Peripheral blood-derived CIK cell preparation method and application of peripheral blood-derived CIK cell in tumor inhibition

Abstract

The invention relates to the technical field of cell biology, and particularly discloses a peripheral blood source CIK cell preparation method and application thereof in tumor inhibition, the peripheral blood source CIK cell preparation method comprises the following steps: separating PBMC from healthy donor peripheral blood, performing IFN-gamma pre-stimulation amplification, transferring into a three-dimensional dynamic culture system, adding IL-2, IL-1alpha and CD3 antibodies, curcumin and resveratrol, and culturing in a three-dimensional dynamic culture system to obtain the peripheral blood source CIK cell. And knocking out a non-tumor chemokine receptor through CRISPR / Cas9 and over-expressing a tumor specific receptor, and finally harvesting the peripheral blood-derived CIK cells. Experiments show that the peripheral blood-derived CIK cells prepared by the method have higher amplification efficiency and tumor cell specific killing ability, and show a remarkable inhibition effect in tumor treatment, in addition, the technical scheme is simple and convenient to operate, controllable in cost and suitable for large-scale production, a novel efficient cell preparation is provided for tumor immunotherapy, and the technical scheme has wide application prospects. Wide clinical application prospects are realized.

Description

Technical Field

[0001] The present application relates to the field of cell biology technology, and more specifically to a method for preparing peripheral blood-derived CIK cells and their use in tumor suppression. Background Art

[0002] Cancer is a major public health threat worldwide, with high morbidity and mortality rates. Among men, the most common cancers are prostate cancer (29%), lung cancer, colorectal cancer, and bladder cancer, respectively. Among women, breast cancer (31%), lung cancer, colorectal cancer, and cervical cancer are the most common. While recent advances in medical technology have enriched cancer treatment options and significantly improved patient survival rates, finding more effective treatments remains a key research priority.

[0003] Among the many tumor immunotherapy methods, cytokine-induced killer cell (CIK) therapy has shown significant advantages. CIK cells are a heterogeneous cell population obtained by stimulating peripheral blood mononuclear cells with multiple cytokines in vitro. Their core effector cells are NK-like T lymphocytes that express both CD3 and CD56. This type of cell has both the powerful specific anti-tumor activity of T lymphocytes and the non-MHC-restricted killing characteristics of NK cells, and has the following outstanding characteristics: (1) strong proliferation ability; (2) broad-spectrum anti-tumor effect; (3) sensitivity to drug-resistant tumor cells; (4) low toxicity to normal hematopoietic cells. Because the content of such effector cells in natural peripheral blood is extremely low, CIK cells expanded in vitro have become one of the preferred options for current tumor immunotherapy.

[0004] However, existing CIK cell preparation technologies still have significant limitations: first, the in vitro expansion efficiency of cells is insufficient, making it difficult to obtain a sufficient number of effector cells; second, the tumor-killing activity of cultured CIK cells needs to be improved; and more importantly, existing treatment options lack specificity and long-term efficacy in tumor suppression. These technical bottlenecks have severely restricted the clinical application of CIK cell therapy. Based on the above statements, this application provides a method for preparing peripheral blood-derived CIK cells and their use in tumor suppression. Summary of the Invention

[0005] To address the shortcomings of existing CIK cell preparation methods, such as low cell expansion efficiency, low tumor cell killing activity, limited effect in tumor treatment, and high cost, this application provides a method for preparing peripheral blood-derived CIK cells and their use in tumor suppression.

[0006] In the first aspect, the present application provides a method for preparing peripheral blood-derived CIK cells, using the following technical solutions:

[0007] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0008] S1. Peripheral blood was collected from healthy donors and PBMCs were isolated by density gradient centrifugation.

[0009] S2, PBMC were cultured at 1-2×10 6 cells / mL in culture medium, and IFN-γ was added for pre-stimulation and cultured until the cell density reached 4-6×10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0010] S3. Initial peripheral blood-derived CIK cells were transferred into a three-dimensional dynamic culture system and cultured with a dynamic perfusion method supplemented with IL-2, IL-1α, CD3 monoclonal antibody, and active ingredients until the cell density reached 1-3 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0011] S4. Modify the enhanced peripheral blood-derived CIK cells using the CRISPR / Cas9 gene editing system delivered by electroporation to knock out non-tumor-associated chemokine receptor genes and overexpress tumor microenvironment-specific chemokine receptor genes to obtain modified peripheral blood-derived CIK cells;

[0012] S5. Continue culturing the transformed peripheral blood-derived CIK cells for 3-5 days, and harvest the cells after washing and resuspending.

[0013] Preferably, the density gradient centrifugation method in step S1 uses Ficoll-Paque PLUS separation medium, and the specific operation steps are: collect 50-100 mL of peripheral blood from a healthy donor, mix the peripheral blood with an equal volume of PBS, slowly add it to the upper layer of 25-55 mL of Ficoll-Paque PLUS separation medium along the wall of the tube, centrifuge at 300-400g for 20-30 minutes at 15-25°C, collect the middle mononuclear cell layer after centrifugation, and wash it 2-4 times with PBS at 200-300g for 5-15 minutes to obtain PBMC.

[0014] Preferably, the culture medium in step S2 is X-VIVO 15 serum-free culture medium, the added concentration of IFN-γ is 500-1000 IU / mL, the pre-stimulation culture time is 3-5 days, and the culture environment is a constant temperature incubator at 35-40°C and 4-6% CO2.

[0015] Preferably, the three-dimensional dynamic culture system in step S3 is a G-Rex bioreactor, the dynamic perfusion flow rate is 0.5-1.5 mL / min, the concentration of IL-2 is 200-500 IU / mL, the concentration of IL-1α is 10-20 ng / mL, the concentration of CD3 monoclonal antibody is 60-100 ng / mL, the concentration of the active ingredient is 30-70 μM, and the culture medium is X-VIVO 15 serum-free medium.

[0016] Preferably, the active ingredients include 10-20 μM curcumin and 20-50 μM resveratrol.

[0017] Preferably, in step S4, electroporation is used to deliver the CRISPR / Cas9 system containing sgRNA targeting CCR5, CXCR2 and CXCR4 and Cas9 protein, and the electroporation parameters are: voltage of 100-300V, capacitance of 500-1000μF, and pulse time of 10-30ms; the non-tumor-related chemokine receptor gene is CCR5, and the tumor microenvironment-specific chemokine receptor genes are CXCR2 and CXCR4.

[0018] Preferably, in step S5, the cells are washed 2-4 times using PBS, and the cells are resuspended in physiological saline containing 5-10% human serum albumin.

[0019] In a second aspect, the present application provides peripheral blood-derived CIK cells prepared using the above-mentioned peripheral blood-derived CIK cell preparation method.

[0020] In a third aspect, the present application provides the use of the above-mentioned peripheral blood-derived CIK cells in the preparation of a drug for inhibiting lung cancer, liver cancer, gastric cancer, colorectal cancer or breast cancer.

[0021] In summary, this application has the following beneficial effects:

[0022] (1) Efficient and stable cell preparation process: Through optimized density gradient centrifugation, the recovery rate and purity of PBMC are significantly improved, laying a high-quality foundation for subsequent peripheral blood-derived CIK cell culture; using X-VIVO 15 serum-free culture medium combined with IFN-γ pre-stimulation, high-density initial peripheral blood-derived CIK cells can be obtained within 3-5 days, shortening the culture cycle while avoiding batch differences and contamination risks caused by serum.

[0023] (2) Significantly enhanced cell proliferation and activity: The three-dimensional dynamic culture system combined with dynamic perfusion technology simulates the in vivo microenvironment to promote intercellular signal transduction, achieving high-density cell proliferation, which is 3-5 times more efficient than traditional static culture. The addition of active ingredients curcumin and resveratrol significantly improves the survival rate and CD4+ T cells of peripheral blood-derived CIK cells through their antioxidant and immunomodulatory effects. 3+ CD 56+ The proportion of double-positive cells was increased, and its killing activity against tumor cells was enhanced.

[0024] (3) Precision-targeted gene editing technology: The CRISPR / Cas9 system is used to knock out the non-tumor-related chemokine receptor CCR5 and overexpress the tumor microenvironment-specific receptors CXCR2 and CXCR4, giving CIK cells the ability to migrate to the tumor site, significantly improving the therapeutic targeting; the electroporation non-viral delivery technology is used to ensure the efficiency of gene editing while avoiding the potential safety risks of viral vectors, meeting the safety and compliance requirements of clinical cell therapy.

[0025] (4) Excellent tumor suppression effect

[0026] By knocking out the CCR5 gene, the migration of peripheral blood-derived CIK cells to non-tumor tissues (such as inflammatory sites) can be reduced, and the off-target rate can be lowered; by overexpressing the CXCR2 gene, the high concentration of IL8 secreted by tumor cells in the tumor microenvironment can be responded to, and the chemotaxis of CIK cells to tumor cells can be promoted; by overexpressing the CXCR4 gene, the CXCL12 secreted by tumor stromal cells can be responded to, and the ability of CIK cells to penetrate vascular barriers and mechanisms can be enhanced; through the above chemokine receptor gene modification, the retention time of peripheral blood-derived CIK cells in the tumor microenvironment in vivo can be prolonged, the local killing effect can be enhanced, and the tumor inhibition effect can be significantly enhanced. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0030] Examples 1-3 provide methods for preparing peripheral blood-derived CIK cells.

[0031] Example 1

[0032] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0033] S1. Collect 50 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top layer of 25 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 300 g for 20 min at 15°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle and wash twice with PBS at 200 g for 5 min to obtain PBMCs.

[0034] S2, PBMC were cultured at 1×10 6 The cells were seeded at a density of 4 × 10 cells / mL in X-VIVO 15 serum-free medium, IFN-γ was added at 500 IU / mL, and pre-stimulated and cultured in a constant temperature incubator at 35°C and 4% CO2 for 3 days until the cell density reached 4 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0035] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 0.5 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 200 IU / mL, the concentration of IL-1α was 10 ng / mL, the concentration of CD3 monoclonal antibody was 60 ng / mL, the concentration of curcumin was 10 μM, and the concentration of resveratrol was 20 μM. The cells were cultured until the cell density reached 1×10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0036] S4. Electroporation was used to deliver a CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 100 V, capacitance of 500 μF, and pulse time of 10 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0037] S5. The transformed peripheral blood-derived CIK cells were cultured for 3 days, washed twice with PBS, and then resuspended in physiological saline containing 5% human serum albumin to harvest the cells.

[0038] Example 2

[0039] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0040] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0041] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0042] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of curcumin was 15 μM, and the concentration of resveratrol was 35 μM. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0043] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0044] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0045] Example 3

[0046] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0047] S1. Collect 100 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it to the upper layer of 55 mL of Ficoll-Paque PLUS separation buffer along the tube wall to form a clear liquid layer. Centrifuge at 400 g for 30 min at 25°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture four times with PBS at 300 g for 15 min to obtain PBMCs.

[0048] S2, PBMC were cultured at 2×10 6 The cells were seeded at a density of 6 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 1000 IU / mL of IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 40°C and 6% CO2 for 5 days until the cell density reached 6 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0049] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.5 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 500 IU / mL, the concentration of IL-1α was 20 ng / mL, the concentration of CD3 monoclonal antibody was 100 ng / mL, the concentration of curcumin was 20 μM, and the concentration of resveratrol was 50 μM. The cells were cultured until the cell density reached 3 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0050] S4. Electroporation was used to deliver a CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 300 V, capacitance of 1000 μF, and pulse time of 30 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0051] S5. The transformed peripheral blood-derived CIK cells were cultured for 5 days, washed 4 times with PBS, and then resuspended in physiological saline containing 10% human serum albumin to harvest the cells.

[0052] Comparative Example 1

[0053] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0054] S1. Collect 75 mL of peripheral blood from a healthy donor, mix the peripheral blood with an equal volume of PBS, centrifuge at 800 g for 25 min at 20°C, and wash three times with PBS at 250 g for 10 min to obtain mixed PBMCs.

[0055] S2, mixed PBMC at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0056] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of curcumin was 15 μM, and the concentration of resveratrol was 35 μM. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0057] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0058] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0059] Comparative Example 2

[0060] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0061] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0062] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in RPMI 1640 medium containing 10% fetal bovine serum, and IFN-γ was added at 750 IU / mL. The cells were pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0063] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The medium was supplemented with 350 IU / mL of IL-2, 15 ng / mL of IL-1α, 80 ng / mL of CD3 monoclonal antibody, 15 μM of curcumin, and 35 μM of resveratrol. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0064] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0065] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0066] Comparative Example 3

[0067] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0068] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0069] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, PBMC expansion cells were obtained;

[0070] S3. Transfer the expanded PBMC cells to the G-Rex bioreactor. Set the dynamic perfusion flow rate to 1.0 mL / min and dynamically perfuse X-VIVO 15 serum-free medium into the G-Rex bioreactor. The medium contains IFN-γ at a concentration of 750 IU / mL, IL-2 at a concentration of 350 IU / mL, IL-1α at a concentration of 15 ng / mL, CD3 monoclonal antibody at a concentration of 80 ng / mL, curcumin at a concentration of 15 μM, and resveratrol at a concentration of 35 μM. Culture the cells until the cell density reaches 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0071] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0072] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0073] Comparative Example 4

[0074] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0075] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0076] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0077] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of curcumin was 15 μM, and the concentration of cordycepin was 35 μM. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0078] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0079] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0080] Comparative Example 5

[0081] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0082] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0083] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0084] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of icariin was 15 μM, and the concentration of resveratrol was 35 μM. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0085] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0086] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0087] Comparative Example 6

[0088] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0089] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it to the upper layer of 40 mL of Ficoll-Paque PLUS separation buffer along the tube wall to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the cell suspension three times with PBS by centrifugation at 250 g for 10 minutes to obtain a PBMC cell suspension.

[0090] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0091] S3. Initial peripheral blood-derived CIK cells were transferred to culture flasks for static culture. Half of the medium was changed every 2 days. The culture medium was X-VIVO 15 serum-free medium. The concentration of IL-2 added to the culture medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of curcumin was 15 μM, and the concentration of resveratrol was 35 μM. The cells were cultured until the cell density reached 2×10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0092] S4. Electroporation was used to deliver the CRISPR / Cas9 system containing sgRNAs targeting CCR5, CXCR2, and CXCR4 and the Cas9 protein. The electroporation parameters were set as follows: voltage of 200 V, capacitance of 750 μF, and pulse time of 20 ms. The enhanced peripheral blood-derived CIK cells were then modified to knock out the non-tumor-associated chemokine receptor gene CCR5 and overexpress the tumor microenvironment-specific chemokine receptor genes CXCR2 and CXCR4, thereby obtaining modified peripheral blood-derived CIK cells.

[0093] S5. The transformed peripheral blood-derived CIK cells were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0094] Comparative Example 7

[0095] The method for preparing peripheral blood-derived CIK cells comprises the following steps:

[0096] S1. Collect 75 mL of peripheral blood from a healthy donor. Mix the peripheral blood with an equal volume of PBS and slowly add it along the tube wall to the top of 40 mL of Ficoll-Paque PLUS separation buffer to form a clear liquid layer. Centrifuge at 350 g for 25 minutes at 20°C. After centrifugation, carefully aspirate the milky white mononuclear cell layer in the middle. Wash the mixture three times with PBS at 250 g for 10 minutes to obtain PBMCs.

[0097] S2, PBMC were cultured at 1.5×10 6 The cells were seeded at a density of 5 × 10 cells / mL in X-VIVO 15 serum-free medium, supplemented with 750 IU / mL IFN-γ, and pre-stimulated and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 days until the cell density reached 5 × 10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells;

[0098] S3. Initial peripheral blood-derived CIK cells were transferred to a G-Rex bioreactor. The dynamic perfusion flow rate was set to 1.0 mL / min. X-VIVO 15 serum-free medium was dynamically perfused into the G-Rex bioreactor. The concentration of IL-2 added to the medium was 350 IU / mL, the concentration of IL-1α was 15 ng / mL, the concentration of CD3 monoclonal antibody was 80 ng / mL, the concentration of curcumin was 15 μM, and the concentration of resveratrol was 35 μM. The cells were cultured until the cell density reached 2 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties;

[0099] S4. The peripheral blood-derived CIK cells with enhanced properties were cultured for another 4 days, washed 3 times with PBS, and then resuspended in physiological saline containing 7.5% human serum albumin to harvest the cells.

[0100] Cell number detection

[0101] 1 mL of peripheral blood-derived CIK cells prepared in Examples 1-3 and Comparative Examples 1-7 were sampled and counted using a trypan blue staining method using a cell counting plate. The cell number and cell survival rate were calculated. The results are shown in Table 1.

[0102] Table 1 Statistics of the number of CIK cells from peripheral blood

[0103]

[0104]

[0105] The data in Table 1 show that the number and survival rate of peripheral blood-derived CIK cells in Examples 1-3 were significantly better than those in Comparative Examples 1-7. Among them, the number of cells in Comparative Example 6 was only 59 million cells / mL, and the survival rate was only 79.2%, indicating that the pre-stimulation, three-dimensional dynamic culture and active ingredient addition methods of the present application can simultaneously improve cell expansion efficiency and survival rate.

[0106] Cell surface antigen detection

[0107] The peripheral blood-derived CIK cells prepared in Examples 1-3 and Comparative Examples 1-7 were sampled and the cell immune phenotypes were determined by flow cytometry. The culture cell density was adjusted to 1×10 6 / mL, 100μL / tube was dispensed into flow test tubes, and labeled with isotype control, anti-CD3-PerCP, and anti-CD56-PE respectively. Cell phenotype was detected by flow cytometry to determine the CD3 expression in peripheral blood CIK cells. 3+ CD 56+ The results of the cell ratios are shown in Table 2.

[0108] Table 2 CD4+ in peripheral blood-derived CIK cells 3+ CD 56+ Cell ratio statistics

[0109] Group <![CDATA[CD 3+ CD 56+ (%)]]> Example 1 59.9 Example 2 65.2 Example 3 61.2 Comparative Example 1 40.4 Comparative Example 2 52.5 Comparative Example 3 50.6 Comparative Example 4 57.6 Comparative Example 5 56.1 Comparative Example 6 38.2 Comparative Example 7 57.9

[0110] The data in Table 2 show that the CD 3+ CD 56+ The cell ratio is significantly higher than that of Comparative Examples 1-7. 3+ CD 56+ The proportion of cells is the highest, and the CD 3+ CD 56+ The proportion of cells was the lowest, CD 3+ CD 56+ CIK cells are the key subpopulation of peripheral blood-derived CIK cells to exert their cytotoxic activity, and their proportion directly affects the immune function. Therefore, it can be seen that the cell culture method of Examples 1-3 is more conducive to improving the cytotoxic activity and immune phenotype advantages of peripheral blood-derived CIK cells.

[0111] In vitro tumor cell killing assay

[0112] K562 tumor cell lines in the logarithmic growth phase were taken and resuspended at a cell concentration of 1×10 5 cells / ml, 5×10 4 cells / ml, 2.5×10 4 cells / ml, 100 μL per well was plated in a 96-well flat-bottom plate, and cultured in a constant temperature incubator at 37°C, 5% CO2 concentration, and 100% humidity for 24 h before use;

[0113] The peripheral blood-derived CIK cells prepared in Examples 1-3 and Comparative Examples 1-7 were resuspended in 1×10 6 cells / ml, were added to the aforementioned 96-well plate at effector-target ratios of 10:1, 20:1, and 40:1. Four replicate wells were set for each concentration, and a blank control group of effector cells that did not react with tumor cells was set. After 48 h of co-culture, 20 μL of MTT solution (5 mg / ml) was added to each well. After further culture for 4 h, the supernatant was discarded by centrifugation, 100 μL of DMSO was added to each well, the absorbance was measured at 570 nm, and the killing rate was calculated. The results are shown in Table 3.

[0114] The formula for the killing rate is as follows: [1-(OD value of the effector target cell group-OD value of the effector cell) / OD value of the target cell-OD value of the blank control group] × 100%;

[0115] Table 3 Statistics of killing rate of peripheral blood-derived CIK cells

[0116]

[0117]

[0118] Table 3 shows the killing rates of K562 tumor cells by peripheral blood-derived CIK cells in different groups at various effector-target ratios. The killing rates of Examples 1-3 were generally higher than those of Comparative Examples 1-7, and the killing rates of each group increased significantly as the effector-target ratio increased from 10:1 to 40:1. Among them, Example 2 had the highest killing rate at all effector-target ratios, reaching 92.5% at 40:1, while Comparative Example 7 had the lowest killing rate, at only 59.1%. This demonstrates that the peripheral blood-derived CIK cells in the Example groups have a stronger ability to kill tumor cells, and that increasing the effector-target ratio enhances the killing effect.

[0119] In vivo tumor suppression assay

[0120] The peripheral blood-derived CIK cells prepared in Examples 1-3 and Comparative Examples 1-7 of the present application were respectively tested for their in vivo tumor inhibition effects.

[0121] Experimental animals and groups

[0122] 120 6-8 week old, female, 18-22 g BALB / c nude mice were randomly divided into 12 groups, 10 mice in each group, and recorded as blank control group, model group, Example 1-3 groups and Comparative Example 1-7 groups.

[0123] Rearing conditions

[0124] SPF-grade environment, 12h / 12h day / night cycle, temperature 22±2℃, humidity 50±10%.

[0125] Mouse modeling

[0126] Except for the blank group, the mice in other groups were modeled as follows: HepG liver cancer cells (5×10 6 The cells were mixed with Matrigel at a ratio of 1:1 and injected subcutaneously into the back of the right hind limb of BALB / c nude mice. Tumor growth was monitored daily and the tumor volume was 110 ± 10 mm. 3 The modeling is considered successful.

[0127] Cell therapy

[0128] The treatment regimens for the blank control group, model group, Example 1-3 groups, and Comparative Example 1-7 groups are as follows:

[0129] (1) Blank control group: healthy BALB / c nude mice injected with PBS;

[0130] (2) Model group: BALB / c nude mice with tumors injected with PBS;

[0131] (3) Example 1 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Example 1;

[0132] (4) Example 2 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Example 2;

[0133] (5) Example 3 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Example 3;

[0134] (6) Comparative Example 1 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 1;

[0135] (7) Comparative Example 2: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 2;

[0136] (8) Comparative Example 3: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 3;

[0137] (9) Comparative Example 4 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 4;

[0138] (10) Comparative Example 5 group: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 5;

[0139] (11) Comparative Example 6: BALB / c nude mice bearing tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 6;

[0140] (12) Comparative Example 7: BALB / c nude mice with tumors were injected with peripheral blood-derived CIK cells prepared in Comparative Example 7.

[0141] According to the above treatment plan, the mice in Example 1-3 and Comparative Example 1-7 were injected into the tail vein, and the amount of injected cells was 5×10 6 cells / mouse, injected once every 3 days, for a total of 6 times; the blank control group and the model group were injected with an equal volume of PBS, injected once every 3 days, for a total of 6 times.

[0142] Monitoring indicators

[0143] (1) Tumor volume: measured every 3 days, and the relative tumor volume (RTV) was calculated, RTV = Vt / V0, where V0 is the initial tumor volume after treatment and Vt is the current tumor volume after treatment;

[0144] (2) Mouse survival rate: Observed for 60 days, tumor size ≥ 2000 mm 3 Or weight loss ≥20% is considered death.

[0145] Experimental results statistics

[0146] After the experiment on each group of mice was completed, the average tumor volume and survival rate of each group of mice were statistically analyzed. The results are shown in Table 4.

[0147] Table 4 Statistics of average tumor volume and survival rate of mice

[0148]

[0149]

[0150] Note: Data are expressed as mean ± standard deviation (Mean ± SD); significant marks: *p < 0.05, **p < 0.01 vs. model group.

[0151] Analysis of the data in Table 4 showed that compared with the blank control group, the average tumor volume of the mice in the model group was significantly increased, and the survival rate was 0. Compared with the model group and the control group 1-7, the average tumor volume of the Example 1-3 group was closer to normal, and the differences were statistically significant (P < 0.05). This shows that after the modeling was completed, the mice were treated with the peripheral blood-derived CIK cells obtained in Examples 1-3 during the cell therapy stage, achieving effective inhibition of tumors in the body and nearly returning to normal levels.

[0152] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing peripheral blood-derived CIK cells, characterized in that: The following steps are involved: S1. Peripheral blood was collected from healthy donors and PBMCs were isolated by density gradient centrifugation. S2, PBMC were cultured at 1-2×10 6 cells / mL in culture medium, and IFN-γ was added for pre-stimulation and cultured until the cell density reached 4-6×10 6 cells / mL, to obtain initial peripheral blood-derived CIK cells; S3. Initial peripheral blood-derived CIK cells were transferred into a three-dimensional dynamic culture system and cultured with a dynamic perfusion method supplemented with IL-2, IL-1α, CD3 monoclonal antibody, and active ingredients until the cell density reached 1-3 × 10 7 cells / mL, and obtain peripheral blood-derived CIK cells with enhanced properties; S4. Modify the enhanced peripheral blood-derived CIK cells using the CRISPR / Cas9 gene editing system delivered by electroporation to knock out non-tumor-associated chemokine receptor genes and overexpress tumor microenvironment-specific chemokine receptor genes to obtain modified peripheral blood-derived CIK cells; S5. Continue culturing the transformed peripheral blood-derived CIK cells for 3-5 days, and harvest the cells after washing and resuspending.

2. The method for preparing peripheral blood-derived CIK cells according to claim 1, wherein: In step S1, the density gradient centrifugation method uses Ficoll-Paque PLUS separation medium. The specific operation steps are as follows: 50-100 mL of peripheral blood from a healthy donor is collected, and the peripheral blood is mixed with an equal volume of PBS. The blood is then slowly added along the wall of the tube to the upper layer of 25-55 mL of Ficoll-Paque PLUS separation medium. The blood is centrifuged at 300-400g for 20-30 minutes at 15-25°C. After centrifugation, the middle mononuclear cell layer is collected and washed 2-4 times with PBS at 200-300g for 5-15 minutes to obtain PBMCs.

3. The method for preparing peripheral blood-derived CIK cells according to claim 1, characterized in that: In step S2, the culture medium is X-VIVO 15 serum-free culture medium, the added concentration of IFN-γ is 500-1000 IU / mL, the pre-stimulation culture time is 3-5 days, and the culture environment is a constant temperature incubator at 35-40° C. and 4-6% CO 2 .

4. The method for preparing peripheral blood-derived CIK cells according to claim 1, wherein: In step S3, the three-dimensional dynamic culture system is a G-Rex bioreactor, the dynamic perfusion flow rate is 0.5-1.5 mL / min, the concentration of IL-2 is 200-500 IU / mL, the concentration of IL-1α is 10-20 ng / mL, the concentration of CD3 monoclonal antibody is 60-100 ng / mL, the concentration of the active ingredient is 30-70 μM, and the culture medium is X-VIVO 15 serum-free medium.

5. The method for preparing peripheral blood-derived CIK cells according to claim 4, characterized in that: The active ingredients include 10-20 μM curcumin and 20-50 μM resveratrol.

6. The method for preparing peripheral blood-derived CIK cells according to claim 1, wherein: In step S4, electroporation is used to deliver the CRISPR / Cas9 system containing sgRNA targeting CCR5, CXCR2 and CXCR4 and Cas9 protein, and the electroporation parameters are: voltage of 100-300V, capacitance of 500-1000μF, and pulse time of 10-30ms; the non-tumor-related chemokine receptor gene is CCR5, and the tumor microenvironment-specific chemokine receptor genes are CXCR2 and CXCR4.

7. The method for preparing peripheral blood-derived CIK cells according to claim 1, wherein: In step S5, the cells are washed 2-4 times with PBS, and the cells are resuspended in physiological saline containing 5-10% human serum albumin.

8. Peripheral blood-derived CIK cells prepared by the method for preparing peripheral blood-derived CIK cells according to any one of claims 1 to 7.

9. Use of the peripheral blood-derived CIK cells according to claim 8 in the preparation of a medicament for inhibiting lung cancer, liver cancer, gastric cancer, colorectal cancer or breast cancer.

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