Instant-use universal NK cell freezing preparation as well as preparation method and use method thereof

Abstract

The invention discloses an instant-use universal NK cell freezing preparation as well as a preparation method and a use method thereof. Umbilical cord blood is separated to obtain mononuclear cells, and a seed bank of NK cells is obtained through in-vitro induction culture and cryopreserved. The cells in the revived seed bank are subjected to secondary large-scale multiplication culture, an NK cell preparation with the purity larger than 90% is obtained, residues in a culture system are removed after multiple times of cleaning, then the cells are resuspended with human serum albumin, a cryopreservation protection solution is added, subpackaging and cryopreservation are conducted, an NK cell cryopreservation preparation bank is obtained, the total amount of cryopreserved cells can reach 1011 orders of magnitude, and the cryopreserved cells can reach 1011 orders of magnitude. And complete cell activity, purity, microorganisms, endotoxin and the like are detected on the stock cells. When required, the NK cell frozen preparation is recovered, the ready-to-use NK cell preparation with high purity and excellent quality can be obtained, the quality of the cell preparation is uniform, and all detection results are perfect. According to the freezing preparation method of the NK cell preparation, required NK cells can be more conveniently and quickly provided for clinical research and disease patients.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering technology, and in particular to a universal NK cell freezing preparation for immediate use, and a preparation method and a use method thereof. Background Art

[0002] NK cells (natural kill cells) are effector cells of the innate immune system. They can recognize tumors and virus-infected cells without antigen presensitization, and play a key role in the body's immune surveillance and immune defense. NK cells participate in anti-tumor immune responses through multiple mechanisms: NK cell surface receptor CD16 kills tumor cells through ADCC effect; NK cells can release perforin and granzyme to activate the apoptosis system to attack tumor cells; NK cells can express death ligand (FASL) to bind to target cell death receptor (FAS) to cause target cell apoptosis; activated NK cells can secrete a variety of cytokines, such as TNF, granulocyte-macrophage colony stimulating factor, IL-15, IL-10, IL-13 and other cytokines to inhibit tumor cell proliferation and kill tumor cells.

[0003] NK cell applications mainly include autologous NK cells and allogeneic NK cells. A large number of clinical experiments have shown that autologous NK cell therapy has a certain therapeutic effect in tumor treatment and has no obvious toxic side effects. However, with the increase of age, the number of NK cells in the human body will gradually decrease, and the activity will also decrease. Especially for the elderly or patients with low immunity, it may not be able to meet the treatment needs. In addition, the preparation process of autologous NK requires separation and amplification steps, which takes a long time. For patients with emergency treatment, it may not be the best choice. The main form of allogeneic NK cell clinical application is fresh preparations. Although the cell activity of fresh preparations is higher, due to the characteristics of living cells, its validity period is short (generally 24 hours), it cannot be used immediately, and there are problems such as delayed product testing results. Therefore, it is urgent to study a frozen preparation library of allogeneic NK cells that can be prepared in batches, used immediately, and tested without delay. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a universal NK cell freezing preparation that can be used immediately and a preparation method and a use method thereof, which can be used immediately and has no hysteresis in detection.

[0005] The specific technical solution adopted by the present invention to solve the technical problem is: a method for preparing a universal NK cell frozen preparation for immediate use, comprising the following steps:

[0006] (1) cryopreserving NK seed bank cells prepared by in vitro trophoblast stimulation and expansion of cord blood mononuclear cells, resuscitating the seed bank, and performing large-scale secondary expansion with trophoblast cells, centrifuging at 400-600 g for 10-15 minutes, discarding the supernatant, and adding a washing solution containing 0.2%-0.4% human albumin by volume of sodium chloride injection for washing, and finally obtaining a cell viability of not less than 95%;

[0007] (2) Resuspend the cell pellet with human albumin at a concentration of 20% by mass to volume, adjust the cell concentration according to the counting results, mix the cell suspension and cryopreservation protection solution at a volume ratio of 2:1, and dispense into cryopreservation tubes, place in a medical low-temperature refrigerator, and randomly select a tube of cells for inspection. After the inspection meets the quality standards, transfer the frozen preparation to a gas phase liquid nitrogen tank for long-term storage after 24 hours to obtain a universal NK cell frozen preparation for immediate use;

[0008] Preparation of the cryopreservation protection solution: 20% human albumin and DMSO are prepared in a volume ratio of 7:3 by mass volume, and stored in a refrigerator.

[0009] Furthermore, in step (2), the cell concentration was adjusted to 7.5×10 7 / ml.

[0010] Further, the cells were divided into 5 ml cryopreservation tubes, with a cryopreservation specification of 4 ml per tube and a final cell concentration of 5×10 7 / ml.

[0011] Furthermore, in step (2), the tests of the cells submitted for inspection include: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, endotoxin, and surface markers.

[0012] The above-mentioned preparation method obtains a universal NK cell frozen preparation that can be used immediately.

[0013] The method of using the above-mentioned ready-to-use universal NK cell frozen preparation is to use a metal bath or a water bath to resuscitate the universal NK cell frozen preparation according to the preparation specifications, and directly use it in clinical research or animal experiments.

[0014] Advantages and technical effects of the present invention: The present invention separates mononuclear cells from umbilical cord blood, obtains a seed bank of NK cells through in vitro induction culture and freezes them. The revived seed bank cells are cultured on a large scale for a second time to obtain an NK cell preparation with a purity greater than 90%. After multiple washings, the residues in the culture system are removed, and then the cells are resuspended with human albumin and cryopreservation protection fluid is added. The cells are packaged and frozen to obtain a NK cell frozen preparation bank, and the total amount of frozen cells can reach 10 11The NK cell preparation can be revived to obtain a ready-to-use NK cell preparation with high purity and good quality. The invention does not require the patient to provide seed cells, which reduces the pain of the patient. It can be used immediately without delay in detection, and can provide the required NK cells for clinical research, animal experiments, and disease patients more conveniently and quickly. The NK cell preparation has high purity, and the cell quality between batches is stable and controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flow cytometry result diagram of the NK seed bank culture on the 7th day in Example 1 of the present invention;

[0016] Figure 2 This is a flow cytometry result diagram of the NK cell cryopreservation preparation library of Example 1 of the present invention;

[0017] Figure 3 This is a flow cytometry result diagram of the NK cell cryopreservation preparation library after resuscitation in Example 1 of the present invention;

[0018] Figure 4 This is a state diagram of the cell killing experiment of the NK cryopreservation preparation in Example 1 of the present invention (20×);

[0019] Figure 5 This is a graph showing the results of a cell killing experiment of a NK cryopreservation preparation according to Example 1 of the present invention;

[0020] Figure 6 This is a flow cytometry result diagram of the NK seed bank cultured on the 7th day in Example 2 of the present invention;

[0021] Figure 7 This is a flow cytometry result diagram of the NK cell cryopreservation preparation library of Example 2 of the present invention;

[0022] Figure 8 This is a flow cytometry result diagram of the NK cell frozen preparation library after resuscitation in Example 2 of the present invention;

[0023] Fig. 9 This is a state diagram of the cell killing experiment of the NK cryopreservation preparation of Example 2 of the present invention (20×);

[0024] Fig.10 This is a graph showing the results of a cell killing experiment of the NK cryopreservation preparation of Example 2 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] The method for preparing the instant universal NK cell frozen preparation of the present invention comprises the following steps:

[0027] (1) cryopreserving NK seed bank cells prepared by in vitro trophoblast stimulation and expansion of cord blood mononuclear cells, resuscitating the seed bank, and performing large-scale secondary expansion with trophoblast cells, centrifuging at 400-600 g for 10-15 minutes, discarding the supernatant, and adding a washing solution containing 0.2%-0.4% human albumin by volume of sodium chloride injection for washing, and finally obtaining a cell viability of not less than 95%;

[0028] (2) Resuspend the cell pellet with human albumin at a concentration of 20% by mass to volume, adjust the cell concentration according to the counting results, mix the cell suspension and cryopreservation protection solution at a volume ratio of 2:1, and dispense into cryopreservation tubes, place in a medical low-temperature refrigerator, and randomly select a tube of cells for inspection. After the inspection meets the quality standards, transfer the frozen preparation to a gas phase liquid nitrogen tank for long-term storage after 24 hours to obtain a universal NK cell frozen preparation for immediate use;

[0029] Preparation of the cryopreservation protection solution: 20% human albumin and DMSO are prepared in a volume ratio of 7:3 by mass volume, and stored in a refrigerator.

[0030] Furthermore, in step (2), the cell concentration was adjusted to 7.5×10 7 / ml.

[0031] Further, the cells were divided into 5 ml cryopreservation tubes, with a cryopreservation specification of 4 ml per tube and a final cell concentration of 5×10 7 / ml.

[0032] Furthermore, in step (2), the tests of the cells submitted for inspection include: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, endotoxin, and surface markers.

[0033] The above-mentioned preparation method obtains a universal NK cell frozen preparation that can be used immediately.

[0034] The method of using the above-mentioned ready-to-use universal NK cell frozen preparation is to use a metal bath or a water bath to resuscitate the universal NK cell frozen preparation according to the preparation specifications, and directly use it in clinical research or animal experiments.

[0035] Specifically:

[0036] (1) Prepare complete culture medium: containing 10% fetal bovine serum by volume and 500 IU interleukin-2 at a final concentration.

[0037] (2) Preparation of NK cell seed bank: 50-100 mL of umbilical cord blood sample was centrifuged by Ficoll density gradient to recover buffy coat cells, washed once with 50-100 mL of sodium chloride injection, and resuspended in complete culture medium to obtain umbilical cord blood mononuclear cells (UBMC). 5 UBMCs were inoculated into T182 cell culture flasks at a concentration of cells / mL; trophoblast cells (APCs) were revived in a metal bath, and APCs were inoculated into the above T182 culture flasks at a ratio of UBMC:APC=1:1-2, mixed and cultured in a 37°C, 5% CO2 incubator; the cell status was observed daily, and the cell suspension was counted every 2-3 days. According to the cell counting results, complete culture medium was added to adjust the cell concentration to (0.4-0.6)x10 9 cells / L; after 7-10 days of culture, the total number of cells reaches (2-4)×10 8 When 5×10 6 The expression of NK cell surface markers (CD3-CD16+ / CD56+) was detected by 1×10 cells. When the ratio reached more than 80%, the number of pure NK cells was calculated. 7 The NK cell seed bank was cryopreserved with 100 CD3-CD16+ / CD56+ cells / mL.

[0038] (3) Preparation of universal NK cell preparation: The NK seed bank cells prepared in the metal bath resuscitation step (2) were heated to (2.5-5)×10 5 The cells were inoculated into T182 culture flasks at a concentration of 1×10 cells / mL. APCs were revived in a metal bath and added to the T182 cell culture flasks containing NK seed cells at a ratio of 1:2-4. The flasks were cultured in a 37°C, 5.0% CO2 incubator. 200 μL of cell suspension was taken for counting every 2-3 days, and complete culture medium was added to adjust the cell concentration to 1×10 9 cells / L, and cultured until the 11th to 14th day, the cell concentration reached (3-7)×10 9 When the concentration of cells / L was 100, all cells were recovered and cryopreserved.

[0039] (4) Preparation of cryopreservation protection solution: 20% (mass-volume ratio) human albumin and DMSO are prepared in a volume ratio of 7:3 and stored in a refrigerator.

[0040] (5) Preparation of washing solution: Sodium chloride injection is added with human albumin at a ratio of 0.2%-0.4%.

[0041] (6) Preparation of universal NK cell frozen preparation library: The cells recovered in step (3) were centrifuged at 400-600 g for 10-15 minutes, the supernatant was discarded, the washing solution was added and washed three times, the cell pellet was resuspended with human serum albumin, and the cell concentration was adjusted to 7.5×10 based on the counting results. 7 / ml, the cell suspension and the cryopreservation protection solution in step (4) were mixed evenly at a volume ratio of 2:1, and filled into 5ml cryopreservation tubes. The cryopreservation specification was 4ml per tube, and the final cell concentration was 5×10 7 / ml. The frozen preparation is placed in a medical low-temperature refrigerator, and a tube of cells is randomly selected for inspection. The inspection indicators include: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, and surface markers. After the inspection meets the quality standards, the universal NK cell frozen preparation is obtained. After 24 hours, the frozen preparation is transferred to a gas phase liquid nitrogen tank for long-term storage.

[0042] (7) After the cells in the NK cell cryopreservation preparation bank meet the quality standards, the universal NK cell cryopreservation preparation is resuscitated in a metal bath according to the preparation specifications and directly used in clinical research or animal experiments.

[0043] Specifically, the relevant preparation and detection examples are as follows

[0044] Example 1

[0045] (1) Prepare complete culture medium: NK immune serum-free culture medium (Youkang / NC11240701) containing 10% fetal bovine serum and a final concentration of 500 IU interleukin-2;

[0046] (2) Isolation and extraction of umbilical cord blood mononuclear cells (UBMC): Place the umbilical cord blood sample into a 250mL centrifuge tube (tube 1) through a blood transfusion device, with a net blood volume of 60mL. Add 60mL of sodium chloride injection to tube 1 and dilute and mix thoroughly. Slowly add the diluted sample to 4 50mL centrifuge tubes containing 15mL of lymphocyte separation solution at a rate of 30mL / tube, balance the centrifugation, and centrifuge at the following speeds: 500g, 19℃, 23min, start slow. After centrifugation, aspirate the buffy coat cells into a new 250mL centrifuge tube, add sodium chloride injection to a total volume of 220mL, balance the centrifugation, and centrifuge at the following speeds: 500g, 19℃, 10min, start max, brake 3. After centrifugation, discard the supernatant, resuspend the cells in 5mL of complete culture medium, with a suspension volume of 5.4mL, and aspirate 200μL of cell suspension. Count: 15.4×10 9 / L, total number of cells 8.316×10 7 .

[0047] (3) Preparation of NK seed bank: Based on the counting results, 1.3 mL of cell suspension was inoculated into a new T182 culture flask, and one APC cell (2×107 / tube) were inoculated into the culture flask, and complete medium was added to a total volume of 50 mL. After mixing, the cells were placed in a 37°C, 5% CO2 incubator for culture. The cell status was observed daily. On the third day of culture, the cells were mixed by pipetting, and 200 μL of the cell suspension was taken for counting. The result was: 0.4×10 9 / L, without any other treatment, the cells were placed in a 37°C, 5% CO2 incubator for further culture. On the fifth day of culture, the cells were mixed by blowing, and 200 μL of the cell suspension was taken for counting. The result was: 1.4×10 9 / L, add 90mL of complete culture medium according to the counting results to adjust the cell concentration to 0.5×10 9 / L, mixed and placed in a 37°C, 5% CO2 incubator for continued culture. On the 7th day of culture, the cells were mixed by blowing and 200 μL of cell suspension was taken for counting. The result was: 2.4×10 9 / L, total number of cells 3.36×10 8 , take 1mL of cell suspension and send it to the test for cell surface markers (see the results in Figure 1 ), and the total number of pure NK cells was calculated based on the flow cytometry results to be 2.81×10 8 Transfer all the cell suspension into a 500mL centrifuge cup, balance and centrifuge, centrifuge program: 500g, 19℃, 10min, start max, brake 3. After centrifugation, discard the supernatant, resuspend the cells in 7mL 1640 culture medium, add 7mL cryopreservation protection solution (cryopreservation protection solution composition is 40% 1640 culture medium, 20% DMSO (ORIGEN / CP70), 40% fetal bovine serum), mix well and count according to the NK cell count of 2×10 7 / 1ml / tube is filled into 14 2ml cryotubes, placed in a programmable cooling box and transferred to a -80℃ refrigerator, and after 24h, transferred to -196℃ liquid nitrogen for storage.

[0048] (4) Preparation of universal NK cell preparation: prepare complete culture medium for use, resuscitate 1 tube of NK seed cells in a metal bath, inoculate in 10 182 culture flasks, resuscitate 20 APCs in a metal bath, inoculate in the above 10 T182 culture flasks at a rate of 2 per bottle, add complete culture medium to each bottle to 50 mL, mix well, and place in a 37°C, 5% CO2 incubator for culture. On the third day, take 1 bottle of cells, pipette and mix well, and take 200uL of cell suspension for counting. The result is: 0.9×10 9 / L, add 50mL complete medium to each bottle, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 5th day, take one bottle of cells, blow and mix well, and take 200uL of cell suspension for counting. The result is: 1.4×10 9 / L, add 100mL complete medium to each bottle, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 7th day, take one bottle of cells, blow and mix well, take 200uL of cell suspension and count the result: 2.3×10 9 / L, all cells were transferred into cell culture bags, and each bag was supplemented with 220mL complete culture medium and 2 APCs. After mixing, they were placed in a 37°C, 5% CO2 incubator for further culture. On the 9th day, 1 bag of cells was taken, mixed, and 200uL of cell suspension was taken for counting. The result was: 2.8×10 9 / L, add 400mL complete culture medium to each bag, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 11th day, take 1 bag of cells, mix well and take 200uL of cell suspension for counting. The result is: 3.2×10 9 / L, add 800ml complete culture medium to each bag, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 14th day, take 1 bag of cells, mix well and take 200uL of cell suspension for counting. The result is: 3.7×10 9 / L, harvest all cells.

[0049] (5) Preparation of cryopreservation protection solution: 20% (mass-volume ratio) human albumin and DMSO were prepared in a volume ratio of 7:3, with a total volume of 382 mL, and stored in a refrigerator.

[0050] (6) Preparation of washing solution: sodium chloride injection containing 0.2-0.4% human albumin by volume;

[0051] (7) Preparation of universal NK cell frozen preparation library: All cells were harvested into a 500 mL centrifuge cup and centrifuged at a balanced level. The centrifugation program was: 500 g, 19 °C, 10 min, start max, brake 3. After centrifugation, the supernatant was discarded, the cells were resuspended in washing solution, and the cells were centrifuged at a balanced level. The cells were washed 3 times in total, and the cell pellet was resuspended with human serum albumin. According to the counting results, a total of 5.73 × 10 10 cells, and the cell concentration was adjusted to 7.5 × 10 7 / ml, mix the cell suspension and cryopreservation protection solution at a volume ratio of 2:1, and fill into 5ml cryopreservation tubes. The cryopreservation specification is 4ml / tube, and the cell concentration is 5×10 7 / ml, a total of 286 tubes. The frozen preparation was placed in a medical low-temperature refrigerator, and one tube of cells was randomly selected for inspection. The test indicators included: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, surface markers (see the results for details). Figure 2 ), and after testing to meet the quality standards, the universal NK cell frozen preparation is obtained. After 24 hours, the frozen preparation is transferred to a gas phase liquid nitrogen tank for long-term storage.

[0052] (8) When using NK cryopreservation preparations, NK cell cryopreservation preparations should be revived in a metal bath according to the preparation specifications and can be directly used in clinical research or animal experiments.

[0053] Related tests

[0054] (I) Count, viability (4h, 24h) and surface marker detection of ready-to-use NK cell cryopreservation preparations after resuscitation

[0055] (1) Resuscitate 1 frozen NK cell preparation in a metal bath. Add the cell suspension to a 50 ml centrifuge tube containing 6 ml of washing solution (sodium chloride injection containing 0.2-0.4% human albumin by volume). Mix well and take 200 ul of the cell suspension for counting and calculating the viability. Take another 5×10 6 Cell surface markers were detected (see Figure 3 ), and the remaining samples were stored at 4°C, and the activity rates were calculated after 4 and 24 hours.

[0056] (2) The total number of NK cells after resuscitation was 1.83×10 8 The viability of the cells was 98% when detected by trypan blue exclusion method; the cell surface markers were detected by flow cytometry, and the results were: the proportion of CD3-CD16CD56+ was not less than 90%, and the proportion of activated NK cells (NKG2D+) was not less than 95%; the cell viability was 94% at 4 hours; and 89% at 24 hours.

[0057] (II) MTT assay for detecting the killing activity of NK cell frozen preparations

[0058] (1) Resuscitate 1 frozen NK cell preparation in a metal bath and adjust the cell density to 3×10 cells using a culture medium containing 10% fetal bovine serum. 5 / mL, inoculated in a 12-well plate, 1mL per well, aspirated the culture medium after 24 hours, added effector cells according to the effector-target ratio of 2:1, 5:1, and 10:1, and each experimental group had 3 replicates, and no cells were added to the zero well, only 1640 culture medium containing 10% FBS was added.

[0059] (2) After 24 hours, the OD value of each group was measured using a microplate reader according to the instructions of the MTT kit. The resuscitated NK cell frozen preparation still had killing ability (see the results). Figure 4 , Figure 5 ).

[0060] Example 2

[0061] (1) Prepare complete culture medium: NK immune serum-free culture medium (Youkang / NC11240701) containing 10% fetal bovine serum and a final concentration of 500 IU interleukin-2;

[0062] (2) Isolation and extraction of umbilical cord blood mononuclear cells (UBMC): Place the umbilical cord blood sample into a 250mL centrifuge tube (tube 1) through a blood transfusion device, with a net blood volume of 60mL. Add 60mL of sodium chloride injection to tube 1 and dilute and mix thoroughly. Slowly add the diluted sample to 4 50mL centrifuge tubes containing 15mL of lymphocyte separation solution at a rate of 30mL / tube, balance the centrifugation, and centrifuge at the following speeds: 500g, 19℃, 23min, start slow. After centrifugation, aspirate the buffy coat cells into a new 250mL centrifuge tube, add sodium chloride injection to a total volume of 220mL, balance the centrifugation, and centrifuge at the following speeds: 500g, 19℃, 10min, start max, brake 3. After centrifugation, aspirate the supernatant and resuspend the cells in 4mL of complete culture medium. The suspension volume is 4.5mL. Aspirate 200μL of cell suspension and count: 13.4×10 9 / L, total number of cells 6.7×10 7 .

[0063] (3) Preparation of NK seed bank: Based on the counting results, 1.5 mL of cell suspension was inoculated into a new T182 culture flask, and one APC cell (2×10 7 / tube) were inoculated into the culture flask, and complete medium was added to a total volume of 50 mL. After mixing, the cells were placed in a 37°C, 5% CO2 incubator for culture. The cell status was observed daily. On the third day of culture, the cells were mixed by pipetting, and 200 μL of the cell suspension was taken for counting. The result was: 0.5×10 9 / L, without any other treatment, placed in a 37℃, 5% CO2 incubator for further culture. On the fifth day of culture, the cells were mixed by blowing, and 200μL of the cell suspension was taken for counting. The result was: 1.6×10 9 / L, add 110mL of complete culture medium according to the counting results to adjust the cell concentration to 0.5×10 9 / L, mixed and placed in a 37°C, 5% CO2 incubator for continued culture. On the 7th day of culture, the cells were mixed by blowing and 200 μL of cell suspension was taken for counting. The result was: 2.2×10 9 / L, total number of cells 3.52×10 8 , take 1mL of cell suspension and send it to the test for cell surface markers (see the results in Figure 6 ), and the total number of pure NK cells was calculated based on the flow cytometry results to be 2.27×10 8 Transfer all the cell suspension into a 500mL centrifuge cup, balance and centrifuge, centrifuge program: 500g, 19℃, 10min, start max, brake 3. After centrifugation, discard the supernatant, resuspend the cells in 5.5mL 1640 culture medium, add 5.5mL cryopreservation protection solution (cryopreservation protection solution composition is 40% 1640 culture medium, 20% DMSO (ORIGEN / CP70), 40% fetal bovine serum), mix well and add 2×10 NK cells.7 / 1ml / tube is filled into 11 2ml cryotubes, placed in a programmable cooling box and transferred to a -80℃ refrigerator, and after 24h, transferred to -196℃ liquid nitrogen for storage.

[0064] (4) Preparation of universal NK cell preparation: prepare complete culture medium for use, resuscitate 1 tube of NK seed cells in a metal bath, inoculate in 10 182 culture flasks, resuscitate 20 APCs in a metal bath, inoculate in the above 10 T182 culture flasks at a rate of 2 per bottle, add complete culture medium to each bottle to 50 mL, mix well, and place in a 37°C, 5% CO2 incubator for culture. On the third day, take 1 bottle of cells, pipette and mix well, and take 200uL of cell suspension for counting. The result: 1.0×10 9 / L, add 50mL complete medium to each bottle, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 5th day, take one bottle of cells, blow and mix well, and take 200uL of cell suspension for counting. The result is: 1.7×10 9 / L, add 100mL complete medium to each bottle, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 7th day, take one bottle of cells, blow and mix well, take 200uL of cell suspension and count the result: 2.9×10 9 / L, all cells were transferred into cell culture bags, and each bag was supplemented with 220mL complete culture medium and 2 APCs. After mixing, they were placed in a 37°C, 5% CO2 incubator for further culture. On the 9th day, 1 bag of cells was taken, mixed, and 200uL of cell suspension was taken for counting. The result was: 2.9×10 9 / L, add 400mL complete culture medium to each bag, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 11th day, take 1 bag of cells, mix well and take 200uL of cell suspension for counting. The result is: 3.3×10 9 / L, add 800ml complete culture medium to each bag, mix well and place in a 37℃, 5% CO2 incubator for further culture. On the 14th day, take 1 bag of cells, mix well and take 200uL of cell suspension for counting. The result is: 3.5×10 9 / L, harvest all cells.

[0065] (5) Preparation of cryopreservation protection solution: 20% (mass-volume ratio) human albumin and DMSO were prepared in a volume ratio of 7:3, with a total volume of 315 mL, and stored in a refrigerator.

[0066] (6) Preparation of washing solution: sodium chloride injection containing 0.2-0.4% human albumin by volume;

[0067] (7) Preparation of universal NK cell frozen preparation library: All cells were harvested into a 500 mL centrifuge cup and centrifuged at a balanced speed. The centrifugation program was as follows: 500 g, 19 °C, 10 min, start at max, brake 3. After centrifugation, the supernatant was discarded, the cells were resuspended in washing solution, and the cells were centrifuged at a balanced speed. The cells were washed 3 times in total, and the cell pellet was resuspended with human albumin. According to the counting results, a total of 4.7348 × 10 10 cells, and the cell concentration was adjusted to 7.5 × 10 7 / ml, mix the cell suspension and cryopreservation protection solution at a volume ratio of 2:1, and fill into 5ml cryopreservation tubes. The cryopreservation specification is 4ml / tube, and the cell concentration is 5×10 7 / ml, a total of 236 tubes. The frozen preparation was placed in a medical low-temperature refrigerator, and one tube of cells was randomly selected for inspection. The test indicators included: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, surface markers (see the results for details). Figure 7 ), and after testing to meet the quality standards, the universal NK cell frozen preparation is obtained. After 24 hours, the frozen preparation is transferred to a gas phase liquid nitrogen tank for long-term storage.

[0068] (8) When using NK cryopreservation preparations, NK cell cryopreservation preparations should be revived in a metal bath according to the preparation specifications and directly used in clinical research or animal experiments.

[0069] Related tests

[0070] (I) Count, viability (0h, 4h, 24h) and surface marker detection of ready-to-use NK cell cryopreservation preparations after resuscitation

[0071] (1) Resuscitate 1 frozen NK cell preparation in a metal bath, add the cell suspension to a 50 ml centrifuge tube containing 6 ml of washing solution (sodium chloride injection containing 0.2-0.4% human albumin by volume), mix well, and take 200 ul of the cell suspension for counting and calculating the viability. 6 Cell surface markers were detected (see Figure 8 ), and the remaining samples were stored at 4°C, and the activity rates were calculated after 4 and 24 hours.

[0072] (2) The total number of NK cells after resuscitation was 1.9×10 8 The viability of the cells was 98% when detected by trypan blue exclusion method; the cell surface markers were detected by flow cytometry, and the results were: the proportion of CD3-CD16CD56+ was not less than 90%, and the proportion of activated NK cells (NKG2D+) was not less than 95%; the cell viability was 93% at 4 hours; and 89% at 24 hours.

[0073] (II) MTT assay for detecting the killing activity of NK cell frozen preparations

[0074] (1) Resuscitate 1 frozen NK cell preparation in a metal bath and adjust the cell density to 3×10 cells using a culture medium containing 10% fetal bovine serum. 5 / mL, inoculated in a 12-well plate, 1mL per well, aspirated the culture medium after 24 hours, added effector cells according to the effector-target ratio of 2:1, 5:1, and 10:1, and each experimental group had 3 replicates, and no cells were added to the zero well, only 1640 culture medium containing 10% FBS was added.

[0075] (2) After 24 hours, the OD value of each group was measured using a microplate reader according to the instructions of the MTT kit. The resuscitated NK cell frozen preparation still had killing ability (see the results). Fig. 9 , Fig.10 ).

[0076] The present invention can be used immediately without any delay in detection, and can provide the required NK cells for clinical research, animal experiments, disease patients, etc. more conveniently and quickly according to different preparation specifications; the NK cell preparation of the present invention has high purity, and the cell quality between batches is stable and controllable.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a universal NK cell frozen preparation for immediate use, characterized in that: The following steps are involved: (1) cryopreserving NK seed bank cells prepared by in vitro trophoblast stimulation and expansion of cord blood mononuclear cells, resuscitating the seed bank, and performing large-scale secondary expansion with trophoblast cells, centrifuging at 400-600 g for 10-15 minutes, discarding the supernatant, and adding a washing solution containing 0.2%-0.4% human albumin by volume of sodium chloride injection for washing, and finally obtaining a cell viability of not less than 95%; (2) Resuspend the cell pellet with 20% human albumin by mass volume, adjust the cell concentration according to the counting result, mix the cell suspension and cryopreservation protection solution at a volume ratio of 2:1, and dispense into cryopreservation tubes, place in a medical low-temperature refrigerator, and randomly select a tube of cells for inspection. After the inspection meets the quality standards, transfer the frozen preparation to a gas phase liquid nitrogen tank for long-term storage after 24 hours to obtain a universal NK cell frozen preparation for immediate use; The cryopreservation protection solution is prepared by mixing 20% ​​human albumin and DMSO in a volume ratio of 7:3 and storing in a refrigerator.

2. The method for preparing the universal NK cell frozen preparation for immediate use according to claim 1, characterized in that: In step (2), the cell concentration was adjusted to 7.5×10 7 / ml.

3. The method for preparing the universal NK cell frozen preparation for immediate use according to claim 2, characterized in that: In step (2), the cells were divided into 5 ml cryopreservation tubes, with a cryopreservation specification of 4 ml per tube and a final cell concentration of 5 × 10 7 / ml.

4. The method for preparing the universal NK cell frozen preparation for immediate use according to claim 1, characterized in that: In step (2), the tests of the cells include: total cell count, viability, aerobic / anaerobic bacteria, fungi, mycoplasma, endotoxin, and surface markers.

5. A universal NK cell frozen preparation ready for use obtained by the preparation method according to any one of claims 1 to 4.

6. The method for using the universal NK cell frozen preparation for immediate use according to claim 5, characterized in that: According to the preparation specifications, use a metal bath or water bath to thaw the universal NK cell frozen preparation and use it directly in clinical research or animal experiments.

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