A culture system and method and application thereof for preparing cord blood NK cell preparation by removing trophoblast cells and producing floccules

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a culture system and method and application thereof for removing floccules that appear when preparing umbilical cord blood NK cell preparations by the trophoblast cell method. The culture system A comprises a serum-free culture medium, a cell expansion reagent, IL-2 and a serum substitute; the serum substitute does not contain fibrinogen. The culture system and method provided by the present invention can eliminate floccules that appear when preparing umbilical cord blood NK cell preparations by the trophoblast cell method, improve the stability and safety of the cell preparation, and solve the problem of clinical use. And the NK cells prepared by the method of the present invention have higher amplification multiples and purity, and stronger biological activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a culture system and method and application thereof for preparing umbilical cord blood NK cell preparations by removing trophoblast cells and producing floccules. Background Art

[0002] In recent years, cell therapy technology has developed rapidly, especially NK cell immunotherapy has been widely used in clinical practice. NK cells are effector cells of the human natural immune system and the main functional cells that monitor malignant cells in the body. They can directly kill target cells without antigen pre-sensitization, so they play an important role in the body's immune response to tumors, early antiviral or intracellular bacterial infections. NK cells account for only 10-20% of peripheral blood mononuclear cells. When the disease occurs, some patients are not suitable for extracting their own peripheral blood for in vitro expansion of NK cells due to the patient's own condition. At this time, it is necessary to select a new source of NK cells. Compared with adult peripheral blood NK cells, NK cells from umbilical cord blood have lower immunogenicity, higher purity, stronger interferon expression ability, and stronger bone marrow homing function.

[0003] There are several major requirements for the in vitro preparation of NK cells, including the number of cell expansion, the purity of NK cells, and the cytotoxicity of NK cells. How to obtain enough high-quality effector NK cells is one of the most core difficulties. The in vitro culture of primary NK cells is generally divided into the cytokine method and the trophoblast method. The trophoblast method can achieve rapid expansion of NK cells, obtain high-purity and high-quality NK cells, and has good stability. However, after using this method to prepare cord blood NK cell preparations, the cell preparation bag is prone to visible flocs in a short period of time, affecting clinical use.

[0004] The reason why floccules appear in NK cell preparation bags within a short period of time is not clear, because this phenomenon may involve a variety of complex factors. For example, (1) Cell morphology and behavior: In some cases, NK cells may proliferate rapidly and aggregate together to form clumps during culture. These clumps may appear visually as floccules, especially when the cell density is high or the culture conditions change. In addition, if NK cells are affected by adverse factors during preparation or storage (such as temperature fluctuations, malnutrition, pollutants, etc.), they may undergo apoptosis or die. Dead cells and their fragments may form flocs, thereby affecting the purity and quality of the preparation. (2) Culture system factors: Certain components in the culture system (such as fibrinogen and other proteins and polysaccharides in autologous fire-killing plasma) may combine with cells or cell fragments under specific conditions (such as temperature, pH value, ionic strength, etc.) to form very strong cross-linked flocs. (3) Solution factors: If the stability of the solution is destroyed during the storage or transportation of NK cell preparations (such as temperature fluctuations, light, vibration, etc.), it may also cause the formation of flocs.

[0005] The appearance of flocs is related to the quality of the preparation, which may be caused by a variety of factors such as cell apoptosis, culture system components, and transportation conditions. Solving this problem can significantly improve the purity, stability, and safety of NK cell preparations, ensuring their effectiveness and reliability in clinical applications. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for removing floccules in preparing umbilical cord blood NK cell preparations by the trophoblast cell method, thereby solving the problem that the appearance of floccules in cell preparations affects clinical use.

[0007] In one aspect, the present invention provides a culture system for NK cells, wherein the culture system comprises a serum replacement.

[0008] Specifically, the serum substitute does not contain fibrinogen.

[0009] Preferably, the amount of serum substitute added is 1%-5% of the rehydration volume, such as 1%, 2%, 4%, 4%, 5%, and any value within the range.

[0010] Specifically, the culture system further includes one or more of serum-free culture medium, cell expansion reagents and cytokines.

[0011] More specifically, the serum-free culture medium may be KBM581, AIM-V, GT-T551 H3 or SCGM.

[0012] More specifically, the cytokines include, but are not limited to, one or more of IL-2, IL-7, IL-12 or IL-15.

[0013] Preferably, the cytokine may be IL-2.

[0014] More preferably, the amount of IL-2 added may be 100-300 IU / mL, such as 100 IU / mL, 200 IU / mL, 300 IU / mL, and any value within the range.

[0015] In another aspect, the present invention provides a method for culturing NK cells, the method comprising the following steps:

[0016] (1) Cell inoculation: Add mononuclear cells into culture system A for culture;

[0017] (2) Medium change: replace the culture system A in step (1) with the culture system B and continue culturing;

[0018] (3) First rehydration: add culture system B to step (2) and continue culturing;

[0019] (4) Second rehydration: add culture system A to step (3) and continue culturing;

[0020] (5) Third rehydration: adding culture system C to step (4) and continuing the culture;

[0021] (6) Harvesting cells;

[0022] The culture system A comprises serum-free culture medium, cell expansion reagent, IL-2 and serum replacement;

[0023] The culture system B comprises serum-free culture medium, IL-2 and serum replacement;

[0024] The culture system C includes serum-free culture medium and IL-2;

[0025] The serum substitute does not contain fibrinogen.

[0026] Specifically, the culture conditions in steps (1) to (5) may be: 2%-5% CO2, 35-37°C.

[0027] Preferably, the culture conditions in steps (1) to (5) may be: 4%-5% CO2, 36-37°C.

[0028] Further preferably, the culture conditions in steps (1)-(5) may be: 5% CO2, 37°C.

[0029] Specifically, the amount of serum replacement added in the culture system A can be 1%-5% v / v, and the amount of IL-2 added can be 100-300 IU / mL.

[0030] Further specifically, in some embodiments, the serum replacement added in the culture system A may be 5% v / v, and the IL-2 added is 200 IU / mL;

[0031] In other embodiments, the serum replacement added in the culture system A may be 1% v / v, and the IL-2 added may be 200 IU / mL;

[0032] Alternatively, in some other embodiments, the above two adding methods are added successively.

[0033] Alternatively, in some other embodiments, plasma may be added first, and then any of the above two methods may be added.

[0034] Specifically, the amount of serum replacement added in the culture system B is 3%-5% v / v, and the amount of IL-2 added is 100-300 IU / mL.

[0035] Further specifically, in some embodiments, the serum replacement added in the culture system A may be 3% v / v, and the IL-2 added may be 200 IU / mL;

[0036] In some other embodiments, in some embodiments, the serum replacement added in the culture system A may be 5% v / v, and the IL-2 added may be 200 IU / mL;

[0037] Alternatively, in some other embodiments, the above two adding methods may be added successively.

[0038] Alternatively, in some other embodiments, plasma may be added first, and then any of the above two methods may be added.

[0039] Specifically, the amount of IL-2 added in the culture system C is 100-300 IU / mL.

[0040] Preferably, the amount of IL-2 added in the culture system C is 200 IU / mL.

[0041] More specifically, the serum substitute is Helios UItraGROTM-Advanced serum substitute, purchased from Helios Bioscience, with the product number HPCFDCGL05.

[0042] In another aspect, the present invention provides the use of the aforementioned culture system or method in the preparation of cord blood NK cell preparations by removing trophoblast cells and producing floccules.

[0043] The technical effects achieved by the present invention are:

[0044] The present invention provides a method for removing flocs in preparing umbilical cord blood NK cell preparations by the trophoblast cell K562 method, thereby solving the problem that the final cell preparation has flocs that affect clinical use; the present invention provides a novel use of a serum substitute, i.e., it is used for preparing umbilical cord blood NK cell preparations by the trophoblast cell K562 method; the serum substitute does not contain fibrinogen, fundamentally solving the problem that the umbilical cord blood NK cell preparations cultured by the trophoblast cell K562 method have flocs visible to the naked eye that affect clinical use; the trophoblast cell K562 method uses a serum substitute to replace umbilical cord blood autologous plasma for culturing NK cells, and the NK cells grow rapidly and stably, and have high purity and biological activity; the serum substitute is of clinical grade and can be used for clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The following are microscope pictures, wherein A is Example 1; B is Comparative Example 1; C is Comparative Example 2; D is Comparative Example 3; and E is Comparative Example 4.

[0046] Figure 2 is the total number of NK cells prepared in Example 1 and Comparative Examples 1-2.

[0047] Figure 3 It is the expansion multiple of NK cells prepared in Example 1 and Comparative Examples 1-2.

[0048] Figure 4 The apoptosis rate of NK cells prepared in Example 1 and Comparative Examples 1-2.

[0049] Figure 5 The content of cell surface markers CD3-CD56+ of NK cells prepared in Example 1 and Comparative Examples 1-2.

[0050] Figure 6 The biological activities of NK cells prepared in Example 1 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0052] The information of the main reagents and materials in the examples of the present invention are as follows:

[0053] NK cell expansion reagent: purchased from Hangzhou Zhongying Biomedical Technology Co., Ltd., item number: ZY-NKZ-0104, used to stimulate NK cell activation and expansion; there are two specifications, A1 is 1mL and A2 is 4mL.

[0054] NK cell serum-free culture medium KBM581: purchased from Corning, catalog number: 88-581-CMF, used for NK cell expansion culture.

[0055] IL-2: Recombinant human interleukin 2, purchased from Sihuan Pharmaceutical Company, is added throughout the culture process to stimulate cell proliferation.

[0056] Helios UItraGROTM-Advanced serum replacement (GMP grade): purchased from HELIOS Bioscience, item number: HPCFDCGL05, added at a concentration of 1-5%, used for NK cell expansion to provide nutrients required for cell growth. Hereinafter referred to as serum replacement.

[0057] Annexin V-APC / PI double staining cell apoptosis detection kit: purchased from Jiangsu KeyGen Biotechnology Co., Ltd., catalog number: KGA1107.

[0058] Annexin V-FITC / PI double staining cell apoptosis detection kit: purchased from Jiangsu KeyGen Biotechnology Co., Ltd., catalog number: KGA1102.

[0059] Calcium gluconate: purchased from Shandong Qidu Pharmaceutical Co., Ltd.

[0060] Autologous inactivated plasma (frozen): The plasma was obtained after centrifugation of umbilical cord blood. After inactivation at 56°C for half an hour, it was centrifuged at 2300g for 15 minutes to harvest the autologous inactivated plasma for comparative experiments.

[0061] Autologous inactivated plasma (8% calcium gluconate): After the umbilical cord blood is centrifuged, plasma is obtained, and 8% calcium gluconate is added. The mixture is mixed by inversion, inactivated at 56°C for half an hour, and then centrifuged at 2300g for 15 minutes to obtain autologous inactivated plasma for comparative experiments.

[0062] Example 1

[0063] In this example, NK cells derived from cord blood were cultured using serum substitutes.

[0064] The overall steps of the specific cell culture method are as follows:

[0065] (1) Day 0 NK cell expansion reagent A1 resuscitation: transfer 10 mL of rewarmed KBM581 medium to a 15 mL centrifuge tube in advance, quickly remove NK cell expansion reagent A1 from liquid nitrogen, and place in a 37°C water bath until completely dissolved (no more than 3 min). After dissolution, quickly transfer the cells to the above 15 mL centrifuge tube, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells with 5 mL of rewarmed KBM581 medium, recorded as A1 cell suspension.

[0066] (2) Day 0 Cell inoculation: Isolate and count mononuclear cells, and take 3×10 7 The cells were resuspended in KBM581 medium that had been rewarmed, and 3×10 7 Add all the cells and the A1 cell suspension in step (1) into a T75 suspension culture flask; add 5% serum replacement to the total volume; then add KBM581 culture medium with a final IL-2 concentration of 200 IU / mL to a total volume of 30 mL and mix well, then culture in a 5% CO2, 37°C incubator;

[0067] (3) Day 3 Centrifugation and medium replacement: On the third day after inoculation, place the cells in the T75 suspension culture flask in a 50 mL centrifuge tube and centrifuge at 300 g for 5 min. Discard the supernatant and resuspend the cells in the rewarmed KBM581 medium. Return the cell suspension to the original T75 suspension culture flask and add 5% serum replacement and KBM581 medium with a final IL-2 concentration of 200 IU / mL to a total volume of 30 mL. Mix well and place in a 5% CO2, 37°C incubator for culture.

[0068] (4) Day 4-6: Add KBM581 culture medium with a final IL-2 concentration of 200 IU / mL every day to maintain the cell concentration at 0.8-1.2×10 6 cells / mL, add 5% serum replacement of the replenishment volume on the 4th and 5th days, and add 3% serum replacement of the replenishment volume on the 6th day. The volume exceeds 100mL and transfer to T182 suspension culture flask;

[0069] (5) Day 7 NK cell expansion reagent A2 recovery: On day 7, transfer 40 mL of rewarmed KBM581 medium to a 50 mL centrifuge tube in advance, quickly remove NK cell expansion reagent A2 from liquid nitrogen, and place in a 37°C water bath until completely dissolved (no more than 3 min). After dissolution, quickly transfer the cells to the above 50 mL centrifuge tube, centrifuge at 300 g for 5 min, discard the supernatant, and resuspend the cells with 10 mL of rewarmed KBM581 medium, recorded as A2 cell suspension. Add the A2 cell suspension to the culture flask, and add KBM581 medium with a final IL-2 concentration of 200 IU / mL to maintain the cell concentration at 0.8-1.2×10 6 cells / mL, and add 1% serum replacement of the replenishment volume, the volume exceeds 400mL, and transfer to the cell culture bag;

[0070] (6) Day 8-10 Cell expansion: Add KBM581 medium with a final IL-2 concentration of 200 IU / mL every day to maintain the cell concentration at 0.8-1.2×10 6 cells / mL;

[0071] (7) Harvesting cells on Day 11: Cells can be harvested on the 11th to 13th day of culture. This time, cells were harvested on Day 11. All cells were collected by centrifugation at 300 g for 8 min, the culture medium was removed, and the cells were washed twice with saline and resuspended with saline to maintain a cell density of 2-3 × 10 7 / mL, take 5mL of cells and place them in a T25 culture flask for observation and photography under a microscope; put the rest of the cell suspension in a 150mL cell preparation bag, store it at 4°C, and observe it at regular intervals.

[0072] (8) Sampling and testing: Starting from the 5th day, 1 mL of cell suspension was collected every day for counting and calculation of the total cell number and expansion multiple. On the 7th, 9th and 11th days, 5 mL of sample was collected for NK cell apoptosis rate, surface markers CD3-CD56+, and biological activity testing.

[0073] The detection method is as follows:

[0074] (a) Expansion fold / total number of cells

[0075] When rehydrating each day, take 1 mL and count using a blood analyzer to calculate the total cell number for that day.

[0076] The formula for calculating the total cell number is: total cell number = cell density × total volume.

[0077] Amplification factor formula: Amplification factor Total number of cells on the day / initial inoculation cell number.

[0078] (b) Cell apoptosis rate

[0079] Cell apoptosis was detected using the FITC / PI double staining apoptosis kit. 2E6 NK cells were taken and evenly placed in two 1.5mL EP tubes, one for the control tube and the other for the experimental tube. The tubes were centrifuged at 300g for 5min, the culture medium was removed, and 1mL PBS was added for washing once. The tubes were centrifuged at 300g for 5min, the supernatant was removed, 500μL apoptosis kit buffer, 5μL FITC and 5μL PI were added to the experimental group, and 500μL apoptosis kit buffer was added to the control group. The cells were incubated at room temperature and away from light for 10min, and detected using a flow cytometer, and the FITC and PI double negative ratios were recorded.

[0080] Apoptosis rate = -b.

[0081] Wherein, a: FITC and PI double negative ratio in the control group; b: FITC and PI double negative ratio in the experimental group.

[0082] (c) Cell surface markers CD3-CD56+

[0083] Flow cytometry was used to detect the CD3-CD56+ ratio of cultured cells. 2E6 NK cells were evenly placed in two 1.5mL EP tubes, one for the control tube and the other for the experimental tube. The cells were centrifuged at 300g for 5min, the culture medium was removed, and 1mL PBS was added to wash once. The cells were centrifuged at 300g for 5min, and 100μL of normal saline was added to resuspend the cells. 5μL of CD3 flow cytometry antibody and 5μL of CD56 flow cytometry antibody were added to the experimental group, and the CD3-negative and CD56-positive ratios were recorded.

[0084] (d) Biological activity detection

[0085] NK: K562 (GFP) = 5:1 incubated for 1 hour, and the APC / PI apoptosis kit was used to detect the apoptosis rate of K562 to detect the killing activity of NK cells. The cells were spread into a 6-well plate, including the K562 (GFP) positive control well, the K562 (GFP) negative control well, the NK cell control well and the NK: K562 (GFP) = 5:1 well, incubated at 37°C, CO25% for 1 hour, collected the cells, centrifuged at 300g for 5 minutes, removed the culture medium, and washed once with 1mL PBS, centrifuged at 300g for 5 minutes, removed the supernatant, added 500μL apoptosis kit buffer and 5μL APC and 5μL PI to the K562 positive control tube and the NK: K562 (GFP) = 5:1 tube, and added 500μL apoptosis kit buffer to the K562 negative control group and the NK control group, incubated at room temperature and away from light for 10 minutes, detected by flow cytometry, and recorded the double negative ratio of APC and PI.

[0086] Killing activity = %.

[0087] Wherein, a: APC and PI double negative ratio in K562 positive control tube; b: APC and PI double negative ratio in NK:K562 (GFP) = 5:1 tube.

[0088] Comparative Example

[0089] A comparative example was set up with reference to Example 1. The differences between the comparative example and Example 1 are shown in Table 1, and the rest are the same as Example 1.

[0090] Table 1

[0091]

[0092] Results and Analysis

[0093] 1. Microscopic examination results

[0094] The results are as follows Figure 1 As shown, the cell preparation prepared in Example 1 had no flocs, and no flocs were visible to the naked eye after being placed at 4°C for 24 hours, while Comparative Examples 1-4 had more flocs under the microscope, and small particles visible to the naked eye appeared after being placed at 4°C for 1 hour, and flocs visible to the naked eye appeared after 2 hours. This shows that only when Helios UItraGROTM-Advanced fibrinogen-free serum substitute is used, the final preparation meets the clinical grade, i.e., has no flocs.

[0095] 2. Amplification times / total number of cells

[0096] The results are as follows Figure 2-Figure 3 As shown, the cell preparation prepared by the serum substitute of Example 1 not only has no flocs, but also has a larger number of cells finally harvested and a higher expansion multiple.

[0097] 3. Cell apoptosis rate

[0098] The results are as follows Figure 4 As shown, the overall apoptosis of the cell preparation prepared with the serum substitute of Example 1 was lower than that of plasma, and the cell viability was higher.

[0099] 4. Cell surface markers CD3-CD56+

[0100] The results are as follows Figure 5 As shown, the cell preparation prepared with the serum substitute of Example 1 had a higher purity, reaching more than 80% on the 7th day.

[0101] 5. Biological activity detection

[0102] The results are as follows Figure 6As shown, the cell preparation prepared with the serum substitute of Example 1 had a higher killing capacity, reaching 80% on the 7th day.

[0103] In summary, Example 1 of the present invention can fundamentally solve the problem that floccules appear in the preparation of cord blood NK cell preparations by the trophoblast cell method, which affects clinical use, and the cord blood NK cells cultured using the method of the present invention have good effects in terms of total cell volume, expansion multiple, apoptosis rate, NK cell purity and biological activity. Therefore, the present invention provides a new and effective method for the preparation of cord blood NK cell preparations in clinical practice.

Claims

1. A method for culturing NK cells, characterized in that: The method comprises the following steps: (1) Cell inoculation: Add mononuclear cells into culture system A for culture; (2) Medium change: replace the culture system A in step (1) with the culture system B and continue culturing; (3) Rehydration on days 4-6: Add culture system B to step (2) and continue culturing; (4) Rehydration on the 7th day: add culture system A to step (3) and continue culturing; (5) Rehydration on days 8-10: Add culture system C to step (4) and continue culturing; (6) Harvesting cells; The culture system A comprises serum-free culture medium, cell expansion reagent, IL-2 and serum substitute; the serum substitute in the culture system A is added in an amount of 1%-5% v / v, and the IL-2 is added in an amount of 100-300 IU / mL; The culture system B comprises serum-free culture medium, IL-2 and serum substitute; the serum substitute added in the culture system B is 3%-5% v / v, and the IL-2 added is 100-300 IU / mL; The culture system C includes serum-free culture medium and IL-2; the amount of IL-2 added in the culture system C is 200 IU / mL; The serum substitute does not contain fibrinogen, and the serum substitute is Helios UItraGROTM-Advanced serum substitute; The rehydration on days 4-6 is as follows: KBM581 medium with a final IL-2 concentration of 200 IU / mL is added every day to maintain the cell concentration at 0.8-1.2×10 6 cells / mL, add 5% v / v serum replacement of the replenishing volume on days 4 and 5, and add 3% v / v serum replacement of the replenishing volume on day 6. The volume exceeds 100 mL and is transferred to a T182 suspension culture flask; The 7th day rehydration is as follows: 40 mL of rewarmed KBM581 medium is transferred to a 50 mL centrifuge tube in advance, NK cell expansion reagent A2 is quickly taken out from liquid nitrogen, and placed in a 37°C water bath until completely dissolved. After dissolution, the cells are quickly transferred to the above 50 mL centrifuge tube, centrifuged at 300 g for 5 min, the supernatant is discarded, and the cells are resuspended with 10 mL of rewarmed KBM581 medium, which is recorded as A2 cell suspension; the A2 cell suspension is added to the culture bottle, and KBM581 medium with a final IL-2 concentration of 200 IU / mL is added to maintain the cell concentration at 0.8-1.2× 10 6 cells / mL, and add 1% serum replacement of the replenishment volume, the volume exceeds 400mL, and transfer to the cell culture bag; The rehydration on days 8-10 is as follows: KBM581 medium with a final IL-2 concentration of 200 IU / mL is added every day to maintain the cell concentration at 0.8-1.2×10 6 cells / mL.

2. The method according to claim 1, characterized in that The culture conditions of steps (1) to (5) are: 2%-5% CO2, 35-37°C.

3. Use of the method according to any one of claims 1-2 in the preparation of cord blood NK cell preparation by removing trophoblast cells in the presence of floccules.

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