Immune cell killing activity detection method based on fluorescently labeled tumor target cells

Abstract

The invention discloses an immune cell killing activity detection method based on fluorescently labeled tumor target cells, and relates to the technical field of detection.The immune cell killing activity detection method comprises the steps that peripheral blood or umbilical cord blood of a healthy volunteer is collected, mononuclear cells are obtained through separation with a human lymphocyte separation medium and induced into immune effector cells, and multiplication culture is conducted; taking the fluorescently-labeled tumor cells as target cells, and co-incubating the immune effector cells and the target cells for a period of time; collecting a cell product after co-incubation is finished, and detecting the number of fluorescently-labeled positive cells in the co-incubation product by using a flow cytometer or a fluorescent cell counter; and calculating the killing rate of the immune effector cells to the target cells based on the detected number of the positive cells. The scheme is simple and convenient to operate, rapid, accurate, low in cost and free of cell damage.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells. Background Technology

[0002] In the era of precision medicine, immunotherapy, as a novel cancer treatment following surgery, radiotherapy, and chemotherapy, has demonstrated significant clinical potential by activating or enhancing the body's immune system's ability to recognize and kill tumor cells. Currently, the types of therapeutic immune cells are becoming increasingly diverse, ranging from traditional effector cells (such as NK cells, γδT cells, CIK cells, and NK92) to genetically engineered cells (such as CAR-T, CAR-NK, TCR-T, and NK92-MI) and tumor-infiltrating lymphocytes (TILs), all of which have become key effector cells in the fight against tumors. Nevertheless, the clinical application of immunotherapy highly depends on the quality control of cell products, encompassing multiple dimensions of quality control including cell identification, effector cell purity, activity, and safety. Cell-killing activity, as a key quality attribute, is an important reference for predicting in vivo treatment efficacy and a core functional indicator directly reflecting the effectiveness of immune cell products.

[0003] Currently, there are many methods for evaluating cell-killing activity. The chromium-51 release assay, considered the "gold standard" for cytotoxicity detection, assesses killing efficiency by measuring the radioactive isotopes released from target cells; however, this method carries the risk of radioactive contamination. The LDH release assay indirectly assesses killing efficiency by detecting the activity of LDH released after cell lysis; however, fluctuations in endogenous LDH in serum can easily lead to false positives, and it requires disrupting cell membrane integrity. Dye labeling methods such as CFSE / Calcein-AM use fluorescent dyes to label target cells and quantify killing activity through changes in fluorescence signals. However, dye labeling efficiency is affected by cellular metabolic state, and phototoxic effects may induce apoptosis, thus interfering with the assessment of the true killing activity of immune cells. Analyzing killing efficiency by antibody-labeled target cell surface antigens or apoptosis markers (such as Annexin V / PI) may result in false positives due to non-specific antibody binding, and requires complex pretreatment steps. Furthermore, its reliance on high-configuration equipment limits its high-throughput application. The Luciferase luminescence method involves introducing the luciferase gene into target cells and co-incubating them with immune cells, then adding the substrate luciferin. The intensity of the chemiluminescence reflects the number of surviving target cells. However, the luminescence reaction only lasts for a few minutes after the substrate is added, requiring immediate detection and resulting in a short operational time. Furthermore, redox substances secreted by effector cells may quench the luminescence signal. In addition, the expensive luciferase substrate is not suitable for high-throughput screening.

[0004] Therefore, the multiple shortcomings of traditional methods for detecting cytotoxic activity in terms of safety (radioactivity), accuracy (false positives / signal attenuation), cost-effectiveness (high-cost reagents / equipment), and ease of operation (complex procedures / time sensitivity) have become a bottleneck restricting the development of the immune cell therapy industry. There is an urgent practical need to develop a novel method for assessing cytotoxic activity that requires no additional antibody labeling or dyes, is simple and rapid to operate, and provides accurate and non-invasive detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells. This method is simple to operate, rapid, accurate, low-cost, and causes no cell damage.

[0006] In order to achieve the objective of this invention, the following solution is proposed: In a first aspect, the present invention provides a method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells, using flow cytometry, comprising the following steps: (1) Preparation of target cells: Tumor cells labeled with fluorescent protein were used as target cells. K562-eGFP target cells in the logarithmic growth phase were obtained, centrifuged and the supernatant was discarded. The cells were resuspended in fresh culture medium and the cell concentration was adjusted to between 1×10⁻⁶. 4 Cells / mL~1×10 7 cells / mL; (2) Preparation of effector cells: peripheral blood or umbilical cord blood of healthy volunteers were collected, and mononuclear cells were separated using human lymphocyte separation medium. These cells were induced into immune effector cells and expanded and cultured. The cultured immune effector cells were taken out, centrifuged, and the supernatant was discarded after centrifugation. Immune effector cell culture medium was added, and the required number of cells was calculated and the cell concentration was adjusted according to the effector-to-target ratio of 40:1 to 0.1:1 to serve as effector cells with different effector-to-target ratios. (3) Plate laying: K562-eGFP cells and immune effector cells were seeded into cell well plates as experimental groups, and single effector cell wells and single target cell wells were set up as control groups. The experimental groups and control groups were placed in a 37℃, 5% CO2 incubator for a certain period of time. (4) Killing detection: After co-incubation, cells from the experimental group and the control group were collected, and the signal intensity of the FITC channel was detected by flow cytometry. The killing rate of immune effector cells was calculated according to the nucleolysis formula.

[0007] Furthermore, the flow cytometry detection procedure is as follows: select the FITC channel, excitation light at 488nm, and emission light at 510-550nm. The gating strategy is: ① Live cell screening: using a dual-parameter scatter plot of FSC / SSC, set an FSC threshold to exclude cell debris (FSC<1000) and an SSC threshold to exclude dead cells and high-particulate impurities, thus delineating the live cell population (P1 gate and P2 gate); ② Target cell identification: within the P2 gate, set a gate based on the fluorescence intensity of the FITC channel, identifying eGFP-positive cells (FITC+>10). 5 The target cells (P3 gate) are defined as K562-eGFP target cells. The number of P3 gated cells is calculated, which is the number of target cells remaining after killing. Blank tubes, single-effect cell tubes, and single-target cell tubes are set up to gate K562-eGFP positive cells.

[0008] Tumor killing rate of immune effector cells = (concentration of eGFP+ cell particles in single effector cell control group - concentration of eGFP+ cell particles in experimental group) / concentration of eGFP+ cell particles in single target cell control group × 100%, where the unit of particle concentration is cells / mL.

[0009] Secondly, based on a fluorescence cell counter, this invention provides another method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells, comprising the following steps: Steps (1) to (3) are the same as in the first aspect. (4) Killing detection: After co-incubation, cells from the experimental group and the control group are collected, mixed by pipetting, and 20µL is drawn into the counting plate and placed on the sample stage of the fluorescence cell counter. The instrument used in this embodiment is the Countstar fully automated cell counter with AO / PI fluorescence channel. After setting the AO / PI fluorescence field detection program, the detection is performed, and the killing rate of immune effector cells is calculated according to the calculation formula.

[0010] Tumor killing rate of immune effector cells = (1 - cell density of experimental group / cell density of single target cell control group) × 100%, cell density unit: cells / mL.

[0011] Further: The detection procedure for the fluorescence cell counter is as follows: set the fluorescence field parameters: AO / PI excitation light 405nm, emission light 515nm-565nm, used to label dead cells (cells that have infiltrated the cell nucleus after cell membrane rupture); diameter recognition range: live K562-eGFP cells with a diameter of 12-15μm (determined by laboratory-specific results obtained after measurement by microscope scale or preoperative backscattering (FSC) calibration by flow cytometry), and set cells with a diameter ≥12μm and FITC+ signal as live target cells.

[0012] Furthermore, before step (1), the procedure includes: removing the K562-eGFP cells to be revived, transferring them to a constant temperature water bath, shaking the cryovial to completely thaw the contents, opening the cryovial cap, transferring the cell suspension to a prepared centrifuge tube containing RPMI 1640 culture medium, mixing well, centrifuging, discarding the supernatant after centrifugation, resuspending the cells in complete culture medium, counting the number of viable cells, and setting the viable cell density at 1×10⁻⁶. 5 Cells / mL~2×10 5 Inoculate cells at a density of 1 × 10⁶ cells / mL. After 48 hours of culture, wait until the cell density does not exceed 1 × 10⁶ cells / mL. 6 When the cell count is ≥95% and the cell viability is ≥95%, passage or experiment can be performed.

[0013] Furthermore, before step (2), the process includes: peripheral blood / umbilical cord blood plasma separation, acquisition of peripheral blood / umbilical cord blood mononuclear cells, cryopreservation of mononuclear cells, revival of cryopreserved peripheral blood / umbilical cord blood mononuclear cells, and the acquisition of immune effector cells (NK cells, γδT cells, CIK cells, TILs, CAR-T, CAR-NK, TCR-T, etc.) through induction, amplification, viral transduction, gene knockout, etc., or preparation through iPSC directed differentiation.

[0014] Furthermore, cell subtype detection was performed on days 10 to 14 of culture, and the proportion of cells positive for cell surface markers was detected by flow cytometry.

[0015] In some embodiments, the co-incubation time of target cells and effector cells is preferably 1 hour, 2 hours, 4 hours, 6 hours or 8 hours, with the most preferred incubation time being 2 hours or 4 hours.

[0016] The beneficial effects of this invention are as follows: 1. Simplified labeling: The target cells endogenously express eGFP or other fluorescent proteins, eliminating the need for antibody labeling or dye loading such as CFSE, avoiding cell damage and fluorescence leakage during the labeling process, and ensuring stable detection signals with low background interference.

[0017] 2. High operational efficiency: The entire process from cell co-incubation to result output takes ≤6 hours, without the need for complicated pretreatment steps, which greatly shortens the experimental time and improves work efficiency.

[0018] 3. Quantitative accuracy: By detecting the absolute value of fluorescence intensity using flow cytometry or analyzing the single-cell diameter-fluorescence dual parameters using a counter, live target cells can be accurately distinguished from dead cells / fragments, eliminating interference from effector cell fluorescence (because effector cells do not express eGFP or other fluorescent proteins), making the detection results more accurate and reliable.

[0019] 4. Cost-effectiveness: It reduces the use of reagents such as antibodies and dyes, and is compatible with conventional laboratory equipment such as fluorescence cell counters, while supporting the detection modes of expensive instruments such as flow cytometers, thus reducing experimental costs.

[0020] 5. Cell compatibility: The entire process involves no chemical treatment steps and will not affect cell metabolism and function, thus more accurately reflecting the killing activity of immune effector cells. Attached Figure Description

[0021] Figure 1 Morphological features of target cells K562-eGFP under a microscope; Figure 2 This image shows the morphological characteristics of effector NK cells under a microscope. Figure 3 The killing effect of NK cells on K562 cells at different effector-to-target ratios is shown in the figure. Figure 4 The images, in order, show the delineated cell population, the screened live cell population, and the target cell identification image. Figure 5 Flow cytometry plots showing the killing effect of NK cells on K562 cells at different effector-to-target ratios. Detailed Implementation

[0022] In some embodiments, the target cells are virus-stable cells; in other embodiments, the target cells are tumor cells, such as K562 cells, Daudi cells, Jurkat cells, MCF-7 cells, A549 cells, or HepG2 cells.

[0023] In some embodiments, effector cells are cells with cytotoxic effects, such as immune cells and immune-engineered cells, including NK cells, T cells, CTL cells, LAK cells, CIK cells, TIL cells, TCR-T cells, DC-CIK cells, CAR-T cells, CAR-NK cells, or NK92, NK92MI, NK92MI-CD16a engineered cells.

[0024] This invention provides a method for detecting the killing activity of immune cells based on fluorescently labeled tumor target cells. Taking the detection of NK cell tumor killing activity as an example, the method specifically includes the following steps: (1) Preparation of target cells: Tumor cells labeled with fluorescent protein were used as target cells. K562-eGFP target cells in the logarithmic growth phase were obtained, centrifuged and the supernatant was discarded. The cells were resuspended in fresh culture medium and the cell concentration was adjusted to between 1×10⁻⁶. 5 Cells / mL~1×10 6 cells / mL; (2) Preparation of effector cells: peripheral blood or umbilical cord blood was collected from healthy volunteers, and mononuclear cells were separated using human lymphocyte separation medium. These cells were induced into immune effector cells and expanded and cultured. The cultured NK cells were taken out, centrifuged, and the supernatant was discarded after centrifugation. The cells were resuspended in RPMI 1640 medium, counted, and the required number of cells was calculated according to the effector-to-target ratio of 20:1, 10:1, 5:1, and 1:1. The cell concentration was adjusted to obtain NK cell suspensions with different effector-to-target ratios. (3) Plate laying: K562-eGFP cells and NK cells were seeded into cell well plates as experimental groups, and single-effect cell wells and single-target cell wells were set up as control groups. The experimental groups and control groups were placed in a 37℃, 5% CO2 incubator for a certain period of time. (4) Killing detection: After co-incubation, remove the cell well plate, collect the cells, and use flow cytometer or fluorescence cell counter to detect eGFP positive cells and calculate the killing rate of effector cells.

[0025] Example 1 This embodiment provides a method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells, which specifically includes the following steps: (1) Preparation of target cells: Obtain K562-eGFP target cells in the logarithmic growth phase, centrifuge at 300g for 5min, discard the supernatant, collect the cell pellet, resuspend in DMEM complete medium, count the number of viable K562-eGFP cells, and adjust the cell concentration to 1×10⁻⁶. 6 cells / mL, 5×10 5 cells / mL, 2×10 5 K562-eGFP cells were prepared at a concentration of [number] cells / mL for later use. Microscopic morphological characteristics of K562-eGFP cells are shown in the image below. Figure 1 As shown.

[0026] (2) Preparation of effector cells: NK cells cultured in Donor 1 for 14 days were obtained, transferred to 50 mL centrifuge tubes, centrifuged at 500 g for 5 min, the supernatant was discarded, and the cells were resuspended in RPMI 1640 complete medium. The cells were counted, and the NK cell concentration was adjusted according to four effector-target ratios of 20:1, 10:1, 5:1, and 1:1 to obtain NK cell suspensions for later use. The morphological characteristics of NK cells under a microscope are shown in the figure below. Figure 2 As shown.

[0027] (3) Plate formation: K562-eGFP cells and NK cells were seeded into 96-well plates. Four experimental groups (A1, A2, A3, A4) with effector-to-target ratios of 20:1, 10:1, 5:1, and 1:1 were set up, along with four control groups (B1, B2, B3, B4) with single-effect cell (NK cell) ratios of 20:0, 10:0, 5:0, and 1:0, and single-target cell (K562-eGFP cell) ratios (C). Each experimental well, single-effect cell control well, and single-target cell control well had three replicates. During plate formation, a ring of empty wells was left around the experimental and control wells, and NaCl (physiological saline) was added to this ring of empty wells. The experimental and control groups were incubated together at 37°C and 5% CO2 for 4 hours. Taking a K562-eGFP concentration of 1e6 cells / mL as an example, the experimental groups are shown in Table 1; the distribution of contents in the 96-well plate is shown in Table 2; the killing effect of NK cells on K562-eGFP cells at different effector-to-target ratios is shown in the figure. Figure 3 As shown.

[0028] Table 1. Grouping of experimental and control groups, and types and volumes of added contents. Table 2. Distribution of contents in a 96-well plate (4) Killing detection: Remove the cell plate, collect experimental and control cells after the reaction, mix thoroughly, and then aspirate 20µL of cell suspension from the experimental group into a counting chamber. Place the chamber in a fluorescence cell counter to detect the viable cell density. Detect the remaining cells using a flow cytometer. Adjust the parameters of FSC, SSC, and FITC, collect fluorescence intensity from at least 6000 target cell channels, and perform a detection time of 60s per well. Calculate the concentration of P3 gate particles in the flow cytometer. Then, based on the flow cytometry results, calculate the effector cell killing rate using the following formula: Killing rate (%) = (Single effector cell control group eGFP) / (Effective cell control group eGFP) / (Effective cell control group eGFP) / Effective cell control group eGFP) + Cell granule concentration - experimental group eGFP + Cell particle concentration / single target cell control group eGFP + Cell granule concentration × 100%. Figure 4 The image shows three sub-plots. The first plot, according to the arrows, shows live cell screening: using FSC-A and SSC-A gates, cell debris (FSC < 1000), dead cells, and high-granularity impurities are excluded by setting a threshold, thus defining the cell population (P1 gate). The second plot shows refining the live cell population: based on the P1 gate, live cell populations are further screened (P2 gate). The third plot shows target cell identification: within the P2 gate, the FITC channel is used to detect eGFP fluorescence intensity, selecting cells with fluorescence signals > 10... 5 eGFP-positive cells were defined as K562-eGFP target cells (P3 phylum). The concentration of K562-eGFP cells was 1×10⁻⁶.6 Cells / mL (cell plate 1), 5×10 5 Cells / mL (cell plate 2), 2×10 5 P3-gated particle concentration (i.e., eGFP) detected by flow cytometry / mL (cell plate 3). + Positive cells and their killing rates are shown in Tables 3, 4, and 5, respectively. Flow cytometry results of the killing effect of Donor 1 NK cells on K562-eGFP cells are shown in Tables 3, 4, and 5, respectively. Figure 5 As shown.

[0029] Table 3. Donor 1 NK Kill Detection Flow Cytometry 1×10⁻⁶ 6 Results table for cells / mL (cell plate 1) As shown in Table 3, in cell plate 1, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 1 NK cells decreased from 97.00% to 76.35%.

[0030] Table 4. Donor 1 NK Killer Detection Flow Cytometry 5×10⁻⁶ 5 Results table for cells / mL (cell plate 2) As shown in Table 4, in cell plate 2, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 1 NK cells decreased from 98.59% to 71.04%.

[0031] Table 5. Donor 1 NK Kill Detection Flow Cytometry 2×10⁻⁶ 5 Results table for cells / mL (cell plate 3) As shown in Table 5, in cell plate 3, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 1 NK cells decreased from 97.83% to 49.45%.

[0032] From Tables 3 to 5 and Figure 4 Comprehensive data analysis shows that: The killing rate of NK cells in cell plates 1–3 increased significantly with increasing effector cell concentration (increased effector-to-target ratio), showing a good dose-dependent relationship, confirming that the method sensitivity meets the detection requirements.

[0033] Within the effect-to-target ratio range of 20:1 to 5:1, the three groups of killing rate results were highly consistent at the same effect-to-target ratio, proving that this method can stably quantify the killing efficacy of NK cells.

[0034] The initial density of target cells significantly affects the stability and accuracy of detection; low-density group (2×10⁻⁶) 5 Although the cell count (CV) within the group was stable, the low-efficiency target ratio resulted in an underestimated kill rate (26.90% and 21.59% lower than the other two groups), possibly due to a weak background signal from eGFP-positive cells, making the detection susceptible to background interference. The low density of effector and target cells also led to poor contact and a lower killing effect. The high-density group (1×10⁻⁶ cells / mL) showed better cytotoxicity. 6 The data fluctuation (CVmax = 8.38%) for cells / mL is speculated to be related to uneven distribution between plates due to cell concentration, resulting in overcrowding during counting. This leads to larger cell clusters formed during cell killing, preventing complete dispersion during counting and thus causing a higher CV. The moderate target cell concentration group (5 × 10⁻⁶) showed a higher CV. 5 The 5×10⁻⁶ cells / mL ratio exhibits the lowest overall variability, stable data (CV ≤ 5% at all effective target ratios), strong data repeatability, and clear trends; therefore, it is recommended to use 5×10⁻⁶ cells / mL. 5 The optimal target cell density was set at 10 cells / mL for subsequent detection of immune killing activity.

[0035] The standardized detection system established in this study enables reproducible quantitative analysis of tumor cell killing activity through target cell / effect cell preparation, co-incubation procedures, and flow cytometry detection, providing a highly sensitive and stable standardized platform for evaluating the efficacy of cell therapy.

[0036] Example 2 In this embodiment, the killing experiment was conducted according to the method of Example 1. A fluorescence cell counter was used to evaluate the tumor cell killing efficacy of Donor1 NK cells. The fluorescence cell counter was used to calculate the tumor killing rate of immune effector cells using the following formula: (1 - experimental group cell density / single target cell control group cell density) × 100%. The viable cell density (i.e., eGFP+ positive cell density) and its killing rate detected by the fluorescence cell counter are shown in Tables 6, 7, and 8, respectively. The difference in killing rate between the fluorescence cell counter and flow cytometry is shown in Table 9.

[0037] Table 6. Donor 1 NK Killer Detection Fluorescent Cell Counter 1×10⁻⁶ 6 Results table per mL As shown in Table 6, when the effector-target ratio of the co-culture group was 20:1, 10:1, 5:1 and 1:1, the killing rate of Donor1 NK cells decreased from 93.81% to 46.95% when the fluorescence cell counter was used to detect the effector-target ratio.

[0038] Table 7. Donor 1 NK cell killing assay using a fluorescent cell counter (5×10⁻⁶) 5 Results table per mL As shown in Table 7, when the effector-target ratio of the co-culture group was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor1 NK cells decreased from 96.21% to 66.74%, as detected by a fluorescence cell counter.

[0039] Table 8. Donor 1 NK Killer Detection Fluorescent Cell Counter 2×10 5 Results table per mL As shown in Table 8, when the effector-target ratio of the co-culture group was 20:1, 10:1, 5:1 and 1:1, the killing rate of Donor1 NK cells decreased from 91.96% to 26.02%, as detected by a fluorescence cell counter.

[0040] Table 9. Differences in killing rate between Donor 1 NK cell counting and flow cytometry. The comprehensive data analysis from Tables 6 to 9 shows that: Fluorescent cell counters and flow cytometry both showed a consistent trend in detecting NK cell killing rate, namely, the killing rate gradually decreased as the effector-target ratio decreased from 20:1 to 1:1, which is consistent with the immunological dose-response relationship.

[0041] When the target cell concentration is 1×10 6 cells / mL and 2×10 5 At a target cell concentration of 5 × 10⁶ / mL, the fluorescence cell counter data showed abnormal CVs at both ends of the 20:1 and 1:1 ratios, suggesting that these two concentration groups may have systematic errors such as background interference or uneven cell distribution. In contrast, the target cell concentration was 5 × 10⁶ / mL. 5 When the number of cells / mL was ≤5%, the CV of all effector-target ratios were ≤5%, consistent with the flow cytometry data, and the difference was within an acceptable range.

[0042] When the target cell concentration is 1×10 6 cells / mL and 2×10 5 When the cell count is 1 / mL, the killing rate detected by the fluorescence cell counter is systematically lower than that of the flow cytometry (∆kill rate = cell counter method - flow cytometry % < 0). The underestimation bias increases as the effector-to-target ratio decreases. Therefore, flow cytometry is preferred as the gold standard detection method for NK cell killing efficacy, especially under conditions of low effector-to-target ratio (≤5:1) or low effector cell concentration.

[0043] Example 3 In this embodiment, the tumor cell killing activity of NK cells from different sources (Donor 2 and Donor 3) was evaluated using the detection methods described in Examples 1 and 2. The killing effect and killing rate of Donor 2 NK cells on K562-eGFP cells are shown in Tables 10 and 11, respectively; the killing effect and killing rate of Donor 3 NK cells on K562-eGFP cells are shown in Tables 12 and 13, respectively. The difference in killing rate detected by fluorescence cell counting and flow cytometry is shown in Table 14.

[0044] Table 10. Donor 2 NK Kill Detection Flow Cytometry 5×10⁻⁶ 5 Results table per mL Table 11. Donor 2 NK cell killing assay using a fluorescent cell counter (5×10⁻⁶) 5 Results table per mL As shown in Tables 10 and 11, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 2 NK cells decreased, from 94.71% / 91.86% to 28.59% / 28.80%.

[0045] Table 12 Donor 3 NK Killer Detection Flow Cytometry 5×10⁻⁶ 5 Results table per mL Table 13 Donor 3 NK Killer Detection Fluorescent Cell Counter 5×10 5 Results table per mL As shown in Tables 12 and 13, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 2 NK cells decreased, from 98.92% / 94.24% to 38.15% / 34.77%.

[0046] Table 14. Differences in NK cell killing rate between fluorescence cell counter and flow cytometry in Donor 2 / 3 NK cell killing assay. The comprehensive data analysis from Tables 10 to 14 shows that: NK cells from different donor sources showed significant differences in cytotoxic activity. At high-efficiency target-to-cell ratios (20:1–10:1), the killing rates of all donors were similar (flow cytometry-based killing rates reached Donor 1:98.59%; Donor 2:94.71%; Donor 3:98.92%). When the target-to-cell ratio decreased to 1:1, Donor 1 exhibited the strongest cytotoxic activity, with a flow cytometry-based killing rate of 71.99% (Donor 2:28.59%; Donor 3:38.15%), suggesting that individual differences among donors significantly affect NK cell efficacy.

[0047] Under uniform experimental conditions (target cell concentration of 5 × 10⁻⁶), 5 The fluorescence cell count (cells / mL) of all donor samples showed high consistency with the results of fluorescence counting and flow cytometry killing rate assays at different effector-to-target ratios. The difference in killing rate between the two methods was within an acceptable range (maximum ≤ |4.68%|), which meets the needs of routine laboratory efficacy evaluation. Although there was a slight systematic underestimation trend in the fluorescence cell count, no error exceeding the permissible range (±5%) was observed, meeting safety requirements. It is recommended that flow cytometry verification be performed on ultra-high activity samples (>95% killing) or data used for critical decision-making to improve accuracy.

[0048] Example 4 In this embodiment, the tumor cell killing activity of γδT (Donor 4) and CIK (Donor 5) cells was evaluated using the detection methods described in Examples 1 and 2. The method specifically includes: (1) Preparation of target cells: Obtain K562-eGFP target cells in the logarithmic growth phase, centrifuge and discard the supernatant, resuspend in fresh culture medium, and adjust the cell concentration to 5×10⁻⁶. 5 cells / mL; (2) Preparation of effector cells: cultured γδT or CIK cells are centrifuged, the supernatant is discarded, RPMI 1640 medium is added to resuspend the cells, count them, and the cell concentration is adjusted according to different effector-target ratios to obtain effector cell suspension. (3) Plate laying: K562-eGFP cells and γδT / CIK cells were seeded into cell well plates as experimental groups, and single-effect cell wells and single-target cell wells were set up as control groups. The experimental groups and control groups were placed in a 37℃, 5% CO2 incubator for 4 hours. (4) Killing detection: Cells were collected from the cell well plates and EGFP-positive cells were detected by flow cytometry or fluorescence cell counting. The killing rate of effector cells was calculated. The killing effect and killing rate of Donor 4 γδT cells on K562-eGFP cells are shown in Tables 15 and 16, respectively; the killing effect and killing rate of Donor 5 CIK cells on K562-eGFP cells are shown in Tables 17 and 18, respectively.

[0049] Table 15. Detection of Donor 4 γδT killing effect by flow cytometry (5×10⁻⁶ cells) 5 Results table per mL Table 16. Donor 4 γδT killing assay using a fluorescent cell counter (5×10⁻⁶) 5 Results table per mL As shown in Tables 15 and 16, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 4 γδ T cells decreased, from 93.64% / 89.20% to 17.41% / 16.95%.

[0050] Table 17 Donor 5 CIK Kill Detection Flow Cytometry 5×10⁻⁶ 5 Results table per mL Table 18 Donor 5 CIK Killer Detection Fluorescent Cell Counter 5×10 5 Results table per mL As shown in Tables 17 and 18, when the effector-target ratio was 20:1, 10:1, 5:1, and 1:1, the killing rate of Donor 5 CIK cells decreased, from 89.59% / 86.87% to 27.44% / 24.40%.

[0051] Table 19. Differences in cell killing rate between Donor 4 / 5 fluorescence cell counter and flow cytometry. The comprehensive data analysis from Tables 15 to 19 shows that: The killing efficiency of different types of immune effector cells varied significantly. γδT cells exhibited strong killing activity under the condition of an efficient target-to-cell ratio (E:T=20:1), with a killing rate of 93.64% measured by flow cytometry; CIK cells in the same group showed a slightly lower killing rate of 89.59%. However, under the condition of a low efficient target-to-cell ratio (E:T=1:1), CIK cells showed strong killing activity, with a killing rate of 27.44% measured by flow cytometry, which was higher than that of γδT cells.

[0052] Under all effector-to-target ratios and for all tested immune effector cell types, the difference in killing rate between fluorescence counting and flow cytometry was ≤|5.00%|, indicating good consistency between the two detection methods. This result demonstrates that fluorescence counting is a feasible alternative to flow cytometry for detecting the killing activity of mainstream immune effector cells such as NK, γδT, and CIK. It provides laboratories lacking flow cytometry equipment or ELISA readers with a low-cost, high-throughput, and highly reliable standardized killing activity detection solution.

[0053] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the protection scope of the present invention.

Claims

1. A method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells, characterized in that, Includes the following steps: S1. Collect peripheral blood or umbilical cord blood from healthy volunteers, separate mononuclear cells using human lymphocyte separation medium, induce them into immune effector cells, and expand and culture them. S2. Fluorescently labeled tumor cells are used as target cells, and immune effector cells are co-incubated with target cells for a period of time. S3. After co-incubation, collect the cell products and use flow cytometry or a fluorescence cell counter to detect the number of fluorescently labeled positive cells in the co-incubation products. S4. Based on the number of positive cells detected, calculate the killing rate of immune effector cells against target cells.

2. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S1, the immune effector cells include one or more of the following: NK cells, T cells, CTL cells, LAK cells, CIK cells, TIL cells, TCR-T cells, DC-CIK cells, CAR-T cells, CAR-NK cells, NK92, NK92MI, and NK92MI-CD16a engineered cells.

3. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S1, the induction and amplification culture lasts for 14 to 20 days.

4. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S2, the fluorescent label includes one or more of eGFP, GFP, RFP, mCherry, tdTomato, mOrange, and EYFP.

5. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S2, the target cells include tumor cell lines, which include one or more of K562 cells, Daudi cells, Jurkat cells, MCF-7 cells, A549 cells, and HepG2 cells.

6. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S2, during co-incubation, the initial target cell density ranges from 1×10⁻⁶ cells / year. 4 Cells / mL~1×10 7 The target-effect ratio is 40:1 to 0.1:

1.

7. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S2, during co-incubation, the initial target cell density ranges from 5 × 10⁻⁶ cells / year. 5 The target-effect ratio is 20:

1.

8. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S2, the total incubation time is from 1 hour to 48 hours.

9. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S3, the fluorescent cell counter is a cell counter equipped with a fluorescent channel for detection.

10. The method for detecting the cytotoxic activity of immune cells based on fluorescently labeled tumor target cells according to claim 1, characterized in that, In step S3, flow cytometry is performed by absolute counting of fluorescently labeled positive tumor target cells in the FITC channel.

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