Preparation method of cryopreserved umbilical cord blood hematopoietic stem cell-derived NK cells

Abstract

The invention discloses a preparation method of NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells. The preparation method comprises the following steps: (1) recovering umbilical cord blood; (2) separating mononuclear cells of umbilical cord blood; (3) induced preparation of NK cells: adding an activation culture medium into the coated culture flask, inoculating the mononuclear cells into the culture flask, adding IL-21, IL-18, an anti-apoptosis agent, BDNF, NGF, TPO and SDF-1, and culturing under the conditions of 5% CO2 and 37 DEG C; supplementing the activated culture medium on the third day and the fifth day of the culture; from the seventh day of culture, supplementing the amplification culture medium every two days; and collecting the cells on the 17th-20th day of culture to obtain the NK cells. According to the method disclosed by the invention, various activating factors are added during activation, so that the cells can be promoted to recover the state before cryopreservation. The NK cells obtained by the method can secrete a large amount of cell factors IFN-gamma, and are the same as the NK cells from fresh umbilical cord blood.

Description

Technical Field

[0001] The invention relates to a method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells, and belongs to the technical field of immune cells. Background Art

[0002] NK cells, full name Natural Killer cells, are an important type of immune cells and are core members of the innate immune system. Their main function is to identify and kill tumor cells and virus-infected cells, and they are the body's first line of defense against tumors and viruses. Currently, NK cells can be obtained through the following ways: peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), NK cell lines, bone marrow, embryonic stem cells (ESCs), etc. Among them, NK cells derived from umbilical cord blood have the following advantages: no pain and risk for donors during collection; contain more immature immune cells, easy to culture and activate; strong proliferation ability and bone marrow homing ability; lower immunogenicity and GVHD risk; stronger adaptability to the tumor microenvironment, can better adapt to the internal environment of different tumors, and improve the treatment effect. These advantages make NK cells derived from umbilical cord blood an important direction in the field of immunotherapy. However, the current difficulty is to use frozen umbilical cord blood hematopoietic stem cells to culture NK cells.

[0003] As we all know, frozen umbilical cord blood cells are very fragile, and the immune cells are even more fragile. During the programmed cooling and thawing process, ice crystal damage may occur on the cell membrane and the membrane of the main organelles, and many receptor proteins on the cell surface may fall off during the freezing and thawing process, which will affect the proliferation of cells. At the same time, when umbilical cord blood cells are thawed, dead cells (mainly granulocytes) are very easy to aggregate and form clumps, making it difficult to separate from living cells, affecting the subsequent culture process. This is not considered in most literature. The steps of extracting mononuclear cells (CBMC) from frozen umbilical cord blood and inducing NK cells are different from those of extracting mononuclear cells from fresh umbilical cord blood. The reason is that the cells become more fragile after freezing, and it is necessary to quickly recover and activate the immune cells in them after extracting mononuclear cells to induce them into NK cells. There are literature reports on the use of 4℃ pre-cooled thawing solution to thaw frozen umbilical cord blood hematopoietic stem cells, but the cells are thawed in a 37℃ water bath and then added with 4℃ pre-cooled thawing solution, which makes the cells (especially the immune cells) more fragile. Invention patent application CN117286101 A discloses a method for efficiently amplifying natural killer cells in cryopreserved umbilical cord blood, which uses animal cell culture medium. There are safety issues with animal-derived components in clinical use, and it is mixed with a large number of red blood cells, which will affect the subsequent growth of immune cells, and there is a problem of low amplification multiples. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells.

[0005] The present invention is achieved through the following technical solutions: A method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells comprises the following steps: (I) Umbilical cord blood resuscitation: Take frozen umbilical cord blood, thaw it quickly in a water bath, add 37°C resuscitation solution (pre-warmed); centrifuge, discard the supernatant, and obtain a blood sample; (ii) Separation of mononuclear cells from umbilical cord blood: separation of mononuclear cells from umbilical cord blood using lymphocyte separation solution, addition of ammonium chloride solution to lyse red blood cells, and washing with a cleaning solution to obtain mononuclear cells; (III) Induction and preparation of NK cells: Add activation medium to the culture flask coated with Lymactin-NK, inoculate umbilical cord blood mononuclear cells into the culture flask, add IL-21, IL-18, anti-apoptotic agent, BDNF, NGF, TPO and SDF-1 (after addition, the concentrations of IL-21, IL-18, anti-apoptotic agent, BDNF, NGF, TPO and SDF-1 are 20 ng / mL, 10 ng / mL, 20mM, 50μg / mL, 50μg / mL, 50μg / mL and 50μg / mL, respectively), and culture in a 5% CO2, 37°C constant temperature incubator; on the 3rd and 5th day of culture, add activation medium; starting from the 7th day of culture, add expansion medium every 2 days; collect cells on the 17th to 20th day of culture to obtain NK cells; The activation medium is composed of KBM581 medium and IL-15, and the concentration of IL-15 is 20 ng / mL; The expansion medium is composed of KBM581 medium and IL-2, and the concentration of IL-2 is 1000 IU / mL.

[0006] Furthermore, in the step (i), the water bath temperature during melting in a water bath is 40°C.

[0007] Furthermore, in step (i), the resuscitation solution is prepared from the following solutions: 140 mL of 0.9% (mg / mL) sodium chloride injection, 50 mL of compound electrolyte solution, and 10 mL of human albumin solution (the concentration of human albumin is 20%, g / L).

[0008] Furthermore, in the step (i), when adding the 37°C resuscitation solution, the solution is added at a constant speed while shaking and the duration is not less than 3 minutes.

[0009] Furthermore, in step (i), the amount of resuscitation solution added is 8 times the volume of the umbilical cord blood.

[0010] Furthermore, in the step (ii), the concentration of the ammonium chloride solution is 0.8% (unit: g / 100 mL).

[0011] Furthermore, in the step (ii), the cleaning solution is prepared by the following method: add 2 mL of DNAseⅠ solution (1 mg / mL) and 1.5 mL of MgCl2 solution (25 mM) to 200 mL of 0.9% sodium chloride solution, mix well, and obtain the cleaning solution.

[0012] Furthermore, the specific operation of step (ii) is as follows: (1) Layering: slowly and evenly add the blood sample to the upper layer of human lymphocyte separation medium at a ratio of 2:1 and centrifuge; (2) Extraction of mononuclear cells: After centrifugation, obvious stratification appears in the centrifuge tube. Aspirate the mononuclear cell layer (buffy coat layer); (3) First wash: add an equal volume of washing solution to the mononuclear cells, centrifuge, discard the supernatant, and obtain the cell pellet; (4) Lysis of red blood cells: dilute the cell pellet with 3 times (volume multiple) of 0.8% ammonium chloride solution, mix well, and lyse at 4°C for 10 min to obtain the lysate. (5) Second wash: Add cleaning solution to the lysis buffer to terminate lysis and wash, centrifuge, discard the supernatant, and obtain the cell pellet; (6) Third wash: Add cleaning solution to wash the cells, centrifuge, and discard the supernatant to obtain mononuclear cells.

[0013] Furthermore, in the step (iii), the specific method of coating is: coating the non-TC-treated T75 culture flask with 4 mL of PBS buffer and 1 mL of Lymactin-NK solution for more than 1 h, removing the coating solution, and washing away the residual coating solution with PBS buffer.

[0014] Furthermore, in the step (iii), the amount of the activation medium added on the third day of culture is: 90% to 110%, preferably 100% of the initial activation medium.

[0015] Furthermore, in the step (iii), the amount of the activation medium added on the fifth day of culture is 350% to 450%, preferably 400%, of the initial activation medium.

[0016] Furthermore, in step (iii), starting from the 7th day of culture, the expansion medium was added every 2 days to adjust the cell density to 2×10 6 / mL.

[0017] NK cells prepared by the above method.

[0018] The method for preparing NK cells derived from frozen cord blood hematopoietic stem cells of the present invention uses a resuscitation solution that has been pre-warmed at 37°C to resuscitate the cord blood cells, and uses a cleaning solution in the washing process, the cleaning solution contains sodium chloride, DNAse and MgCl2 to prevent cell agglomeration. An ammonium chloride solution is added to lyse red blood cells to reduce the impact of red blood cells on subsequent culture. After obtaining a sufficient number of cord blood mononuclear cells in good condition and good cell potential using this method, multiple activation factors (anti-apoptotic agents, BDNF, NGF, SDF-1, TPO, etc.) are added during activation to promote the cells to return to the state before freezing, so that they are induced and activated, and finally NK cells are obtained through culture.

[0019] The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells of the present invention has the following advantages: (1) The present invention uses a resuscitation protection solution that has been pre-warmed at 37°C to resuscitate umbilical cord blood cells, which can better protect cells compared to the resuscitation protection solution that has been pre-cooled at 4°C reported in the literature.

[0020] (2) After being frozen, the umbilical cord blood is relatively fragile after being resuscitated. It is not suitable for long-term separation by the Ficoll method in a laboratory environment, which will affect the enrichment of mononuclear cells and the final state of the cells. However, if it is not separated by the Ficoll method, a large number of red blood cells will remain. After many attempts, the present invention uses the Ficoll method to extract mononuclear cells from umbilical cord blood. The washing solution used in the washing process of extracting mononuclear cells from umbilical cord blood contains sodium chloride, DNAse and MgCl2, which can prevent cell agglomeration and protect the enrichment of mononuclear cells and the final state of the cells. Adding ammonium chloride solution to lyse red blood cells before centrifugation greatly reduces the impact of red blood cells on subsequent culture.

[0021] (3) Compared with fresh cord blood mononuclear cells, the most important thing about extracting mononuclear cells from frozen cord blood is to restore their cell state to the state of fresh cells as much as possible. Based on the conventional method of culturing NK cells, the present invention adds some cytokines (such as anti-apoptotic agents, BDNF, NGF, SDF-1, TPO, etc.) on the day of activation. The added anti-apoptotic agents play an important role in the cell culture process. They maintain the survival and function of cells by inhibiting the apoptotic process. Apoptosis is a form of programmed cell death that is essential for removing damaged or no longer needed cells. However, in cell culture, excessive apoptosis may lead to a decrease in the number of cells, affecting the experimental results. In the cell culture process, the use of anti-apoptotic agents can help maintain the stable state of cells.

[0022] (4) The subsequent culture of the present invention only uses KBM581 culture medium plus IL-2 expansion culture medium, which greatly improves the expansion multiple of NK cells resuscitated from frozen cord blood and maintains their phenotypes the same as fresh cord blood NK cells while saving costs.

[0023] (5) In addition, the preparation method of the present invention is a pure factor culture method without trophoblast cells. The reagents used are all commercially available reagents, do not require sterilization, do not contain animal-derived ingredients, and can reach clinical grade.

[0024] The preparation method of NK cells derived from cryopreserved cord blood hematopoietic stem cells of the present invention provides an additional option for depositors who have stored cord blood (but not used for treatment or not within the scope of indications of cord blood hematopoietic stem cell treatment technology), and can choose to culture NK cells. With the development of technology, the potential of NK cell therapy will be greatly improved, and the types of diseases treated by NK cells will also increase accordingly. For society, public cord blood banks need to be matched when used, but there are also situations where they cannot be used, which will result in a relatively large waste of resources; the present invention can solve the current status of public blood banks, while improving the amplification efficiency and yield of NK cells, reducing the cost of treatment, so that more patients can afford this treatment, so that more cord blood can be fully utilized, and the market prospects are broad. In short, NK cell therapy derived from cord blood, as an emerging immunotherapy, has great potential and broad application prospects. Through continuous research and technological innovation, overcoming existing technical bottlenecks can further promote the development of NK cell therapy and bring more treatment hope to patients. At the same time, this will also promote the development of the biopharmaceutical industry and bring positive impacts to the social economy.

[0025] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Schematic diagram of flow cytometry phenotyping results.

[0027] Figure 2 : Schematic diagram of cell killing experiment results, where A: A549 cells; B: K562 cells; C: MCF-7 cells.

[0028] Figure 3 : Schematic diagram of cytokine production results. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0030] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0031] The sources of the main experimental reagents used in the present invention are as follows: Reagents: KBM581 culture medium (CORNING), human lymphocyte separation medium (Ficoll, Tianjin Haoyang Biological Products Co., Ltd.), sodium chloride injection (Chenxin Pharmaceutical), compound electrolyte injection (Shijiazhuang Siyao), and human serum albumin (Jetbelin).

[0032] Cytokines and antibodies: IL-15, IL-21, IL-18 (all purchased from Sino Biological Pharmaceuticals); human recombinant IL-2 (Intercon); Lymactin-NK (purchased from Besso); deoxyribonuclease I (DNAse I), MgCl2 solution (both purchased from Solebao); anti-apoptotic agent (Biyuntian), BDNF (Tongli Haiyuan), NGF (Punosai), SDF-1 (Punosai), TPO (Punosai); CD3, CD56, 7-AAD (all purchased from BD).

[0033] CFSE proliferation kit, Annexin V-FITC / PI apoptosis kit, Human IFN-gamma ELISAKit detection kit (enzyme-linked immunosorbent assay) (Lianke Biotechnology).

[0034] The main experimental consumables used in the present invention are as follows: 250 mL centrifuge tube (Jie Te), 50 mL centrifuge tube (Jie Te), 10 mL pipette (Biscellide), 25 mL pipette (Jie Te), 5 mL pipette (Jie Te), T-75 cm 2 Cell culture flasks (Jet), 24-well plates, and 96-well concave bottom culture plates (CORNING).

[0035] Experiment 1 Cultivation of NK cells using cryopreserved umbilical cord blood Here are the steps: 1. Umbilical cord blood resuscitation Take a frozen umbilical cord blood bag (25 mL, containing cryopreservation solution) and quickly thaw it in a 40°C water bath; after thawing, cut the blood bag with sterile scissors, pour the umbilical cord blood containing cryopreservation solution into a 250 mL centrifuge tube, and add 37°C resuscitation solution (previously warmed in a 37°C incubator for more than 1 h) to the centrifuge tube while shaking. The whole process should not be less than 3 minutes, and the amount of resuscitation solution added should be 8 times the volume of the umbilical cord blood; immediately centrifuge (840 g, 10 min, RT, speed increase 9, speed decrease 5) after adding the resuscitation solution, discard the supernatant, and obtain the blood sample.

[0036] The resuscitation solution is prepared from the following solutions: 140 mL of 0.9% sodium chloride injection, 50 mL of compound electrolyte injection, and 10 mL of human albumin solution (the concentration of human albumin is 20%).

[0037] 2. Isolation of mononuclear cells from umbilical cord blood (1) Sample layering: blood sample was slowly and evenly added to the upper layer of human lymphocyte separation solution (15 mL pre-loaded in a 50 mL centrifuge tube and stored at room temperature) at a ratio of 2:1, and centrifuged (935 g, 20 min, RT, speed increase 1, speed decrease 1); (2) Extraction of mononuclear cells: After centrifugation, obvious stratification appears in the centrifuge tube. Aspirate the mononuclear cell layer (buffy coat layer); (3) First wash: add an equal volume of washing solution to the mononuclear cells to a final volume of 45 mL, centrifuge (935 g, 7 min, RT, 9% speed, 9% speed), discard the supernatant, and obtain the cell pellet; The cleaning solution is prepared by the following method: add 2 mL of DNAseⅠ solution (1 mg / mL) and 1.5 mL of MgCl2 solution (25 mM) to 200 mL of 0.9% sodium chloride injection, mix well, and obtain; (4) Lysis of red blood cells: dilute the cell pellet with 3 times (volume multiple) of 0.8% ammonium chloride solution, mix well, and lyse at 4°C for 10 min to obtain the lysate. (5) Second wash: Add washing solution to 45 mL of the lysate to terminate lysis and wash, centrifuge (336 g, 8 min, RT, 9% ascent, 9% as deceleration), discard the supernatant, and obtain the cell pellet; (6) Third wash: Add 45 mL of washing solution to wash the cells, centrifuge (336 g, 8 min, RT, 9% ascent, 9% as deceleration), discard the supernatant, and obtain mononuclear cells.

[0038] (III) Induction and preparation of NK cells (1) Add 10 ml of activation medium to the coated culture flask and culture mononuclear cells at a rate of 4.0×10 6The cells were plated at a density of 10 cells / mL; IL-21 (20 ng / mL), IL-18 (10 ng / mL), anti-apoptotic agent (20 mM), BDNF (50 μg / mL), NGF (50 μg / mL), TPO (50 μg / mL) and SDF-1 (50 μg / mL) were added and cultured in a 5% CO2, 37°C constant temperature incubator; The specific method of coating is: coating a non-TC-treated T75 culture flask with 4 mL of PBS buffer and 1 mL of Lymactin-NK solution for more than 1 h, removing the coating solution, and washing away the residual coating solution with PBS buffer; The activation medium is composed of KBM581 medium and IL-15, and the concentration of IL-15 is 20 ng / mL; (2) On the third day of culture, add 10 mL of activation medium; (3) On the 5th day of culture, add 40 mL of activation medium; (4) Starting from the 7th day of culture, samples were taken and counted every 2 days, and expansion medium was added to adjust the cell density to 2×10 6 / mL or so; The expansion medium is composed of KBM581 medium and IL-2, and the concentration of IL-2 is 1000 IU / mL; (5) Collect cells on the 17th to 20th day of culture to obtain NK cells for phenotypic and functional analysis.

[0039] Experiment 2 NK cell phenotype analysis 1. NK cell identification (A) Take the NK cell suspension cultured for 19 days in Experiment 1 and perform the following operations: (1) Transfer 1 mL of cell suspension to a 1.5 mL centrifuge tube. Centrifuge at 300 g for 5 minutes at 4°C. Carefully aspirate and discard the supernatant.

[0040] (2) Wash the cells with an appropriate amount of PBS buffer, centrifuge at 4°C, 300 g for 5 min, carefully aspirate and discard the supernatant.

[0041] (3) Resuspend the cells in pre-cooled PBS buffer and adjust the final cell concentration to 1×10 7 cells / mL and mix by gently pipetting.

[0042] (4) Take 100 μL of cell suspension as the blank control group, take 100 μL of cell suspension as the parallel control group, and take 200 μL as the experimental group to add FITC-CD3 and PE-CD56 antibodies (both purchased from BD Company), and incubate at 4°C for 35 minutes.

[0043] (5) Add an appropriate amount of PBS buffer to wash the cells, centrifuge at 4°C and 300 g for 5 min, carefully aspirate and discard the supernatant.

[0044] (6) Resuspend the cells in 500 μL of PBS buffer and test on an analyzer.

[0045] (B) Flow cytometry collection (1) Turn on the SA3800 full-spectrum flow cytometer in advance to preheat and perform a self-check on the device.

[0046] (2) Create a new experiment: Select the "Preparation" wizard tab to enter the experiment preparation interface, click the "Experiment Template" button, select "Blank Template", enter the name information in the Name text box, and click "Create Experiment" to create a new experiment template.

[0047] (3) Place the Sample-S sample tube, click "Preview", set the "Fluorescence PMT Voltage" value so that the maximum value of Intensity_H is around 105, and click "Stop" to unload the Sample-S sample tube.

[0048] (4) Place the unstained sample tube, click "Preview", set other parameters, and do not use FSC to set the threshold when performing absolute counting! (5) Collect cells from each group: Place the sample tube to be tested, click "Preview", and when the Flow condition status changes to Stable, click "Acquire" to collect. After the collection begins, the same parameters must be used for the same group of experiments. If there are any changes, all the parameters must be collected again.

[0049] (C) Flow cytometry results analysis (1) Add the markers used and their corresponding fluorescent signals.

[0050] (2) Import the corresponding fluorescence curve into the database. For new fluorescence signals, a positive control tube should be set up separately to establish the curve.

[0051] (3) Select the Unstained group, circle the main cell population, and set it as gate A. Set FSC-H / FSC-A to display the cells in gate A and circle the single cell population D.

[0052] (4) Set CD3 / CD56 to indicate cells in gate D, and set gate E: E-Q1 to CD3 - CD56 + NK cells.

[0053] (5) Save the data, clean and close the instrument.

[0054] (II) CFSE proliferation response (NK cell killing experiment) To determine the cytotoxicity of NK cells (effector cells, E), several human cancer cell lines (target cells, T) were used for killing assays, including lung cancer cell line (A549), breast cancer cell line (MCF-7), and leukemia cell line (K562). The specific procedures are as follows: (1) On the 12th day of culture, tumor cell lines K562, A549, and MCF-7 cells were revived and divided into three groups according to the type of tumor cell line; (2) On the 17th day of culture, prepare the CFSE working solution according to the kit instructions; (3) Collect tumor cell lines separately, centrifuge (300 g, 10 min, RT, 9% acceleration, 9% deceleration), and discard the supernatant; (4) Wash twice by centrifugation in PBS buffer (300 g, 10 min, RT, 9% ascent and 9% as deceleration), and discard the supernatant; (5) Add 2 mL of PBS buffer to resuspend the tumor cell lines and take samples for counting; (6) Take tumor cell line cells (2×10 7 ), add PBS buffer to the volume of 2 mL, add 10 μL CFSE working solution (0.5 mmol / L), incubate at 37°C for 15 min, and add serum to terminate the reaction; (7) Wash twice by centrifugation in PBS buffer (300 g, 10 min, RT, 9% speed, 9% speed), discard the supernatant, and wash off the CFSE molecules attached to the surface; (8) Resuspend NK cells and tumor cell line cells in NK culture medium, take samples and count them respectively, and adjust the cell density of tumor cell line and NK cells to 1×10 7 / mL; 96-well concave bottom culture plate, add 100 μl NK cells (1×10 6 ) and 10 μl of CFSE-labeled tumor cell lines (1 × 10 5 ) Add NK medium to each well to 200 μl. Incubate in a 37°C, 5% CO2 incubator for 24 hours; Flow cytometry was used to analyze the cell status of tumor cell lines, and each group of experiments had three parallel controls; The NK culture medium is composed of KBM581 culture medium and IL-2, and the concentration of IL-2 is 1000 IU / mL.

[0055] (III) Cytokine production experiment: The secretion of cytokine IFN-γ in the culture supernatant of NK cells on the 17th day of culture was measured using an ELISA kit, and KBM581 culture medium was used as a blank control.

[0056] (IV) Experimental results (1) Flow cytometry phenotyping results: Figure 1 As shown, the test results are: CD3 - CD56 + Cell ratio: 97.51%, CD3 + CD56 + Cell ratio: 0.29%, CD3 + CD56 - Cell ratio: 0.10%, CD3 - CD56 - Cell ratio: 2.10%.

[0057] The flow cytometry results showed that the NK cells revived from the frozen umbilical cord blood hematopoietic stem cells had a relatively good flow cytometry phenotype, CD3 - CD56 + The cell ratio was 97.51%, which was no different from the phenotype of fresh NK cells derived from umbilical cord blood.

[0058] (2) Cell killing experiment results: Figure 2 As shown, the results show that the NK cells cultured in the present invention have a higher killing ratio in the three cell lines and have a broader spectrum of killing ability.

[0059] (3) Cytokine production results: Figure 3 As shown, it can be seen that the cryopreserved umbilical cord blood-derived NK cells cultured in the present invention can secrete a large amount of cytokine IFN-γ, which is no different from the NK cells derived from fresh umbilical cord blood.

[0060] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells, It is characterized in that The following steps are involved: (I) Umbilical cord blood resuscitation: Take frozen umbilical cord blood, thaw it in a water bath, add 37°C resuscitation solution, centrifuge, discard the supernatant, and obtain a blood sample; (ii) Separation of mononuclear cells from umbilical cord blood: separation of mononuclear cells from umbilical cord blood using lymphocyte separation solution, addition of ammonium chloride solution to lyse red blood cells, and washing with a cleaning solution to obtain mononuclear cells; (III) Preparation of NK cell induction: Activation medium was added to the culture flask coated with Lymactin-NK, mononuclear cells were seeded into the culture flask, IL-21, IL-18, anti-apoptotic agent, BDNF, NGF, TPO and SDF-1 were added, and the concentrations of IL-21, IL-18, anti-apoptotic agent, BDNF, NGF, TPO and SDF-1 were 20 ng / mL, 10 ng / mL, 20 mM, 50 μg / mL, 50 μg / mL, 50 μg / mL and 50 μg / mL respectively; the cells were placed in a 5% CO 2 , 37°C; on the 3rd and 5th days of culture, add activation medium; starting from the 7th day of culture, add expansion medium every 2 days; on the 17th to 20th day of culture, collect cells to obtain NK cells; The activation medium is composed of KBM581 medium and IL-15, and the concentration of IL-15 is 20 ng / mL; The expansion medium is composed of KBM581 medium and IL-2, and the concentration of IL-2 is 1000 IU / mL.

2. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In step (i), the resuscitation solution is prepared from the following solutions: 140 mL of 0.9% sodium chloride solution, 50 mL of compound electrolyte solution, and 10 mL of 20% human albumin solution.

3. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, Features: In the step (i), when adding the 37° C. resuscitation solution, the solution is added at a constant speed while being shaken for a duration of not less than 3 minutes; and / or: the amount of the resuscitation solution added is 8 times that of the umbilical cord blood.

4. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In step (ii), the cleaning solution is prepared by adding 2 mL of 1 mg / mL DNAse I solution and 1.5 mL of 25 mM MgCl to 200 mL of 0.9% sodium chloride solution. 2 Solution, mix well, you have it.

5. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that The specific operation of step (ii) is as follows: (1) Sample laying: blood sample is slowly and evenly added to the upper layer of human lymphocyte separation medium at a ratio of 2:1 and centrifuged; (2) Extraction of mononuclear cells: After centrifugation, distinct stratification will appear in the centrifuge tube. Aspirate the mononuclear cell layer. (3) First wash: add an equal volume of washing solution to the mononuclear cells, centrifuge, discard the supernatant, and obtain the cell pellet; (4) Lysis of red blood cells: dilute the cell pellet with 3 times (volume multiple) of 0.8% ammonium chloride solution and lyse at 4°C for 10 min to obtain the lysate. (5) Second wash: Add cleaning solution to the lysis buffer to terminate lysis and wash, centrifuge, discard the supernatant, and obtain the cell pellet; (6) Third wash: Add cleaning solution to wash the cells, centrifuge, and discard the supernatant to obtain mononuclear cells.

6. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In the step (iii), the specific method of coating is: coating the non-TC-treated T75 culture flask with 4 mL of PBS buffer and 1 mL of Lymactin-NK solution for more than 1 h, removing the coating solution, and washing away the residual coating solution with PBS buffer.

7. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In the step (iii), the amount of the activation medium added on the third day of culture is 90% to 110% of the initial activation medium.

8. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In the step (iii), the amount of the activation medium added on the fifth day of culture is 350% to 450% of the initial activation medium.

9. The method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to claim 1, It is characterized in that In step (iii), starting from the 7th day of culture, the expansion medium was added every 2 days to adjust the cell density to 2×10 6 / mL.

10. NK cells prepared by the method for preparing NK cells derived from cryopreserved umbilical cord blood hematopoietic stem cells according to any one of claims 1 to 9.

Images