High-purity nkt cell preparation, preparation method and cryopreservation control method

Abstract

The application discloses a high-purity NKT cell preparation and a preparation method and a cryopreservation regulation method, and belongs to the fields of cell biology technology and immunotherapy. The method systematically solves multiple challenges of low purity, insufficient amplification efficiency, poor cell viability, high cost and difficulty in large-scale production of NKT cells in the prior art through a unique secondary cryopreservation process, innovative application of platelet lysate and specific induction of alpha-galactosylceramide (alpha-GalCer). The prepared NKT cell preparation has the advantages of high purity, large amplification fold, strong cell function activity and good safety, and provides a high-quality and standardized cell source for clinical applications such as tumor immunotherapy, and has great industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the fields of cell immunotherapy and biotechnology, specifically relating to a high-purity NKT cell preparation and its preparation method and cryopreservation regulation method using a two-stage culture method combining trophoblast cell stimulation and α-galactosylceramide (α-GalCer). Background Technology

[0002] Natural killer T cells (NKT cells) are a unique subset of T lymphocytes, possessing characteristic surface markers and functional properties of both T cells and NK cells. NKT cells recognize lipid antigens presented by CD1d molecules through their T cell receptors (TCRs), rapidly secrete large amounts of cytokines (such as IFN-γ, IL-4, and IL-10), and exhibit cytotoxic activity that directly kills tumor cells. Based on the composition of the TCR chain and immunological function, NKT cells can be divided into type I NKT cells (also known as iNKT cells, expressing a constant TCRVα14-Jα18 chain) and type II NKT cells (expressing diverse TCRs). Among them, iNKT cells, due to their powerful immunomodulatory function and anti-tumor activity, have become an important research subject in the field of cell immunotherapy.

[0003] However, NKT cells constitute an extremely low proportion of peripheral blood mononuclear cells (PBMCs), accounting for only about 0.01%-1% of total T cells, which severely limits their clinical application. Therefore, developing efficient in vitro expansion technology for NKT cells is a key bottleneck in realizing NKT cell clinical therapy.

[0004] Currently, the main methods for in vitro expansion of NKT cells include the following categories: (1) Cytokine stimulation method. This method directly stimulates the expansion of NKT cells in PBMCs by adding cytokines such as IL-2, IL-15, and IL-7. However, since the proportion of NKT cells in PBMCs is extremely low and the selective stimulation effect of cytokines on NKT cells is limited, this method often results in low expansion efficiency and poor purity. The expansion products are mixed with a large number of conventional T cells and NK cells, making it difficult to obtain high-purity NKT cell preparations.

[0005] (2) Direct stimulation with α-GalCer. α-GalCer is a potent NKT cell-specific agonist that can selectively activate iNKT cells by binding to CD1d molecules. Studies have shown that α-GalCer can effectively induce the activation and proliferation of NKT cells. However, the expansion efficiency of α-GalCer stimulation alone is still limited. Early studies showed that the purity of NKT cells after expansion was only 0.59%-45.45%, with an average of about 11%, which is difficult to meet the dual requirements of cell quantity and purity for clinical applications.

[0006] (3) Feeder cell co-culture method. This method utilizes genetically modified feeder cells (such as the K562 cell line) to provide co-stimulatory signals and cytokines, significantly enhancing the expansion efficiency of target cells. Genetically modified K562 cells can achieve thousands to tens of thousands of times expansion of NK cells in vitro by overexpressing membrane-bound cytokines (such as mbIL-15 and mbIL-21) and co-stimulatory molecules (such as 4-1BBL). However, traditional feeder cell methods are mainly for NK cell expansion, and their direct application to NKT cell expansion suffers from insufficient selectivity.

[0007] (4) Dendritic cell (DC) loaded with α-GalCer stimulation method. This method first prepares DC cells loaded with α-GalCer, and then co-cultures them with PBMCs to induce NKT cell expansion. For example (CN120796187A), this method can provide strong specific stimulation, but the operation process is complicated, requires the isolation and culture of autologous DC cells, the preparation cycle is long and the cost is high, and the expansion efficiency is greatly affected by the quality of donor DCs.

[0008] The main problems with existing technologies include: ① low amplification efficiency, making it difficult to obtain a large number of NKT cells required for clinical treatment; ② insufficient purity, with a low proportion of NKT cells in the amplification products, and mixed cells may affect the treatment effect or safety; ③ complex operation procedures, long preparation cycle, and inconvenience for clinical application; ④ poor batch-to-batch stability, making it difficult to control product quality.

[0009] Therefore, there is an urgent need in this field to develop a method for preparing NKT cells that is easy to operate, has high amplification efficiency, high purity, and good batch stability to meet the needs of clinical applications. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a high-purity NKT cell preparation and its preparation and cryopreservation regulation method. The invention employs a two-stage culture strategy of trophoblast cell stimulation combined with α-GalCer, combined with an optimized cryopreservation and thawing process, which can efficiently prepare a high-purity, high-activity, and high-yield NKT cell preparation suitable for clinical immunotherapy applications.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a high-purity NKT cell preparation, comprising the following steps: (1) The isolated peripheral blood mononuclear cells were used as seed cells for the first cryopreservation, and the cryopreservation time was 24-36 hours; (2) Recovery steps (1) The frozen seed cells are co-cultured with the IL-21 NK cell expansion system for primary stimulation expansion. After 3-5 days of culture, they are cryopreserved for a second time for 24-36 hours. (3) Recovery step (2) The frozen cells are added to the culture medium for secondary stimulation and expansion to obtain NKT cell preparation.

[0012] Furthermore, the ratio of the number of seed cells to the number of cells in the IL-21 NK cell expansion system is 1:(1-4).

[0013] Furthermore, the concentration of α-galactosylceramide in step (3) is 80-120 ng / mL.

[0014] Furthermore, the cryopreservation solution used for the first and second cryopreservations consisted of 1640 culture medium, platelet lysate, and dimethyl sulfoxide in a volume ratio of 7:2:1.

[0015] Furthermore, during the secondary stimulation amplification process in step (3), the cell concentration is periodically monitored and adjusted to (0.6-1) × 10⁻⁶ cells / year by supplementing with culture medium. 9 / L; when the culture volume exceeds 100mL, transfer to a cell culture bag for further culture; when the culture volume reaches 2000mL and the total cell count reaches 3×10⁶ cells / L; 9 The cells are harvested at the above time.

[0016] Furthermore, the primary stimulation amplification in step (2) and the secondary stimulation amplification in step (3) are both carried out in a complete culture medium containing IL-2.

[0017] Furthermore, the primary stimulation amplification medium used in step (2) is a serum-free culture medium for immune cells containing IL-2, and the secondary stimulation amplification medium used in step (3) is a serum-free culture medium for immune cells containing IL-2 and 80-120 ng / mL α-galactosylceramide.

[0018] The high-purity NKT cell preparation obtained by the above preparation method.

[0019] A cryopreservation regulation method for preparing high-purity NKT cell preparations involves first cryopreserving seed cells for 24-36 hours, then co-culturing the seed cells with an IL-21 NK cell amplification system for primary stimulation and amplification, freezing the amplification products for 24-36 hours, and then thawing them before performing secondary stimulation and amplification with α-galactosylceramide.

[0020] The beneficial effects of this invention are: (1) High purity: A key feature of the method of this invention is the use of a programmed double cryopreservation strategy. Specifically, the seed cells are cryopreserved for the first time on the day of isolation and thawed after 24 hours. After thawing, the cells are stimulated and cultured for 3 days, then cryopreserved for the second time, maintained for another 24 hours, and then thawed again for subsequent expansion culture. This specific cryopreservation operation can effectively interrupt the continuous expansion rhythm of NK cells, weaken their competitive growth advantage when co-cultured with NKT cells, and thus significantly promote the selective expansion of NKT cells. Through the two-stage strategy of pre-expansion of feeder cells combined with selective stimulation by α-GalCer, NKT cells can be specifically expanded, and the purity of the final product NKT cells (CD3+CD56+ or CD3+Vα24+) can reach 60%, which is significantly higher than that of traditional methods.

[0021] (2) High yield: Genetically modified trophoblast cells provide sufficient co-stimulatory signals and cytokines, combined with α-GalCer specific stimulation, which can achieve NKT cell expansion of hundreds to thousands of times, and the total cell expansion can reach more than 100 times.

[0022] (3) Good batch stability: Standardized operating procedures and controllable process parameters ensure the consistency of product quality in different batches.

[0023] (4) Simple operation: Compared with complex methods such as DC loading α-GalCer, the operation process of this invention is simpler, the cycle is controllable, and it is easy to promote in clinical practice. Attached Figure Description

[0024] Figure 1 This is a flow cytometry diagram of PBMC cells isolated in Example 1 of the present invention.

[0025] Figure 2 This is a flow cytometry diagram of the cell preparation from Example 1 of the present invention.

[0026] Figure 3 This is a flow cytometry comparison of NKT cell purity between seed cells and formulation cells in Example 1 of this invention.

[0027] Figure 4 This is the amplification curve of the cell preparation in Example 1 of the present invention.

[0028] Figure 5This is a flow cytometry diagram of the control group cells in Example 2 of the present invention.

[0029] Figure 6 This is a flow cytometry diagram of the experimental group cells in Example 2 of the present invention.

[0030] Figure 7 This is the cell expansion curve of the control group in Example 2 of the present invention.

[0031] Figure 8 This is a cell expansion curve diagram of the experimental group in Example 2 of the present invention.

[0032] Figure 9 This is a flow cytometry plot of IFN-γ levels in the control group cells in Example 2 of this invention.

[0033] Figure 10 This is a flow cytometry diagram of IFN-γ levels in the experimental group cells in Example 2 of this invention.

[0034] Figure 11 This is a comparison diagram of IFN-γ levels in the experimental group and the control group in Example 2 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] The method for preparing the high-purity NKT cell preparation of the present invention includes the following steps: (1) The isolated peripheral blood mononuclear cells were used as seed cells for the first cryopreservation, and the cryopreservation time was 24-36 hours; (2) Recovery steps (1) The frozen seed cells are co-cultured with the IL-21 NK cell expansion system for primary stimulation expansion. After 3-5 days of culture, they are cryopreserved for a second time for 24-36 hours. (3) Recovery step (2) The frozen cells are added to the culture medium for secondary stimulation and expansion to obtain NKT cell preparation.

[0037] Furthermore, the ratio of seed cells to IL-21 NK cell expansion system is 1:(1-4). Furthermore, the concentration of α-galactosylceramide in step (3) is 80-120 ng / mL.

[0038] Furthermore, the cryopreservation solution used for the first and second cryopreservations consisted of 1640 culture medium, platelet lysate, and dimethyl sulfoxide in a volume ratio of 7:2:1.

[0039] Furthermore, during the secondary stimulation amplification process in step (3), the cell concentration is periodically monitored and adjusted to (0.6-1) × 10⁻⁶ cells / year by supplementing with culture medium. 9 / L; when the culture volume exceeds 100mL, transfer to a cell culture bag for further culture; when the culture volume reaches 2000mL and the total cell count reaches 3×10⁶ cells / L; 9 The cells are harvested at the above time.

[0040] Furthermore, the primary stimulation amplification in step (2) and the secondary stimulation amplification in step (3) are both carried out in a complete culture medium containing IL-2.

[0041] Furthermore, the primary stimulation amplification medium used in step (2) is a serum-free culture medium for immune cells containing IL-2, and the secondary stimulation amplification medium used in step (3) is a serum-free culture medium for immune cells containing IL-2 and 80-120 ng / mL α-galactosylceramide.

[0042] The high-purity NKT cell preparation obtained by the above preparation method.

[0043] Seed cells were cryopreserved once for 24-36 hours, and then co-cultured with an IL-21 NK cell expansion system for primary stimulation and expansion. The expansion products were then cryopreserved for 24-36 hours, thawed, and then subjected to secondary stimulation and expansion with α-galactosylceramide.

[0044] Example 1: Preparation process of high-purity NKT cell preparation (1) Preparation of complete culture medium: The serum-free culture medium for immune cells (Youkang / NC0101) contains 10% fetal bovine serum (Gibco / A5256701) and 500 IU / mL interleukin-2 (Nearshore / GMPCD66).

[0045] (2) Seed cell extraction: Peripheral blood samples were transferred to a 250mL centrifuge tube (tube 1) via a transfusion set. The net blood volume was measured to be 32mL. 28mL of sodium chloride injection solution was added to tube 1 and mixed thoroughly. The diluted sample was then slowly added at a rate of 30mL / tube to two 50mL centrifuge tubes containing lymphocyte separation solution (Dakewei / 7912011) (each centrifuge tube contained 15mL of lymphocyte separation solution). The centrifuge was balanced and centrifuged using the following program: 500g, 19℃, 23min, start slow, stop slow. After centrifugation, the white membrane cells were carefully aspirated and transferred to a new 250mL centrifuge tube. Sodium chloride injection solution was added to a total volume of 200mL. The centrifuge was balanced and centrifuged using the following program: 500g, 19℃, 10min, start max, stop 3. During centrifugation, prepare cryopreservation buffer (add 1 mL of 1640 medium and 1 mL of platelet lysate (Viva cell / C8010-0500) to a 50 mL centrifuge tube, and slowly add 0.5 mL of DMSO (Wak / Wak-DMSO-70) to the same tube, then mix well). After centrifugation, discard the supernatant and resuspend the cells in 4 mL of complete medium, resulting in a 4.5 mL suspension. Count the cells in 300 μL of the suspension and send it for flow cytometry analysis. Flow cytometry results: NKT cells (CD3+CD56+) accounted for 2.85% (see...). Figure 1 ), Count result: 7.9 × 10 9 / L, total cell count: 3.56×10 7 Centrifuge the cell suspension at 300g for 5 minutes, resuspend the cells in 0.5mL of 1640 medium (Gibco / C22400500BT), slowly add 0.5mL of cryopreservation solution to the resuspended cells, mix well, fill into a 2mL cryovial, and freeze at -80℃ for 24 hours.

[0046] (3) Primary stimulation to expand cells: ① Add 60 mL of complete culture medium to one T182 culture flask and warm it for later use; ② Resuscitate seed cells in a metal bath and add them to the above culture flask using a Pasteur pipette, then mix well; ③ Resuscitate IL-21NK cell expansion system (Hangzhou Zhongying) in a metal bath, count the cells, and take 3 × 10⁻⁶ cells. 7 Add the cells to a culture flask, mix well, and incubate at 37°C with 5% CO2. ④ On day 3 of culture, mix thoroughly by pipetting and take 200 μL for cell counting; the result is 1.0 × 10⁻⁶ cells / mL. 7 / L, collect all cell suspension into a 250mL centrifuge tube, balance the centrifugation (300g, 8min, 19℃, start max, stop 3). During centrifugation, prepare cell cryopreservation solution (preparation method as above). After centrifugation, discard the supernatant, resuspend in 1mL of 1640 medium, add 1mL of cryopreservation solution, mix well, and fill into two 2mL cryopreservation tubes at 1mL / tube. Store at -80℃ for 24 hours.

[0047] (4) Secondary cell expansion: ① Add 60 mL of complete culture medium to one T182 culture flask and warm to room temperature; ② Prepare α-GalCer (MCE / KRN7000) solution: Dissolve 1 mg of dry powder in a centrifuge tube containing 10 mL of DMSO, with a solution concentration of 1 × 10⁻⁶. 5 ng / mL, hereinafter referred to as G solution ③ Metal bath revival step 3 The two frozen cells were added to the above culture flask by Pasteur pipette and mixed well; ④ Add 60uLG solution to the culture flask, mix well and then put it in a 37℃, 5% CO2 incubator for culture.

[0048] (5) After culturing for 3 days, the cells were mixed by pipetting and sampled for counting. The result was: 0.5 × 10⁻⁶. 9 / L, without any other treatment, mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0049] (6) After culturing for 5 days, the cells were mixed by pipetting and sampled for counting. The result was 1.5 × 10⁵ cells / day. 9 / L, add 30mL of complete culture medium based on the cell count results to adjust the cell concentration to 1×10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0050] (7) After culturing for 7 days, the cells were mixed by pipetting and sampled for counting. The result was 1.6 × 10⁻⁶ cells / day. 9 / L, add 54mL of complete culture medium based on the cell count results to adjust the cell concentration to 1×10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0051] (8) After culturing for 9 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁹ cells / day. 9 Transfer the cells to a cell culture bag at a concentration of 1 × 10⁹ / L, add 115 mL of complete culture medium based on the cell count, and adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0052] (9) After culturing for 11 days, the cells were mixed by pipetting and sampled for counting. The result was 2.1 × 10⁻⁶ cells / day. 9 Based on the cell count results, add 285 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0053] (10) After culturing for 13 days, the cells were mixed by pipetting and sampled for counting. The result was 2.3 × 10⁻⁶ cells / day. 9 Based on the cell count results, add 707 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0054] (11) After culturing for 15 days, the cells were mixed by pipetting and then sampled for counting. The result was 2.5 × 10⁻⁶ cells. 9 / L, total cell count: 3.129×10 9 Take 2 mL of cell suspension (5 × 10⁻⁶ cells / mL). 6 Surface markers were detected in NKT cells (CD3+CD56+), and the proportion of NKT cells was 76.9% (see [link to data]). Figure 2 The remaining cell suspension was collected into four 500mL centrifuge cups. After centrifugation at 500g for 10 min, the supernatant was discarded. The cells were resuspended in two 500mL washing buffers (each buffer consisted of 500mL sodium chloride injection solution plus 1mL human serum albumin (Baxter, concentration 0.2%)). After resuspending the cells, the mixture was balanced, and the cells were washed three times by centrifugation at 500g for 10 min. The cells were then resuspended in 200mL of preservation solution (194mL sodium chloride injection solution plus 6mL human serum albumin, concentration 3%) to prepare the NKT cell preparation. Amplification curves are shown in [Figure number missing]. Figure 4 The cells entered a sustained rapid growth phase after day 7, and the culture period was 15 days, during which the total cell number increased from the initial 3 × 10⁻⁶. 7 Amplified to 3.129×10 9 The amplification factor was 104.3 times. Compared with seed cells, the purity of the formulation cells was increased by 74.05 percentage points (see...). Figure 3 NKT cells expanded by approximately 2410 times.

[0055] Example 2: Comparative Study of this Method and Traditional Methods (1) Preparation of complete culture medium: The serum-free culture medium for immune cells (Youkang / NC0101) contains 10% fetal bovine serum (Gibco / A5256701) and 500 IU / mL interleukin-2 (Nearshore / GMPCD66).

[0056] (2) Seed cell extraction: Peripheral blood samples were transferred to a 250mL centrifuge tube (tube 1) via a transfusion set. The net blood volume was measured to be 66mL. 54mL of sodium chloride injection solution was added to tube 1 and mixed thoroughly. The diluted sample was then slowly added at a rate of 30mL / tube to four 50mL centrifuge tubes containing lymphocyte separation solution (Dakewei / 7912011) (each centrifuge tube contained 15mL of lymphocyte separation solution). The centrifuge was balanced and centrifuged using the following program: 500g, 19℃, 23min, start slow, stop slow. After centrifugation, the white membrane cells were carefully aspirated and transferred to a new 250mL centrifuge tube. Sodium chloride injection solution was added to a total volume of 200mL. The centrifuge was balanced and centrifuged using the following program: 500g, 19℃, 10min, start max, stop 3. During centrifugation, a cryopreservation buffer was prepared (1 mL of 1640 medium and 1 mL of platelet lysate (Vivacell / C8010-0500) were added to a 50 mL centrifuge tube, and 0.5 mL of DMSO (Wak / Wak-DMSO-70) was slowly added to the same tube and mixed well). After centrifugation, the supernatant was discarded, and the cells were resuspended in 4 mL of complete medium, resulting in a suspension volume of 7.4 mL. 300 μL of the cell suspension was aspirated for cell counting, and the result was 8.5 × 10⁻⁶ cells / mL. 9 / L, total cell count: 6.29×10 7 Take 3.6 mL (3 × 10⁻⁶) 7 (Number of cells) were seeded into a T182 culture flask, and 60 μL of LG medium (prepared as in Example 1) and 56.4 mL of complete culture medium were added. After mixing, the flask was incubated at 37°C in a 5% CO2 incubator (control group). The remaining cell suspension was centrifuged at 300g for 5 minutes, and 0.5 mL of 1640 medium (Gibco / C22400500BT) was used to resuspend the cells. Another 0.5 mL of cryopreservation solution was slowly added to the resuspended cells, and after mixing, the mixture was filled into a 2 mL cryovial and stored at -80°C for 24 hours (experimental group).

[0057] (3) Subsequent culture of the control group: (3.1) After culturing for 3 days, no obvious cell clusters were observed under a microscope. No other treatment was performed. The cells were then placed in a 37°C, 5% CO2 incubator for further culturing.

[0058] (3.2) After culturing for 5 days, the cells were mixed by pipetting and sampled for counting. Result: 1.0 × 10⁵ 9 / L, without any other treatment, mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0059] (3.3) After culturing for 7 days, the cells were mixed by pipetting and sampled for counting. The result was 1.4 × 10⁻⁶. 9 / L, add 24mL of complete culture medium based on the cell count results to adjust the cell concentration to 1×10⁹ / L. 9Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0060] (3.5) After culturing for 9 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁹ / L. 9 / L, add 67mL of complete culture medium based on the cell count results to adjust the cell concentration to 1×10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0061] (3.6) After culturing for 11 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁻⁶. 9 / L, to cells Transfer the cells to a cell culture bag, add 121 mL of complete culture medium based on the cell count results, and adjust the cell concentration to 1 × 10⁻⁶. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0062] (3.7) After culturing for 13 days (D1), the cells were mixed by pipetting and sampled for counting. The result was 1.9 × 10⁻⁶. 9 Based on the cell count results, add 245 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0063] (3.8) After culturing for 15 days, the cells were mixed by pipetting and sampled for counting. The result was 2.1 × 10⁻⁶. 9 Based on the cell count results, add 569 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0064] (3.9) After culturing for 17 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁻⁶. 9 Add 176uL (914mL) of complete culture medium to a total volume of 2000mL, mix well, and incubate at 37℃ in a 5% CO2 incubator.

[0065] (3.10) After culturing for 20 days, the cells were mixed by pipetting and sampled for counting. The result was 1.7 × 10⁻⁶. 9 / L, total cell count: 3.4×10 9 Take 2.9 mL of cell suspension (5 × 10⁻⁶ cells / mL). 6 Surface markers were detected in NKT cells (CD3+CD56+), and the results showed that the proportion of NKT cells was 35.97% (see [link to data]). Figure 5The remaining cell suspension was collected into four 500ml centrifuge beakers. After centrifugation at 500g for 10 minutes, the supernatant was discarded. The cells were resuspended in two 500ml washing buffers (each buffer consisted of 500ml sodium chloride injection solution plus 1ml human serum albumin, with a human serum albumin concentration of 0.2%). After resuspending the cells, the mixture was balanced, and the cells were washed three times by centrifugation at 500g for 10 minutes. The cells were then resuspended in 200ml of preservation solution (preservation solution consisted of 194ml sodium chloride injection solution plus 6ml human serum albumin, with a human serum albumin concentration of 3%) to prepare the NKT cell preparation. The amplification curve is shown in [Figure number missing]. Figure 7 The cells entered a sustained rapid growth phase after day 9, and the culture period was 20 days, during which the cell number increased from the initial 3 × 10⁶ cells / day. 7 Amplified to 3.4 × 10 9 The amplification factor was 113 times.

[0066] (4) Subsequent cultivation of the experimental group: (4.1) Primary stimulation for cell expansion: ① Add 60 mL of complete culture medium to one T182 culture flask and warm to room temperature; ② Resuscitate seed cells in a metal bath and add them to the culture flask using a Pasteur pipette, then mix well; ③ After counting the IL-21 NK cell expansion system, take 3 × 10⁻⁶ cells. 7 Add the cells to a culture flask, mix well, and incubate at 37°C in a 5% CO2 incubator. On day 3 of culture, mix thoroughly by pipetting and take 200 μL for cell counting; the result is 1.1 × 10⁻⁶ cells / mL. 7 / L, collect all cell suspension into a 250mL centrifuge tube, balance the centrifugation (300g, 8min, 19℃, start max, stop 3). During centrifugation, prepare cell cryopreservation solution (preparation method as above). After centrifugation, discard the supernatant, resuspend in 1mL of 1640 medium, add 1mL of cryopreservation solution, mix well, and fill into two 2mL cryopreservation tubes at 1mL / tube. Store at -80℃ for 24 hours.

[0067] (4.2) Secondary cell expansion: ① Add 60 mL of complete culture medium to one T182 culture flask and warm it for later use; ② Prepare G solution, the method is the same as in Example 1; ③ Resuscitate the two frozen cells in step 3 in a metal bath, add them to the above culture flask with a Pasteur pipette and mix well; ④ Add 60 μL of LG solution to the culture flask, mix well and then place it in a 37°C, 5% CO2 incubator for culture.

[0068] (4.3) After culturing for 3 days, the cells were mixed by pipetting and sampled for counting. The result was 1.2 × 10⁻⁶. 9 / L, add 12mL of complete culture medium according to the counting results, mix well and place in a 37℃, 5% CO2 incubator for continued incubation.

[0069] (4.4) After culturing for 5 days, the cells were mixed by pipetting and sampled for counting. The result was 1.6 × 10⁻⁶. 9 / L, add 43mL of complete culture medium based on the cell count results to adjust the cell concentration to 1×10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0070] (4.5) After culturing for 7 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁻⁶. 9 Transfer the cells to a cell culture bag at a concentration of 1 × 10⁹ / L, and add 92 mL of complete culture medium based on the cell count results to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0071] (4.6) After culturing for 9 days, the cells were mixed by pipetting and sampled for counting. The result was 2.1 × 10⁹ / L. 9 Based on the cell count results, add 228 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0072] (4.7) After culturing for 11 days, the cells were mixed by pipetting and sampled for counting. The result was 2.5 × 10⁻⁶. 9 Based on the cell count results, add 655 mL of complete culture medium to adjust the cell concentration to 1 × 10⁹ / L. 9 Mix well and place in a 37℃, 5% CO2 incubator for further incubation.

[0073] (4.8) After culturing for 13 days, the cells were mixed by pipetting and sampled for counting. The result was 2.3 × 10⁻⁶. 9 / L, add 910mL of complete culture medium to a total volume of 2000mL according to the counting results, mix well and place in a 37℃, 5% CO2 incubator for continued incubation.

[0074] (4.9) After culturing for 15 days, the cells were mixed by pipetting and sampled for counting. The result was 1.8 × 10⁻⁶. 9 / L, total cell count: 3.6×10 9 Take 2.8 mL of cell suspension (5 × 10⁻⁶ cells / mL). 6 Surface markers were detected in NKT cells (CD3+CD56+), and the proportion of NKT cells was 63.79% (see [link to data]). Figure 6The remaining cell suspension was collected into four 500ml centrifuge beakers. After centrifugation at 500g for 10 minutes, the supernatant was discarded. The cells were resuspended in two 500ml washing buffers (each buffer consisted of 500ml sodium chloride injection solution plus 1ml human serum albumin, with a human serum albumin concentration of 0.2%). After resuspending the cells, the mixture was balanced, and the cells were washed three times by centrifugation at 500g for 10 minutes. The cells were then resuspended in 200ml of preservation solution (preservation solution consisted of 194ml sodium chloride injection solution plus 6ml human serum albumin, with a human serum albumin concentration of 3%) to prepare the NKT cell preparation. The amplification curve is shown in [Figure number missing]. Figure 8 The cells entered a sustained rapid growth phase after day 7, and the culture period was 15 days, during which the cell number increased from the initial 3 × 10⁶ cells / day. 7 Amplified to 3.6 × 10 9 The amplification factor was 120 times.

[0075] Example 3: Flow cytometry detection of IFN-γ levels in NKT cells (1) Take 1×10 cells from each of the experimental group and the control group in Example 2. 6 Add cells to the corresponding centrifuge tube, add leukocyte activation mixture and Golgi inhibitor (BD / 550583) at a concentration of 10ug / mL, mix thoroughly with cells and incubate at 37℃ in a 5% CO2 incubator for 4 hours.

[0076] (2) After incubation, remove the cells, centrifuge and discard the supernatant (1200 rpm, 3 min). Resuspend in 1% PBS buffer, add fluorescently labeled anti-human CD3-FITC (BD / 555916) and anti-human CD56-PE antibody (BD / 556647), and incubate at 4°C in the dark for 30 minutes.

[0077] (3) After incubation, centrifuge and discard the supernatant, add fixation buffer, and incubate at room temperature for 30 minutes. After incubation, centrifuge to remove the fixative, add permeabilization buffer, and incubate at room temperature for 15 minutes.

[0078] (4) Resuspend the infiltrated cells in staining buffer and add fluorescently labeled anti-human IFN-γ-PE-Cy7 antibody (Thermo / 25-7319-41). Mix thoroughly and incubate at 4°C in the dark for 30-60 minutes. After incubation, centrifuge and wash the cells to remove unbound antibodies.

[0079] (5) Resuspend the stained cells in flow cytometry detection buffer and place them in the flow cytometry sample tube. Use the flow cytometer with the appropriate fluorescence channel set up to collect data. Flow cytometry results are shown below. Figure 9-10In the control group, the proportion of CD3+ cells was 52.44%, of which 4.35% were CD56+ cells highly expressing IFN-γ; in the experimental group, the proportion of CD3+ cells was 86%, of which 38.38% were CD56+ cells highly expressing IFN-γ. The IFN-γ level in the experimental group was significantly higher than that in the control group (see...). Figure 11 ).

[0080] In summary, the content of this invention is not limited to the above-mentioned embodiments. Those skilled in the art can easily propose other embodiments within the technical guiding principles of this invention, but such embodiments are all included within the scope of this invention.

Claims

1. A method for preparing a high-purity NKT cell preparation, characterized in that, Includes the following steps: (1) The isolated peripheral blood mononuclear cells were used as seed cells for the first cryopreservation, and the cryopreservation time was 24-36 hours; (2) Recovery steps (1) The frozen seed cells are co-cultured with the IL-21 NK cell expansion system for primary stimulation expansion. After 3-5 days of culture, they are cryopreserved for a second time for 24-36 hours. (3) Recovery step (2) The frozen cells are added to the culture medium for secondary stimulation and expansion to obtain NKT cell preparation.

2. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the number of seed cells to the number of cells in the IL-21 NK cell expansion system is 1:(1-4).

3. The preparation method according to claim 1, characterized in that, In step (3), the concentration of α-galactosylceramide is 80-120 ng / mL.

4. The preparation method according to claim 1, characterized in that, The cryopreservation solution used for the first and second cryopreservations consisted of 1640 culture medium, platelet lysate, and dimethyl sulfoxide in a volume ratio of 7:2:

1.

5. The preparation method according to claim 1, characterized in that, During the secondary stimulation amplification process in step (3), the cell concentration is monitored periodically and adjusted to (0.6-1) × 10⁻⁶ cells / day by supplementing with culture medium. 9 / L; when the culture volume exceeds 100mL, transfer to a cell culture bag for further culture; when the culture volume reaches 2000mL and the total cell count reaches 3×10⁶ cells / L; 9 The cells are harvested at the above time.

6. The preparation method according to claim 1, characterized in that, The primary stimulation amplification in step (2) and the secondary stimulation amplification in step (3) are both carried out in a complete culture medium containing IL-2.

7. The preparation method according to claim 1, characterized in that, The primary stimulation amplification medium used in step (2) is a serum-free culture medium for immune cells containing IL-2, and the secondary stimulation amplification medium used in step (3) is a serum-free culture medium for immune cells containing IL-2 and 80-120 ng / mL α-galactosylceramide.

8. The high-purity NKT cell preparation obtained by the preparation method according to any one of claims 1-7.

9. A method for cryopreservation regulation for preparing high-purity NKT cell preparations, characterized in that, Seed cells were cryopreserved once for 24-36 hours, and then co-cultured with an IL-21 NK cell expansion system for primary stimulation and expansion. The expansion products were then cryopreserved for 24-36 hours, thawed, and then subjected to secondary stimulation and expansion with α-galactosylceramide.

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