Method for detecting impurity cells in iPSC-derived iNK cells

Abstract

The present invention relates to a method for detecting impurity cells in iNK cells derived from iPSC. Specifically, the present invention provides a method for quantitatively analyzing impurity cells in iPSC-derived NK products, according to the types of impurity cells in the differentiation process of iNK cells obtained by single-cell sequencing, a multicolor flow analysis method is established by a specific fluorescent antibody combination, and all impurity cell types are simultaneously identified in one test, and their proportions can be quantitatively analyzed. The flow analysis method of the present invention can detect all impurity cells in iNK cells at one time, including mast cells, NKT cells, T cells, monocytes and macrophages, and the test method is simple, low cost, and high test sensitivity, and has application prospects in the field of quality detection of iNK cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for detecting impurity cells in iNK cells derived from iPSCs. Background Art

[0002] iPSC is a type of stem cell that can theoretically differentiate into any type of human cell. iPSC-derived iNK cells have the potential to become a universal mass-produced cell product that can treat solid tumors and autoimmune diseases. However, in the process of iPSC differentiation into iNK cells, non-NK impure cells may be produced. Differentiating impure cells will not only affect the purity of the cells, but may also pose safety risks, such as the GvHD reaction that may be caused by CD3-positive cells. Studies have reported that when TCRαβ is injected into the human body, + T cells (a subtype of CD3-positive cells) were controlled at 5×10 4 / kg, the risk of GvHD can be limited. Therefore, the detection method of impurity cells in iPSC-derived iNK cells is an extremely important safety quality control method.

[0003] At present, the detection methods for impurity cells in iPSC-derived NK products mainly include single-cell sequencing and digital PCR (ddPCR). Single-cell sequencing can identify the impurity cell type in the product when the impurity cell type is unknown, but its detection process is long, expensive, and has low sensitivity. The ddPCR method requires the design of primers or the selection of antibody combinations to detect impurity cells only after the impurity cell type is clear, and the ddPCR method is limited by the number of primer combinations and cannot identify multiple impurity cells in one test.

[0004] Therefore, there is an urgent need in the art to develop a highly sensitive, simple and low-cost method for detecting impurity cells in NK products. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting impurity cells in NK cell products by flow cytometry with high sensitivity, simple process and low cost.

[0006] In a first aspect of the present invention, a method for detecting the quality of NK cell products differentiated from induced pluripotent stem cells (iPSCs) is provided, the method comprising the steps of:

[0007] A. Take samples of NK cell products differentiated from iPSC;

[0008] B. Detecting the content of impure cells in the sample by flow cytometry, wherein the reagents used in the flow cytometry include an antibody combination: anti-CD3 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD14 antibody and anti-FcεRIa antibody.

[0009] In another preferred embodiment, each type of antibody in the antibody combination carries a different fluorescent label.

[0010] In another preferred embodiment, the fluorescently labeled antibody combination is: CD3-BV421 antibody, CD56-BV605 antibody, CD19-PE-Cy7 antibody, CD14-PE antibody, FcεRIα-FITC antibody or FcεRIa-APC antibody.

[0011] In another preferred embodiment, the reagents used in the flow cytometry further include: Fc receptor blocking agent, and dyes for distinguishing live cells from dead cells.

[0012] In another preferred embodiment, the Fc receptor blocking agent is Human TruStain FcX TM .

[0013] In another preferred embodiment, the dye includes: Live / Dead-Zombie NIR.

[0014] In another preferred embodiment, in step A, sampling is performed at the following stages of iPSC differentiation: hematopoietic stem cell differentiation stage, NK cell differentiation stage, and / or NK cell expansion stage.

[0015] In another preferred embodiment, in step A, the NK cell sample is sampled from the end of the NK cell differentiation stage to the NK cell expansion stage.

[0016] In another preferred embodiment, in step A, the NK cell sample is sampled at the end of the NK cell differentiation stage, and the end of the differentiation stage is about day 28-32 after the start of iPSC differentiation.

[0017] In another preferred embodiment, in step A, the NK cell sample is sampled during the NK cell expansion stage, which is about day 32-42 after the start of iPSC differentiation.

[0018] In another preferred embodiment, in step A, the NK cell sample is sampled at the end of the NK cell expansion phase, and the end of the expansion phase is about day 38-42 after the start of iPSC differentiation.

[0019] In another preferred embodiment, the impurity cells include: mast cells, natural killer T cells (NKT cells), T cells, macrophages, monocytes, precursor B cells, and B cells.

[0020] In another preferred embodiment, step B comprises:

[0021] B1. Use CD3 / CD56 combination to set gates to encircle NKT and T cells, where CD3 + CD56 + The cells are marked as NKT cells, CD3 + CD56 - The cells were labeled as T cells;

[0022] B2. In CD3 - CD56 - Under the gate, set the gate with CD19 / FSC combination, among which CD3 - CD56 - CD19 + The cells were labeled as B cells and precursor B cells;

[0023] B3. In CD3 - CD56 - Under the gate, set the gate based on CD14 / FSC combination, where CD3 - CD56 - CD14 + The cells marked were monocytes and macrophages;

[0024] B4. In CD3 - CD56 - Under the gate, set the gate with FcεRIa / FSC combination, among which CD3 - CD56 - FcεRIa + The cell marker mast cells.

[0025] In another preferred embodiment, the step B1 further includes the following steps:

[0026] B0. Circle the main cell population of interest and remove dead cells, cell debris and adherent cells.

[0027] In another preferred embodiment, the FSC / SSC combination is used to set a gate to circle out the main target cell population and remove dead cells and cell debris that have undergone cell shrinkage.

[0028] In another preferred embodiment, the FSC-A / FSC-H combination is used to set a gate, circle out single cells, and remove adherent cells.

[0029] In another preferred embodiment, a dye for distinguishing between live cells and dead cells is used to distinguish between live cells and dead cells, thereby removing dead cells mixed in the main target cell population.

[0030] In another preferred embodiment, when the method is used for detection, the minimum limit of quantification (LOQ) of detection of various types of impurity cells in the sample is ≤0.05%.

[0031] In another preferred example, when the method is used for detection, the lowest limit of quantification (LOQ) of NKT cells and T cells in the sample is ≤0.05%, preferably ≤0.02%, for example 0.0125%.

[0032] In another preferred example, when the method is used for detection, the lowest limit of quantification (LOQ) of the detection of precursor B cells and B cells in the sample is ≤0.05%, preferably ≤0.02%, for example 0.012%.

[0033] In another preferred embodiment, when the method is used for detection, the lowest limit of quantification (LOQ) of monocytes and macrophages in the sample is ≤0.05%, for example, 0.046%.

[0034] In another preferred example, when the method is used for detection, the lowest limit of quantification (LOQ) of mast cell detection in the sample is ≤0.05%, preferably ≤0.04%, for example 0.032%.

[0035] In another preferred embodiment, the method further comprises the steps of:

[0036] C. Determine whether the NK cell product meets the quality standards;

[0037] Among them, for qualified NK cell products, the number of T cells and NKT cells should account for ≤0.25% of the total number of cells in the NK cell product.

[0038] In another preferred embodiment, for a qualified NK cell product, the number of CD3 positive cells should be less than 5×10 4 / kg body weight of a patient to be administered, wherein the CD3-positive cells include T cells and NKT cells.

[0039] In another preferred embodiment, when the patient weighs 50 kg, the infusion volume of the NK cell product is 1×10 9 When the number of CD3 positive cells is less than or equal to 0.25% of the total number of cells in the NK cell product.

[0040] In a second aspect of the present invention, a kit is provided, comprising:

[0041] Antibody combination: anti-CD3 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD14 antibody and anti-FcεRIa antibody; and

[0042] Instructions or labels, wherein the instructions or labels indicate that the kit is used for detecting impurity cells in NK cell products differentiated from iPSCs by flow cytometry.

[0043] In another preferred embodiment, the antibodies carry different types of fluorescent labels.

[0044] In another preferred embodiment, the fluorescently labeled antibody combination is: CD3-BV421 antibody, CD56-BV605 antibody, CD19-PE-Cy7 antibody, CD14-PE antibody, FcεRIα-FITC antibody or FcεRIa-APC antibody.

[0045] In another preferred embodiment, the kit further comprises: an Fc receptor blocking agent and a dye for distinguishing live cells from dead cells.

[0046] In another preferred embodiment, the Fc receptor blocking agent is Human TruStain FcX TM .

[0047] In another preferred embodiment, the dye includes: Live / Dead-Zombie NIR and the like.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0049] The beneficial effects of the present invention include:

[0050] 1. The present invention determines the types of impurity cells in iPSC-derived iNK cells, and specifically provides a fluorescent antibody combination for flow cytometry analysis, which can be used to detect various types of impurity cells in iNK cells.

[0051] 2. The flow cytometry method of the present invention has a simple process for detecting iNK impurity cells and can detect the proportion of all impurity cells at one time. It is easy to operate and low in cost.

[0052] 3. The flow cytometry method of the present invention has high sensitivity in detecting iNK impurity cells, and the detection limit of each type of impurity cells is lower than 0.05%, which can meet the requirements for iNK quality detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1A A schematic diagram of the differentiation and expansion process of iPSC-derived iNK cells is shown.

[0054] Figure 1B The image shows the differentiation and expansion process of iPSC-derived iNK cells and the single-cell sequencing results of iNK02 differentiation stage Day 32 cells.

[0055] Figure 2A The figure shows the results of establishing a flow cytometry method to detect the types and proportions of impurity cells in iNK cells.

[0056] Figure 2B The specificity validation results of the flow cytometry method are shown.

[0057] Figure 3A The sensitivity of flow cytometry for detecting T cells and NKT cells was demonstrated.

[0058] Figure 3B The sensitivity of flow cytometry for detecting B cells is shown.

[0059] Figure 3C The sensitivity of flow cytometry for detecting monocytes and macrophages is shown.

[0060] Figure 3D The sensitivity of flow cytometry for detecting mast cells was demonstrated. DETAILED DESCRIPTION

[0061] After extensive and in-depth research, the inventors have provided a method for quantitatively analyzing the detection of impurity cells in iPSC-derived NK products. Specifically, the present invention first obtains the types of impurity cells in the differentiation process of iNK cells by single-cell sequencing, and establishes a flow analysis strategy based on the type of impurity cells. A multicolor flow analysis method is established through a specific fluorescent antibody combination to simultaneously identify all impurity cell types in one test, and can quantitatively analyze their proportions. It has been verified that the flow analysis method of the present invention can detect all impurity cells in iNK cells at one time, including mast cells, NKT cells, T cells, monocytes and macrophages, and the test method is simple, low cost, and high test sensitivity.

[0062] The present invention has been completed on this basis.

[0063] iPSC-derived NK cells

[0064] Natural killer (NK) cells are important immune cells in the body and are the first line of defense against tumors and pathogens. NK cells play a key role in the body's nonspecific anti-tumor response. A large number of basic and clinical studies have applied NK cells to the treatment of hematopoietic malignancies (leukemia, lymphoma, multiple myeloma, etc.) and solid tumors (such as melanoma, ovarian cancer, and lung cancer, etc.).

[0065] Currently, NK cells used in clinical treatment are mainly derived from human tissues such as umbilical cord blood and peripheral blood. These naturally derived NK cells have high heterogeneity, high preparation costs, low yields, and their preparation cycles are limited by the accessibility of human tissues.

[0066] As used herein, the terms “iPSC-derived NK cells”, “NK cells differentiated from iPSCs” and “iNK cells” are used interchangeably and all refer to NK cells differentiated from iPSCs in vitro.

[0067] iPSC (induced pluripotent stem cells) can differentiate into NK cells, i.e. iNK cells. iPSC is a cell type with characteristics similar to those of embryonic stem cells. It was first reported by the Yamanaka Laboratory of Kyoto University in 2006. By introducing a set of specific transcription factors into terminally differentiated somatic cells such as fibroblasts, these cells can be reprogrammed into pluripotent stem cells. These transcription factors include Oct3 / 4, Sox2, Klf4 and c-Myc, which work together to reset the gene expression pattern of cells and return them to a state similar to that of embryonic stem cells.

[0068] iPSC (induced pluripotent stem cell)-derived NK cells have some unique characteristics that make them show great potential in the field of immunotherapy.

[0069] First, iPSC-derived NK cells have anti-tumor and anti-viral functions similar to natural NK cells in the body. They can fight tumors and viral infections by recognizing and killing target cells. In addition, these cells can secrete a variety of cytokines, further enhancing their immunomodulatory effects. Secondly, iPSC technology can obtain pluripotent stem cells from the patient himself and obtain NK cells by inducing differentiation. A significant advantage of this method is that it reduces the risk of immune rejection, and the anti-tumor activity and safety of these cells can be further optimized through gene editing technology. In general, iPSC-derived NK cells have significant advantages in safety, effectiveness, and personalized treatment.

[0070] Impure cells in iNK cells

[0071] iPSCs can be induced to differentiate into iNK cells under certain conditions. Specifically, iPSCs can aggregate together to form embryoid bodies (EBs). Under the culture conditions of adding special cytokines, EBs gradually grow larger and gradually differentiate into hematopoietic stem cells. After about 28 days of culture, EBs can differentiate into NK cells, which can be further activated and amplified. Ultimately, one starting iPSC cell can differentiate into 1,000-10,000 iNK cells.

[0072] The production process of iPSC-derived iNK is generally divided into two major stages, namely the induction differentiation stage of iPSC to iNK (the 24th to 32nd day is the selection point at the end of differentiation) and the subsequent iNK expansion stage (the 35th to 42nd day is the selection point at the end of expansion). During the whole process, the cell types mainly change in the differentiation stage. After the differentiation stage, the NK cell expansion stage is entered, and no new impurity cell types are generated. After the expansion stage, the purity of NK cells can reach more than 99%, and the number of impurity cells will decrease, which may make some impurity cells difficult to detect. Therefore, selecting the cell population at the end of the differentiation stage for cell type detection can obtain the most accurate results.

[0073] In the present invention, single cell sequencing is performed on samples at the end of the NK differentiation stage to obtain impurity cell types in iNK cells. Preferably, the end of the NK differentiation stage is from the 28th to the 32nd day after the iPSC begins to differentiate, and more preferably the 32nd day.

[0074] According to the single-cell sequencing results, after removing cell types with extremely low content and not in the main differentiation pathway, the results showed that the main impurity cell types in iPSC-derived iNK were: CD3-positive cells (T cells, NKT cells), mast cells, macrophages, monocytes, and precursor B cells.

[0075] CD3 positive cells (T cells, NKT cells) have been verified to be related to graft-versus-host disease (GvHD); mast cells are related to the body's allergic reactions. When mast cells are induced by antigens, they cause the aggregation of FcεRI receptor molecules on the cell surface and release inflammatory factors. Therefore, CD3 positive cells (T cells, NKT cells) and mast cells are impurity cells that need to be paid special attention to in iNK products.

[0076] For clinical iNK products, the required number of T cells and NKT cells should be controlled to less than 5×10 4 / kg. When the patient weighs 50kg, the iNK cell infusion volume is 1×10 9 When the proportion of T cells and NKT cells in NK cell products is ≤0.25%, the corresponding proportion of T cells and NKT cells in NK cell products should be ≤0.25%. Therefore, in the method for detecting impurity cells, the detection limit of at least T cells and NKT cells should be less than 0.25%.

[0077] iNK impurity cell flow cytometry detection method of the present invention

[0078] In the present invention, a flow analysis method for detecting impurity cells and their proportions in iNK cells is provided, which can detect mast cells, NKT cells, T cells, monocytes and macrophages at one time and obtain the proportion of each impurity cell. The method specifically comprises the steps of:

[0079] For the NK cell samples differentiated from iPSC, the content of impurity cells in the samples was detected by flow cytometry.

[0080] iNK cell samples are obtained from the end of the NK differentiation stage to the expansion stage, and the specific sampling time point can be flexibly determined according to the actual situation. For example, when it is necessary to detect the situation of impurity cells after the end of the NK differentiation stage, sampling can be performed on about the 28th to 32nd day after the start of iPSC differentiation; or, when it is necessary to detect the situation of impurity cells after the end of the NK expansion stage, sampling can be performed on about the 35th to 42nd day after the start of iPSC differentiation.

[0081] Based on the markers on the surface of impurity cells, the antibody combination used for flow cytometry analysis was determined: anti-CD3 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD14 antibody and anti-FcεRIa antibody. + CD56 + Characterization of NKT cells, CD3 + CD56 - Characterize T cells; CD3 - CD56 - CD19 + Characterizes B cells and precursor B cells; CD3 - CD56 - CD14 + Characterize monocytes and macrophages; and CD3 - CD56 - FcεRIa + Characterization of mast cells.

[0082] In order to detect various cell types at one time, different antibodies are labeled with different fluorescent groups. For multicolor flow cytometry, the selection of multiple fluorescent markers needs to meet the following conditions: one fluorescent substance is selected for each detection channel, and the fluorescent substances between channels can be matched at will; the overlap of the spectra of various fluorescent substances selected should be minimized; fluorescence itself has different strengths, and the appropriate fluorescence should be selected according to the strength of antigen expression and grouping. For example, if the antigen expression is weak or the grouping is not obvious, it is recommended to choose strong fluorescence. On the contrary, if the antigen expression is strong or the grouping is obvious, it is recommended to choose weak fluorescence.

[0083] In an exemplary embodiment of the present invention, the above-mentioned antibodies are paired with the following types of fluorescent labels: CD3-BV421, CD56-BV605, CD19-PE-Cy7, CD14-PE, FcεRIα-FITC or FcεRIa-APC, wherein BV421, BV605, Cy7, PE, FITC, and APC represent different fluorescent groups, respectively.

[0084] In order to remove the influence of dead cells, cell debris and adherent cells on the detection accuracy in flow cytometry analysis, Fc receptor blocking agents, dyes for distinguishing live cells from dead cells, etc. can be optionally added to the flow cytometry detection reagent.

[0085] In order to verify the feasibility and accuracy of the method of the present invention, the specificity and sensitivity of the method were tested. The results showed that the method of the present invention can still clearly distinguish positive cells in the presence of other negative cells. In addition, the present invention tested the minimum limit of quantification LOQ of various types of impurity cells, that is, the minimum quantifiable value for low-concentration samples while ensuring the accuracy of the results. The results showed that the minimum limit of quantification LOQ for each type of impurity cell detection was lower than 0.05%, which has a high sensitivity and can meet the requirements for iNK cell quality detection.

[0086] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0087] Example 1: Types of impure cells in iNK differentiation

[0088] In this example, iPSCs (named as iP02) provided by Shanghai Oriental Hospital were used, and differentiated and cultured to obtain an iNK cell population, named as iNK02.

[0089] The schematic diagram of the differentiation culture process is shown in Figure 1A , among which days 24-32 are the NK differentiation stage, and days 35-42 are the NK expansion stage.

[0090] The cell types in the cell population mainly change during the differentiation stage. After the differentiation stage, the cell types remain basically unchanged. After the expansion stage, the number of impurity cells will decrease, which may make it difficult to detect some impurity cells. Therefore, the cell population at the end of the differentiation stage was selected for cell type detection. The cells on day 32 of the iNK02 differentiation stage were used for NK cells (CD56 + ) after negative selection and then sent to Xuzhenda Biological Company for single-cell sequencing.

[0091] Test results such as Figure 1B As shown. Since sorting cannot completely remove CD56 +Cells, so there are still some NK cells in the single-cell sequencing results. In addition to NK cells, the cells detected by single-cell sequencing also include mast cells, macrophages, monocytes, precursor B cells, natural killer T cells (NKT), erythrocytes and fibroblasts. The proportion of each cell group is shown in Table 1.

[0092] Table 1 Single-cell sequencing results

[0093]

[0094] From the above results, we can see that the main types of impurity cells contained in the iNK cell population differentiated from iPSC are: mast cells, macrophages, monocytes, precursor B cells, NKT cells, nucleated erythrocytes and fibroblasts. The proportion of nucleated erythrocytes and fibroblasts is extremely low, and considering that they are not the cells that may be produced in the main path of iPSC-induced differentiation into iNK, these two cells are no longer considered in the subsequent study.

[0095] Example 2: Detection of impurity cells in iNK cell population by flow cytometry

[0096] After the impurity cell types are known, this embodiment provides a method for one-time detection of the impurity cell types using flow cytometry and verifies its feasibility.

[0097] For the iNK02 cell population obtained in Example 1, the types and proportions of multiple impurity cells were detected at one time by flow cytometry. The fluorescent antibody combinations and product numbers used were as follows: CD3-BV421 (317344), CD56-BV605 (362538), CD19-PE-Cy7 (302216), CD14-PE (301806), FcεRIα-FITC (334608) or FcεRIa-APC (334612), Zombie NIR (423106) and Human TruStain FcX TM (422302), are commercial reagents purchased from Biolegend, Inc. Among them, TruStain FcX TM Used to block cells and reduce interference from non-specific binding of antibodies; Zombie NIR TM Cell life and death detection dyes can exclude dead cells and reduce cell autofluorescence.

[0098] The steps of flow cytometry are as follows:

[0099] The cell samples were centrifuged and resuspended in DPBS to 1×107 / mL; add 100μL sample cell suspension to the corresponding wells of the 96-well plate; add 100μL DPBS to each sample well, and gently pipette 3~4 times to mix; centrifuge at 300g for 5min, shake the plate, and discard the supernatant; add 100μL of the prepared Zombie NIR premix to each sample well, and gently pipette 6~8 times to mix; incubate at room temperature for 10min; add 100μL DPBS to each sample well, and gently pipette 6~8 times to mix; centrifuge at 300g for 5min, shake the plate, and discard the supernatant; add 50μL of the prepared Fc Block premix to each sample well, and gently pipette 6~8 times to mix; incubate at room temperature for 10min; add 50μL of the prepared extracellular antibody mixture (see Table A below for the specific component ratio) to the corresponding wells, and gently pipette 6~8 times to mix; incubate at room temperature, away from light for 20min; add 100μL DPBS, 300×g Centrifuge for 5 min, shake the plate, and discard the supernatant; add 200 μL DPBS respectively, centrifuge at 300×g for 5 min, shake the plate, and discard the supernatant; add 200 μL DPBS, gently pipette 6 to 8 times to mix, and resuspend the sample; use flow cytometer to collect samples.

[0100] Table A

[0101]

[0102] The analysis strategy of flow cytometry results is as follows:

[0103] a. FSC / SSC combination, circle the main target cell population, remove dead cells and cell debris that have cell shrinkage;

[0104] b. Under the previous gate, use the FSC-A / FSC-H combination to circle out single cells and remove adherent cells;

[0105] c. Under the previous gate, Live / Dead-Zombie NIR / FSC, circle out live cells and remove dead cells mixed in the main target cell population;

[0106] d. Under the previous gate, CD3 / CD56, circle NK / NKT / T cells, CD3 - CD56 + NK cells, CD3 + CD56 + NKT cells, CD3 + CD56 - T cells;

[0107] e. on CD3 - CD56 - Gate, CD19 / FSC combination, circle B and Pre-B cells;

[0108] f. In CD3 - CD56 - Gate, CD14 / FSC combination, monocytes and macrophages are circled;

[0109] g. In CD3 - CD56 - Gate on the FcεRIa / FSC combination and circle mast cells.

[0110] The flow cytometry results are as follows Figure 2A As shown in Table 2. Flow cytometry revealed that various types of impurity cells were detected in different proportions in the iNK cells at the end of differentiation (day 28), while the iNK cells at the end of amplification (day 42) had a higher purity, with only a small number of NKT cells detected, and other impurity cell types were basically not detected. The impurity cell types were the same as the single-cell sequencing results.

[0111] Table 2 Proportion of cells detected by flow cytometry

[0112]

[0113] Example 3: Specificity verification of flow cytometry

[0114] In order to verify whether the flow cytometry method of the present invention can accurately identify negative or positive cells in the presence of other types of cells, the following experiments were performed:

[0115] Undifferentiated iPSC cells (iP02) do not express CD3 / CD14 / CD19 / FcεRIα, Nalm6 cells do not express CD56, and PBMC express all of the above cell markers; PBMC were stained with antibodies against CD3 / CD14 / CD19 / FcεRIα / CD56, iP02 were stained with CD3 / CD14 / CD19 / FcεRIα, and Nalm6 cells were stained with CD56, and the analysis results were superimposed.

[0116] The results are as follows Figure 2B The results showed that the above antibodies can accurately identify positive and negative cells, and can still clearly distinguish positive cells when other negative cells are mixed in.

[0117] Example 4: Sensitivity test of flow cytometry

[0118] In this example, the sensitivity of the method was confirmed by a spike-in experiment. In the NK cells that were confirmed to be free of the above-mentioned impurity cell types by multiple tests, PBMCs were used as positive cells and spiked in proportion to confirm the sensitivity of the method of the present invention for detecting CD3 positive cells (including T cells and NKT cells), B cells, monocytes and macrophages, and mast cells. The specific operation is as follows:

[0119] PBMCs were used as positive cells and mixed into negative cells at different ratios for staining. The sensitivity of the method was determined based on the linear results and the recovery rate between the measured value and the theoretical value. 2 ≥0.95, LOQ recovery rate is 70%~130%, and coefficient of variation CV≤20%. Figure 3A to Figure 3D And as shown in Table 3.

[0120] The results of CD3 positive cell detection are as follows Figure 3A The results show that the linear R 2 =0.999, and when the theoretical value of CD3 positive cell positive rate was 0.0125%, the recovery rate was 93.33%, and the coefficient of variation CV = 13.58%, which met the acceptable standard of LOQ. Therefore, the minimum limit of quantification LOQ of CD3 positive cell detection was 0.0125%.

[0121] Table 3-1 CD3 positive cell detection sensitivity

[0122]

[0123] The results of B cell detection were as follows Figure 3B As shown in Table 3-2. The results show that CD3 - CD56 - CD19 + Cells in the range of 0.012% to 14.9%, linear R 2 =0.999, and in CD3 - CD56 - CD19 + When the theoretical value of cell positive rate was 0.032%, the recovery rate was 98.13% and the coefficient of variation CV = 14.43%, which met the acceptable standard of LOQ. - CD56 - CD19 + The lowest limit of quantification (LOQ) for positive cell detection was 0.012%.

[0124] Table 3-2 CD3 - CD56 - CD19 + Cell detection sensitivity

[0125]

[0126] The results of monocyte and macrophage detection are as follows Figure 3C As shown in Table 3-3. The results show that CD3 - CD56 -CD14 + Cells in the range of 0.046% to 2.92%, linear R 2 =0.998, and in CD3 - CD56 - CD14 + When the theoretical value of cell positive rate was 0.046%, the recovery rate was 86.96% and the coefficient of variation CV = 0%, which met the acceptable standard of LOQ. - CD56 - CD14 + The lowest limit of quantification (LOQ) for positive cell detection was 0.046%.

[0127] Table 3-3 CD3 - CD56 - CD14 + Cell detection sensitivity

[0128]

[0129] Mast cell test results Figure 3D As shown in Table 3-4. The results show that CD3 - CD56 - FcεRIα + The cells were in the range of 0.032% to 2.04%, and the linear R 2 =1, and in CD3 - CD56 - FcεRIα + When the theoretical value of cell positive rate was 0.032%, the recovery rate was 86.75% and the coefficient of variation CV = 9.62%, which met the acceptable standard of LOQ. - CD56 - FcεRIα + The lowest limit of quantification (LOQ) for positive cell detection was 0.032%.

[0130] Table 3-4 CD3 - CD56 - FcεRIα + Cell detection sensitivity

[0131]

[0132] The above results show that the flow cytometry method of the present invention has high sensitivity for the detection of T cells and NKT cells, B cells, monocytes and macrophages, and mast cells, and the minimum limit of quantification LOQ is lower than 0.05%, which can meet the requirements for iNK cell quality detection.

[0133] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting the quality of NK cell products differentiated from induced pluripotent stem cells (iPSCs), characterized in that: The method comprises the steps of: A. taking a NK cell sample differentiated from iPSC; wherein the NK cell sample is taken from the end of the NK cell differentiation stage to the NK cell expansion stage; B. detecting the content of impurity cells in the sample by flow cytometry, wherein the reagents used in the flow cytometry include an antibody combination: anti-CD3 antibody, anti-CD56 antibody, anti-CD19 antibody, anti-CD14 antibody and anti-FcεRIa antibody; Wherein, step B comprises: B1. Use CD3 / CD56 combination to set gates to encircle NKT and T cells, where CD3 + CD56 + The cells are marked as NKT cells, CD3 + CD56 - The cells were labeled as T cells; B2. In CD3 - CD56 - Under the gate, set the gate with CD19 / FSC combination, among which CD3 - CD56 - CD19 + The cells were labeled as B cells and precursor B cells; B3. In CD3 - CD56 - Under the gate, set the gate based on CD14 / FSC combination, where CD3 - CD56 - CD14 + The cells marked were monocytes and macrophages; B4. In CD3 - CD56 - Under the gate, set the gate with FcεRIa / FSC combination, among which CD3 - CD56 - FcεRIa + The cell marker mast cells.

2. The detection method according to claim 1, characterized in that Each type of antibody in the antibody panel carries a different fluorescent label.

3. The detection method according to claim 1, characterized in that In step A, the NK cell sample is sampled during the NK cell expansion stage, which is 32-42 days after the start of iPSC differentiation.

4. The detection method according to claim 1, characterized in that The impurity cells are: mast cells, natural killer T cells (NKT cells), T cells, macrophages, monocytes, precursor B cells, and B cells.

5. The detection method according to claim 1, characterized in that Before step B1, the following steps are also included: B0. Circle the main cell population of interest and remove dead cells, cell debris and adherent cells.

6. The detection method according to claim 1, characterized in that When the method is used for detection, the minimum limit of quantification (LOQ) of various impurity cells in the sample is ≤0.05%.

7. The detection method according to claim 1, characterized in that Also includes the steps: C. Determine whether the NK cell product meets the quality standards; Among them, for qualified NK cell products, the number of T cells and NKT cells should account for ≤0.25% of the total number of cells in the NK cell product.

8. The detection method according to claim 1, characterized in that The NK cell sample is sampled at the end of the NK cell expansion phase, which is 38-42 days after the start of iPSC differentiation.

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