FL1 (FITC) and FL3 (PC5.5) channels. The CFSE+7-AAD+ population represents dead cells in the target cell population, while the CFSE+7-AAD- population represents live cells in the target cell population. Absolute cell counting is performed using absolute counting beads. Bead populations can be obtained by gating using other fluorescence channels of the flow cytometer (such as PB450 or V1) and SSC-A.

[0029] The "absolute counting microspheres" used in this application are tiny particles (typically made of latex or polymer) of known, fixed concentration, with fluorescent labels on their surface, enabling stable identification by flow cytometry. By being analyzed alongside target cells, the "relative proportion" of cells measured by the instrument can be converted into the "absolute number" per unit volume (e.g., per microliter, per milliliter). Specific embodiments may include Trucount™ Tubes, True™ Absolute Counting Beads, Flow-Count™ Fluorospheres, CountBright™ Absolute Counting Beads, KnownConcentration Beads, AccuCheck Counting Beads, Spherotech, and Sigma-Aldrich.

[0030] In a first aspect, the present invention provides a kit for detecting cell density and / or viability, the kit comprising CFSE dye, dead cell dye, and absolute counting microspheres.

[0031] In some embodiments, the concentration of CFSE in the CFSE dye is 0.5 µM-10 µM; preferably, the concentration of CFSE is 0.5 µM, 0.7 µM, 0.9 µM, 1.0 µM, 1.2 µM, 1.5 µM, 1.8 µM, 2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM, 4.0 µM, 4.5 µM, 5.0 µM, 7 µM, 8 µM, 9 µM, or 10 µM. In some embodiments, the dead cell dye is selected from 7-AAD dye, propidium iodide, DAPI, SYTOX® series dyes, fixed reactive dyes, and near-infrared reactive dyes; preferably, the dead cell dye is 7-AAD dye. In some embodiments, the absolute counting microspheres include light scattering counting microspheres or fluorescence counting microspheres.

[0032] In some embodiments, the concentration of 7-AAD in the 7-AAD dye is 0.5-1.5 µg / mL; In some embodiments, the concentration of 7-AAD is 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 1.1 µg / mL, 1.2 µg / mL, 1.3 µg / mL, 1.4 µg / mL, or 1.5 µg / mL; In some embodiments, the volume of the fluorescent counting microspheres is 3 μL, 4 μL, 5 μL, 6 μL, or 7 μL.

[0033] In some embodiments, the dead cell dye includes 7-AAD dye, propidium iodide, DAPI, SYTOX® series (such as SYTOX Blue, Green, Red, NIR), fixed reactive dye, and near-infrared reactive dye; preferably, the dead cell dye is 7-AAD dye; In some embodiments, the absolute counting microspheres include light scattering counting microspheres or fluorescence counting microspheres.

[0034] In a second aspect, the present invention provides a method for detecting cell density and / or viability, the method comprising: (1) Expose the feeder cells to the CFSE dye in the above kit; the concentration of the CFSE dye is 0.5 µM-10 µM; (2) After step (1), remove CFSE dye from the environment; (3) After step (2), the feeder cells are cultured for 0-21 days under suitable conditions; (4) After step (3), the cultured feeder cells are added to the absolute counting microspheres as described in any of the above and exposed to the dead cell dye environment as described in any of the above.

[0035] In some embodiments, the number of cultivation days is 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0036] In some embodiments, the method further includes (5) after step (4), detecting the number of live cells, the number of dead cells, and / or the absolute count of microspheres, and calculating the live cell density or viability.

[0037] In some embodiments, the cell density = ((number of live cells / number of absolute count microspheres) × absolute count microsphere concentration × absolute count microsphere volume) / (dead cell dye volume + absolute count microsphere volume). In some embodiments, the cell density = ((number of live cells / number of absolute count microspheres) × absolute count microsphere concentration × absolute count microsphere volume × 1000) / (volume of dead cell dye + volume of absolute count microspheres). In some embodiments, the viability rate = number of live cells / (number of live cells + number of dead cells).

[0038] In some embodiments, the dead cells are a population that is CFSE-positive and 7-AAD-positive; In some embodiments, the live cells are a population that is CFSE positive and 7-AAD negative; In some embodiments, the live cells are a population that is CFSE positive and 7-AAD negative; In some embodiments, the absolute count microspheres are a population that is positive for SSC-A and PB450, or a population that is positive for SSC-A and V1; In some embodiments, the positivity or negativity of the CFSE is determined by the FITC gate; In some embodiments, the positivity or negativity of the 7-AAD is determined by a PC5.5 gate. In some embodiments, the absolute count microspheres are determined by the positive gate of the SC-A and PB450 channels, or by the positive gate of the SSC-A and V1 channels; In some embodiments, the positivity or negativeness of the CFSE is determined by the FITC gate; in some embodiments, the positivity or negativeness of the 7-AAD is determined by the PC5.5 gate; preferably, the absolute count microspheres are determined by the SSC-A and PB450 or V1 gates. Alternatively, the determination is made by flow cytometry; most preferably, the flow cytometer is a Beckman or Miltenyi.

[0039] In some embodiments, the CFSE is determined by the fluorescence reception signal of the FL1 (FITC) channel.

[0040] The 7-AAD is determined by the fluorescence receiving signal of the FL3 (PC5.5) channel.

[0041] In some embodiments In some embodiments, the fluorescence counting microspheres are determined by a fluorescence channel (such as PB450 or V1) and an SSC-A fluorescence receiving signal.

[0042] In some embodiments, in step (1), the temperature of the exposure is 34.5-40.5°C; In some embodiments, the temperature is 34.5°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or 40.5°C. In some embodiments, in step (1), the CFSE dye environment The concentration of CFSE is 0.5 µM-10 µM; in some embodiments, the concentration of CFSE is 0.5 µM, 0.7 µM, 0.9 µM, 1.0 µM, 1.2 µM, 1.5 µM, 1.8 µM, 2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM, 4.0 µM, 4.5 µM, 5.0 µM, 7 µM, 8 µM, 9 µM, or 10 µM. In some embodiments, in step (1), the exposure time is 5-15 min; the time is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min. In some embodiments, in step (1), the cell density is 1E5-1E7 cells / mL; in some embodiments, the cell density is 1E5 cells / mL, 1E6 cells / mL, or 1E7 cells / mL. In some embodiments, step (1) further includes the step of mixing the cells with CFSE dye; In some embodiments, the removal method in step (2) is centrifugation; in some embodiments, the relative centrifugal force is 300-400g; in some embodiments, the centrifugal force is 300g, 310g, 320g, 330g, 340g, 350g, 360g, 370g, 380g, 390g, or 400g; in some embodiments, the centrifugation time is 3-7min; in some embodiments, the centrifugation time is 3min, 4min, 5min, 6min, or 7min. In some embodiments, step (2) includes the removal step comprising dilution, centrifugation, and resuspension; in some embodiments, the dilution factor is 5-15 times; in some embodiments, the factor is 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times; in some embodiments, the diluted solution is a culture medium; in some embodiments, the culture medium is RPMI 1640 complete culture medium; in some embodiments, the resuspension solution is a culture medium; in some embodiments, the culture medium is RPMI 1640 complete culture medium. In some embodiments, in step (3), the cell density is 2E4-2E7 cells / mL; in some embodiments, the cell density is 2E4 cells / mL, 5E4 cells / mL, 1E5 cells / mL, 2E5 cells / mL, 5E5 cells / mL, 1E6, 2E6, 5E6, 1E7, 2E7 cells / mL; In some embodiments, the conditions in step (3) include a temperature of 34.5-40.5°C; in some embodiments, the temperature is 34.5°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or 40.5°C. In some embodiments, in step (3), the conditions include a carbon oxide concentration of 5.0 ± 0.5%; In some embodiments, in step (3), the conditions include the culture medium being RPMI 1640 complete culture medium; In some embodiments, in step (3), the number of days of cultivation is 1-21 days.

[0043] In some embodiments, in step (3), the number of days of cultivation is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days; In some embodiments, in step (4), the concentration of dead cell dye in the dead cell dye environment is 0.5-1.5 µg / mL; in some embodiments, the concentration of dead cell dye is 0.5 µg / mL, 0.6 µg / mL, 0.7 µg / mL, 0.8 µg / mL, 0.9 µg / mL, 1.0 µg / mL, 1.1 µg / mL, 1.2 µg / mL, 1.3 µg / mL, 1.4 µg / mL, or 1.5 µg / mL. In some embodiments, the exposure time in step (4) is 5-15 min; in some embodiments, the exposure time is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min. In some embodiments, in step (4), the temperature of the exposure is 2-8°C; the temperature is 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C. In some embodiments, step (4) involves a light-avoidance operation; In some embodiments, step (4) further includes the step of mixing the cultured cells, absolute count microspheres, and the dead cell dye.

[0044] In some embodiments, the trophoblast cells include K562 cells, Daudi cells, RPMI-8866 cells, Jurkat cells, EBV-LCL cells, autologous or allogeneic PBMCs, bone marrow mesenchymal stem cells, CD34+ hematopoietic progenitor cells derived from umbilical cord blood / peripheral blood, placental-derived primary trophoblast cells, amnion / chorionic mesenchymal cells, or their transgenic cell lines.

[0045] In some embodiments, the K562 cells are K562 trophoblast cells.

[0046] Thirdly, this invention provides the application of the above-mentioned kit in detecting the cell density and viability of feeder cells.

[0047] In some embodiments, the trophoblast cells include K562 cells, Daudi cells, RPMI-8866 cells, Jurkat cells, EBV-LCL cells, autologous or allogeneic PBMCs, bone marrow mesenchymal stem cells, CD34+ hematopoietic progenitor cells derived from umbilical cord blood / peripheral blood, placental-derived primary trophoblast cells, amnion / chorionic mesenchymal cells, or their transgenic cell lines.

[0048] In some embodiments, the trophoblast cells are revived cells.

[0049] In some embodiments, the revived cells are revived after being exposed to -20°C.

[0050] In some embodiments, the revival temperature is 37.0 ± 0.5 °C.

[0051] On the other hand, this application provides the application of the kit in detecting the cell density or viability of feeder cells.

[0052] In some embodiments, the application is for the preparation of related products.

[0053] In some embodiments, the feeder cells are K562 feeder cells or K562 cells.

[0054] In some embodiments, the method further includes the step of mixing the cultured cells, absolute counting microspheres, and / or the dead cell dye.

[0055] To achieve the above objectives, the present invention adopts the following technical solution: (1) After the feeder cells are revived, cell counting and viability testing are performed.

[0056] (2) Use a certain concentration of CFSE dye to stain and label feeder cells. After labeling, quantitatively transfer feeder cells into 96-well culture plates.

[0057] (3) Samples of feeder cells in the 96-well plate were taken and tested on the day of plate laying (i.e., D0), D7, D14 and D21 respectively.

[0058] (4) Add a certain amount of absolute counting magnetic beads and live / dead dye to the cell suspension in each well, mix thoroughly, and then use a flow cytometer for detection.

[0059] (5) The labeled feeder cells can be circled by FITC (CFSE), and then double-gating is performed by FITC (CFSE) and PC5.5 (7-AAD) to obtain the proportion of live cells in the labeled feeder cells. The density of the feeder cells can be calculated by absolute counting microspheres.

[0060] In this application, the K562 feeder cells were prepared by irradiation of soybeans from Jiangsu Puxin Biotechnology Co., Ltd., catalog number: HG-FEC002-GMP-2. The K562 cells were purchased from ATCC, catalog number CCL-43.

[0061] Example 1 This invention provides a flow cytometry method for absolute counting and cell proliferation detection using dual-dye labeling with CFSE and 7-AAD. CFSE (Carboxyfluorescein Succinimidyl Ester) is a cell membrane-penetrating green fluorescent dye used to label target cells for analysis; 7-AAD (7-aminoactinomycin D) is a fluorescent dye that binds to the DNA of membrane-damaged cells and is used to label dead cells in the cell population. Both CFSE and 7-AAD are excited by 488 nm excitation light and generate fluorescence reception signals in the FL1 (FITC) and FL3 (PC5.5) channels, respectively. The CFSE+7-AAD+ population represents dead cells in the target cell population, while the CFSE+7-AAD- population represents live cells in the target cell population. Flow cytometry absolute counting is performed using absolute counting microspheres (Beads). The Bead population can be obtained by gating using other fluorescence channels of the flow cytometer (such as PB450 or V1) and SSC-A.

[0062] The specific method is as follows: 1. Preparation before the experiment 1.1 Preparation of RPMI 1640 complete medium: 89 V / V% RPMI 1640 + 10 V / V% FBS + 1 V / V% a mixture of mycin-streptomycin-amphotericidal B (100×). V / V% refers to percentage volume.

[0063] 1.2. Preparation of CFSE stock solution (1 mM): Take one vial of CFSE reagent, add 900 μL of DMSO, mix thoroughly, and aseptically aliquot. Store at -20°C for 6 months. Before use, dilute the CFSE stock solution 1:500 with PBS to prepare 1×CFSE staining working solution (2 µM). Prepare fresh before use and avoid repeated freeze-thaw cycles.

[0064] 2. Cell sample resuscitation 2.1 After removing the K562 feeder cell test sample (prepared by irradiation by Jiangsu Puxin Biotechnology Co., Ltd., catalog number: HG-FEC002-GMP-2) and K562 cells (K562 cells were used as positive control reference cells (also known as K562 positive control or K562 positive control reference cells), purchased from ATCC, catalog number CCL-43) from liquid nitrogen, they were transferred to the laboratory using a liquid nitrogen transport box and placed in a water bath at 37.0±0.5℃ for recovery.

[0065] 2.2 Prepare a 15 mL centrifuge tube, add 9 mL of preheated RPMI 1640 complete culture medium, and use a pipette to transfer the recovered cell suspension to the centrifuge tube. Centrifuge at 300 g for 5 minutes.

[0066] 2.3 Discard the supernatant, resuspend the cell pellet in RPMI 1640 complete medium, gently pipette to mix, and take 0.3-0.5 mL of cell suspension for cell counting.

[0067] 3. Cell labeling and plating (This step can be skipped if the test cells are labeled with GFP autofluorescent tags.) 3.1. Take a suspension of 1E6 cells based on the counting results and centrifuge at 300 g for 5 minutes.

[0068] 3.2. Discard the supernatant, resuspend the cell pellet in RPMI 1640 complete medium, centrifuge at 300 g for 5 minutes, and repeat the washing once.

[0069] 3.3. Discard the supernatant, add 1 mL of the prepared 1×CFSE staining working solution (2 μL) to the cell pellet, and gently mix with a pipette to ensure that the cell density is controlled at 1E6 cells / mL during CFSE staining, so that all cells can be stained evenly.

[0070] 3.4. Wrap the centrifuge tube with aluminum foil and incubate it in a water bath at 37.0±0.5℃ for 15 minutes. Shake the bottom of the centrifuge tube every 5 minutes or so to ensure uniform cell staining.

[0071] 3.5. After incubation, wipe the centrifuge tubes with 75% alcohol for disinfection and transfer them to a biosafety cabinet. Open the centrifuge tube cap, add 10 mL of RPMI 1640 complete culture medium, gently pipette to mix, and place the centrifuge tube in a centrifuge. Centrifuge at 300 g-400 g for 5 minutes.

[0072] 4. Cell plating 4.1. Discard the supernatant, add 5 mL of RPMI 1640 complete culture medium to resuspend the cell pellet, mix thoroughly by pipetting and transfer to a sterile sample loading trough.

[0073] 4.2. Take a 96-well U plate and use a multi-channel pipette to add 200 μL of cell suspension to each well. Set up 4 sampling time points (D0, D7, D14, D21). Use feeder cells (K562 feeder cells) as the experimental group and K562 positive control reference cells as the control group. Set up 3 replicates for each time point.

[0074] 4.3. After the samples are added, the 96-well plate is placed in an incubator at 37.0±1.0℃ and 5.0±0.5% carbon dioxide concentration for incubation.

[0075] 5. Cell sample collection and detection 5.1. Follow the startup procedure and complete the quality control procedures according to the operating documents of Beckman Coulter or Miltenyi flow cytometer.

[0076] 5.2. Samples were collected and analyzed on days 0, 7, 14, and 21 after culture. The cell suspension in the 96-well plate was mixed by pipetting and transferred to 1.5 mL centrifuge tubes.

[0077] 5.3. Remove CountBright Beads (Thermofisher, catalog number C36950) from the refrigerator and mix thoroughly by pipetting with a 1 mL pipette (gently pipetting, being careful not to create air bubbles). Add 5 μL of CountBright beads to each tube for both the experimental and control groups (the final concentration is 0.94 x 10⁻⁶). 3 (beads / μL).

[0078] 5.4. Add 2 μL of 7-AAD (final staining concentration of 1 µg / mL) to each tube of the experimental group and 2 μL of PBS to each tube of the control group. Vortex to mix and incubate in a refrigerator at 2-8℃ in the dark for 10 minutes.

[0079] 5.5. After incubation, prepare for flow cytometry testing. Before each sample loading, the flow cytometer tube must be thoroughly shaken and mixed.

[0080] 5.6. For instrumental analysis, set the sample loading to be by volume or by Beads Events. Collect 100 μL of volume or 2000 Beads Events for each sample. Use gating to delineate the locations of Beads, Live cells, and Dead cells populations. Both CFSE and 7-AAD cell dyes are excited by 488 nm excitation light and generate fluorescence reception signals in the FL1 (FITC) and FL3 (PC5.5) channels, respectively. The CFSE+7-AAD+ population represents the dead cells in the target cell population, while the CFSE+7-AAD- population represents the live cells in the target cell population. Flow cytometry absolute counting is performed using absolute counting microspheres (Beads). Bead populations can be delineated by gating through the PB450 fluorescence channel (or V1 channel) and SSC-A channel of the flow cytometer.

[0081] After the lab session, record the Events and percentages for each gate. The gate logic is as follows: Figure 1 As shown; Schematic diagram of the gate is as follows Figure 2 and Figure 3 As shown.

[0082] 5.7. Calculation Formula

[0083] Note: 1) "Conc." refers to the CountBright beads concentration (beads / μL), which can be obtained from the COA of this batch of beads reagent. In this experiment, the bead concentration was 0.94 x 10⁻⁶. 3 1) beads / μL; 2) “V” is the volume of added CountBrightbeads (μL), and the volume of added beads in this experiment is 5 μL; 3) “202” is the total volume of cell suspension and added 7-AAD (100×) 202 μL.

[0084] Viability rate (%) = Live cells / (Live cells+Dead cells).

[0085] 6. Data Plotting 6.1 Using GraphPad Prism software, generate a trend graph of live cell density and cell viability (e.g., Figure 4 (As shown).

[0086] Example 2: Key Parameter Optimization CFSE staining concentration is a key parameter affecting the entire methodology cycle. The fluorescence signal produced by CFSE-labeled feeder cells gradually decreases with cell apoptosis, while the CFSE signal in positive control cells (such as the wild-type K562 cell line) also decreases due to cell proliferation during culture. At day 0, excessively high CFSE signals may exceed the flow cytometry threshold. Therefore, optimizing the CFSE staining concentration is essential to ensure that the labeling signals of both cell lines can be effectively received by the flow cytometer during the culture period from day 0 to day 21.

[0087] 1. CFSE concentration optimization steps 1.2 Prepare CFSE stock solution according to the instructions, and use K562 cells as positive control cells for concentration testing and optimization experiments.

[0088] 2.2 Take one CFSE reagent, add 900 μL of DMSO, mix thoroughly, and aseptically dispense to obtain CFSE stock solution. Dilute the CFSE stock solution with PBS to 100×, 200×, 500×, 1000×, and 2000× respectively (as shown in Table 2) to obtain CFSE dilutions of different concentrations.

[0089] After resuscitation and passage culture, K562 cells were adjusted to a density of 1E6 cells / mL using PBS and aliquoted into 1mL K562 cell suspensions for later use.

[0090] Add 1 mL of K562 cell suspension with the above-mentioned CFSE dilutions at different concentrations to make the final CFSE concentrations 10 µM, 5 µM, 2 µM, 1 µM and 0.5 µM respectively (as shown in Table 2). Incubate in a 37°C water bath for 15 minutes, shaking the bottom of the centrifuge tube every 5 minutes or so to ensure uniform cell staining.

[0091] Table 2

[0092] 2.3 After staining, add 10 mL of RPMI 1640 complete culture medium and gently mix by pipetting.

[0093] 2.4 Place the centrifuge tube in the centrifuge and centrifuge at 400 g for 5 minutes.

[0094] 2.5 Discard the supernatant, add an appropriate amount of RPMI 1640 complete medium to resuspend the cell pellet, and adjust the cell density to 2E5 cells / mL.

[0095] 2.6 Transfer the cell suspensions stained with different concentrations of CFSE to a sterile sample loading well, prepare a 96-well U plate, and add 200 μL of cell suspension to each well.

[0096] 2.7 Place the 96-well U-plate in an incubator at 37.0±1℃ and 5.0±0.5% carbon dioxide for 2-4 hours.

[0097] 2.8 After the incubation is completed, remove the 96-well U-plate from the CO2 incubator.

[0098] 2.9 Flow cytometers of different brands (Beckman and Miltenyi) were used for instrument detection. CFSE staining was excited by 488 nm excitation light and generated a fluorescence receiving signal in the FL1 (FITC) channel. The voltage of the FL1 channel of the instrument was adjusted to ensure that the peak of the negative cell population was in the appropriate position, and cell samples stained with different concentrations of CFSE were processed for instrument detection.

[0099] 2.10 CFSE Concentration Optimization Results K562 cell samples stained with different CFSE concentrations were processed using Miltenyi and Beckman flow cytometers, respectively. The chromatograms are shown below. Figure 5 ( Figure 5 (CFSE spectra obtained from Miltenyi (left) and Beckman (right) flow cytometers). Comparing the CFSE peak positions analyzed by different instruments, it can be found that when the CSFE stock solution is diluted at least 500× (including 500× and 1000× dilutions), the fluorescence signal peaks produced by CFSE are complete, with maximum fluorescence intensity and the cell population position does not exceed the fluorescence threshold range (horizontal axis). Under these conditions, the CFSE staining effect is the best.

[0100] Example 3: Optimization of Culture Time After the culture of feeder cells begins, samples need to be taken at different time points to confirm their growth status. Optimizing the sampling time can not only reduce the amount of experimental samples used, but also reduce experimental costs and testing workload.

[0101] 1. Specific operations 1.1 Take the K562 feeder cells and K562 cells (K562 cells as positive reference cells (also known as K562 positive control or K562 positive control reference cells) described in step 2.1 of Example 1, and perform the experimental operations according to the steps 3-5 of Example 1. Collect samples and detect cell density on days 0, 1, 2, 3, 5, 7, 10, 14 and 21 after culture.

[0102] 1.2 Changes in cell density at different culture times, as shown in the figure. Figure 5As shown in the figure, by comparing the density changes of the two cell lines at different culture times, it can be found that the feeder cells consistently showed a decreasing density, indicating no proliferation; while the K562 positive control cells showed an increasing density from D0 to D7, indicating proliferation. After D7, considering the aging of the culture medium, the cell density began to decrease. Therefore, D7 is a suitable time point for determining whether cells are proliferating, and can be used for cell sampling and detection. To continuously monitor cell status, D14 and D21 can also be used as subsequent time points for sampling and detection. In summary, D0, D7, and D14 (or D21) can be used as detection time points for this method.

[0103] Example 4: Method Performance Testing Specificity detection: Cells labeled with CFSE can be specifically identified by flow cytometry, and a cell population appears in the target area.

[0104] Experimental procedure: (1) Take the K562 Feeder cell test sample described in 2.1 of Example 1 and divide it into 3 groups. One group (K562 Feeder cells + CFSE group) is subjected to CFSE labeling (the experimental operation is described in 3-5 of Example 1); the other two groups (K562 Feeder cells + PBS group and K562 Feeder cells + culture medium group) are not subjected to CFSE labeling (the experimental operation is described in 3-5 of Example 1, the only difference being that PBS and RPMI 1640 complete culture medium are used to replace CFSE dye respectively). The proportion of CFSE+ population is circled in the data analysis, and the circle is referenced as follows. Figure 7 As shown.

[0105] (2) K562 cells (purchased from ATCC, catalog number CCL-43) as described in 2.1 of Example 1 were used as a positive control reference. The experimental procedures were performed according to the descriptions in 3-5 of Example 1. After the test, the proliferation trend curve was plotted, and the results are as follows. Figure 8 The diagram shows the density changes of the positive control cells (K562), the viability changes of the positive control cells (K562), the density changes of the feeder cells (K562), and the viability changes of the feeder cells (K562); CFSE is also summarized. + (%), the results are shown in Table 1).

[0106] The specificity results are shown in Table 1 and Figure 8As shown in the experimental results, the proportion of CFSE+ in the K562 feeder cell group labeled with CFSE was 99.7%, while the proportion of CFSE+ in the CFSE-labeled groups was 0.0%, indicating that the labeled cells can be specifically detected by the instrument. K562 cells, used as a positive control, showed a tendency for cell proliferation before day 7, but proliferation began to weaken after day 7, consistent with general cell growth characteristics. The K562 feeder cells remained in a non-proliferating state, meeting the quality standards for feeder cells.

[0107] Table 1 Specificity Detection Results

[0108] Accuracy: CFSE-labeled K562 Feeder cells test sample A: The sample was prepared according to steps 2-3 in Example 1 to obtain CFSE-labeled K562 Feeder cells test sample A.

[0109] Unlabeled K562 Feeder cells test sample B: The sample was prepared according to step 2 of Example 1 to obtain unlabeled K562 Feeder cells test sample B.

[0110] CFSE-labeled K562 Feeder cells (sample A) and unlabeled K562 Feeder cells (sample B) were prepared. After adjusting the cell density, samples A and B were mixed at a volume ratio of 3:1 to prepare three portions, resulting in Sample 1; samples A and B were mixed at a volume ratio of 1:1 to prepare three portions, resulting in Sample 2; and samples A and B were mixed at a volume ratio of 1:3 to prepare three portions, resulting in Sample 3. After preparing the samples A and B, Sample 1, Sample 2, and Sample 3, they were analyzed using a flow cytometer (the experimental procedures were performed according to the description in Sections 3-5 of Example 1, the only difference being that the sampling time point was only D0). The accuracy results are shown in Table 3.

[0111] Table 3 Accuracy test results Note: Recovery rate calculation formula: Recovery rate (%) = Detected value / Theoretical value × 100%.

[0112] Repeatability: K562 feeder cell test sample: prepared by irradiation by Jiangsu Puxin Biotechnology Co., Ltd., as shown in step 2 of Example 1.

[0113] Six K562 feeder cell samples were taken for labeling and detection (the experimental procedures were performed according to steps 3-5 in Example 1, the only difference being that the sampling time point was D0), and the coefficient of variation (CV) between the viable cell densities in the six samples was calculated. The repeatability test results are shown in Table 4.

[0114] Table 4 Repeatability test results

[0115] Note: The formula for calculating the coefficient of variation (CV) is: CV(%) = standard deviation SD / mean × 100%.

[0116] Intermediate precision: K562 feeder cell test sample: prepared by irradiation by Jiangsu Puxin Biotechnology Co., Ltd., as shown in step 2 of Example 1.

[0117] Two experimenters labeled the same batch of K562 feeder cell samples (the experimental operation was carried out according to steps 3-5 in Example 1, the only difference being that the sampling time point was only D0). The coefficient of variation (CV) between the viable cell densities of 6 samples from each experimenter and between the viable cell densities of 12 samples from the two experimenters were calculated. The intermediate precision test results are shown in Table 5.

[0118] Table 5 Intermediate Precision Test Results

[0119] Note: The formula for calculating the coefficient of variation (CV) is: CV(%) = standard deviation SD / mean × 100%.

[0120] Durability: K562 feeder cell test sample: prepared by irradiation by Jiangsu Puxin Biotechnology Co., Ltd., as shown in step 2 of Example 1.

[0121] Six samples of K562 feeder cells were taken for testing (the experimental operation was carried out according to steps 3-5 in Example 1, the only difference being that the sampling time point was D0). Beckman CytoFLEX and Miltenyi MQ 10 were used for testing, and the coefficient of variation (CV) between the viable cell densities obtained by different instruments was calculated. The robustness test results are shown in Table 6.

[0122] Table 6 Durability Results

[0123] Note: The formula for calculating the coefficient of variation (CV) is: CV(%) = standard deviation SD / mean × 100%.

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting cell density and / or viability, characterized in that, The kit comprises CFSE dye, dead cell dye and absolute counting microspheres.

2. The kit of claim 1, wherein The concentration of CFSE in the CFSE dye is 0.5 µM-10 µM.

3. The kit of claim 1 or 2, wherein The dead cell dye is selected from 7-AAD dye, propidium iodide, DAPI, SYTOX® series dye, fixed active dye, near-infrared active dye; preferably, the dead cell dye is 7-AAD dye; And / or, the absolute counting microspheres comprise light scattering counting microspheres or fluorescent counting microspheres.

4. A method of detecting cell density and / or viability, characterized in that, The method comprises: (1) exposing feeder cells to the CFSE dye environment in the kit of any one of claims 1-3; the concentration of CFSE in the CFSE dye environment is 0.5 µM-10 µM; (2) after step (1), removing the CFSE dye in the environment; (3) after step (2), culturing the feeder cells under suitable conditions for 0-21 days; (4) after step (3), adding the cultured feeder cells to the absolute counting microspheres in the kit of any one of claims 1-3 and exposing them to the dead cell dye environment in the kit of any one of claims 1-3.

5. The method of claim 4, wherein, The method further comprises (5): after step (4), detecting the number of live cells, the number of dead cells and / or the number of absolute counting microspheres, and calculating the cell density or viability.

6. The method of claim 5, wherein, The cell density = ((number of live cells / number of absolute counting microspheres) x concentration of absolute counting microspheres x volume of absolute counting microspheres) / (volume of dead cell dye + volume of absolute counting microspheres); And / or, the viability = number of live cells / (number of live cells + number of dead cells).

7. The method of any one of claims 4-6, wherein, The dead cells are a population that is CFSE positive and 7-AAD positive; And / or, the live cells are a population that is CFSE positive and 7-AAD negative; And / or, the absolute counting microspheres are a population that is SSC-A and PB450 positive, or a population that is SSC-A and V1 positive; Preferably, the positivity or negativity of CFSE is determined by FITC gate; Preferably, the positivity or negativity of 7-AAD is determined by PC5.5 gate; Preferably, the absolute counting microspheres are determined by SSC-A and PB450 channel positive gate, or by SSC-A and V1 channel positive gate; More preferably, the determination is by flow cytometry; most preferably, the flow cytometry is Beckman or Miltenyi.

8. The method of any one of claims 4-7, wherein, In step (1), the exposure temperature is 34.5-40.5℃; And / or, in step (1), the concentration of CFSE in the CFSE dye environment is 0.5 µM-10 µM; And / or, in step (1), the exposure time is 5-15 min; And / or, in step (1), the density of feeder cells is 1E5-1E7 cells / mL; And / or, in step (1), it further comprises the step of mixing feeder cells with CFSE dye; And / or, in step (2), the removal is by centrifugation; And / or, in step (2), the removal step comprises dilution, centrifugation and resuspension; and / or, in the step (3), the density of the cells is 2E4-2E7 cells / mL; and / or, in the step (3), the condition includes temperature is 34.5-40.5℃; and / or, in the step (3), the condition includes carbon dioxide concentration is 5.0%; and / or, in the step (3), the condition includes medium is RPMI 1640 complete medium; and / or, in the step (3), the culture time is 1-21 days; and / or, in the step (4), the concentration of the dead cell dye in the dead cell dye environment is 0.5-1.5 µg / mL; and / or, in the step (4), the exposure time is 5-15 min; and / or, in the step (4), the exposure temperature is 2-8℃; and / or, in the step (4), the operation is in dark.

9. The method according to claims 4-8, characterized in that, The feeder cells are selected from K562 cells, Daudi cells, RPMI-8866 cells, Jurkat cells, EBV-LCL cells, autologous or allogeneic PBMCs, bone marrow mesenchymal stem cells, CD34+ hematopoietic progenitor cells from umbilical cord blood / peripheral blood, primary trophoblast cells from placenta, amniotic membrane / chorion mesenchymal cells, or transgenic cell lines thereof.

10. Use of the kit of any one of claims 1-3 in detecting the cell density or viability of feeder cells.

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