Preparation method of human placenta-derived NK cells

Abstract

The invention relates to the technical field of immune cells, in particular to a preparation method of human placenta-derived NK cells. The preparation method comprises the following steps: S1, separating placenta tissues; s2, placenta tissue digestion; S3, mononuclear cell separation; S4, NK cell sorting; and 5, NK cell multiplication culture.

Description

Technical Field

[0001] This invention relates to the field of immune cell technology, specifically to a method for preparing human placental NK cells. Background Technology

[0002] Natural killer (NK) cells are core effector cells of the body's innate immune system. They can specifically recognize and kill tumor cells, virus-infected cells, and abnormally proliferating cells without prior antigen sensitization. They also possess immunomodulatory functions, making them one of the most promising immune cells for tumor immunotherapy, antiviral infection therapy, and autoimmune disease treatment. The advantage of NK cells lies in their ability to recognize and kill tumor cells without antigen sensitization or MHC restriction, playing a crucial role in the body's anti-tumor and early antiviral immune responses. However, the low proportion and limited number of NK cells in the body restrict their application. Currently, in vitro expansion of NK cells mainly employs the following methods: first, using immunomagnetic beads for cell sorting to obtain purified NK cells from peripheral blood mononuclear cells, followed by in vitro amplification culture with added factors; second, using feeder cell methods to expand primary NK cells; and third, simply stimulating NK cell expansion with a combination of cytokines. The first method requires drawing a large amount of peripheral blood from the patient and often only expands the cell count by a few dozen times. The second method can obtain a large number of high-purity NK cells, but it is prone to contamination by foreign cell lines. The third method has better safety and high clinical application value, but the expansion fold is not high and is unstable. In addition, conventional two-dimensional culture has low space utilization and poor uniformity of the cell environment, making it difficult to improve cell expansion efficiency. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a method for preparing human placental NK cells.

[0004] This invention is achieved through the following technical solution: A method for preparing human placental NK cells includes the following steps: S1. Placental tissue isolation: Under aseptic conditions, fresh placental tissue is taken and repeatedly rinsed 3-5 times with PBS buffer containing 1% penicillin-streptomycin. The amnion is then peeled off, and the placental chorionic villus tissue is taken. The tissue is cut into 1-3 mm pieces. 3 The fragments were washed 2-3 times with PBS buffer, centrifuged at 500 g for 5 min at 4℃, and the supernatant was discarded to obtain placental tissue homogenate. S2. Placental tissue digestion: The placental tissue homogenate obtained in step S1 was digested with a compound enzyme solution. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum was immediately added to terminate the digestion. The mixture was filtered through a 200-mesh sieve, and the filtrate was collected. The cells were washed twice with PBS buffer, resuspended in PBS buffer, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 Cells / mL were used to obtain a cell suspension; S3. Mononuclear cell isolation: The cell suspension obtained in step S2 is slowly stacked along the tube wall above the surface of an equal volume of human lymphocyte separation solution. Centrifuge at 4°C and 500 g for 20 min, aspirate the mononuclear cells in the middle white membrane layer, wash twice with PBS buffer, centrifuge at 4°C and 600 g for 8 min, discard the supernatant, and obtain placental mononuclear cells. S3. NK cell sorting: Placental mononuclear cells obtained in step S3 were collected and sorted using an NK cell negative selection kit and an immunomagnetic bead negative selection method to obtain CD56 cells. + CD3 - Placental NK cells; S4. NK cell expansion culture: Resuspend the NK cells obtained in step S3 in complete culture medium and adjust the cell concentration to 1×10⁻⁶. 6 Cells were seeded at a density of 1-1.5 × 10⁶ cells / mL into culture flasks pre-coated with 5-10 μg / mL CD16 antibody and incubated at 37°C in a 5% CO₂ incubator. Complete culture medium was added every 2-3 days to maintain a cell concentration of 1-1.5 × 10⁶ cells / mL. 6 Cells were cultured at a density of 1 cell per mL for 14 days before being collected.

[0005] Further, the specific operation of the digestion process described in step S2 is as follows: the placental tissue homogenate and the compound enzyme solution are mixed at a volume ratio of 1:3-5, and placed in a constant temperature incubator at 37℃ and 5% CO2 for 30-40 min of shaking digestion, and gently blown to mix once every 10 min.

[0006] Further, in step S2, the composite enzyme solution is a serum-free DMEM medium containing type I collagenase, type IV collagenase, and DNase I; wherein the final concentration of type I collagenase is 0.8-1.2 mg / mL, the final concentration of type IV collagenase is 0.3-0.7 mg / mL, and the final concentration of DNase I is 15-25 μg / mL.

[0007] Further, in step S4, the complete culture medium is prepared by adding IL-2 500-800 IU / mL, IL-15 20-40 ng / mL, IL-12 5-10 ng / mL, IL-18 10-20 ng / mL, IL-21 5-10 ng / mL, and porous microspheres 2-3 mg / mL to the basal culture medium Lonza X-VIVO 15 serum-free culture medium.

[0008] Furthermore, the method for preparing the porous microspheres includes the following steps: (1) Wash and dry the honeysuckle, pulverize it through an 80-100 mesh sieve to obtain honeysuckle powder. Add the honeysuckle powder to deionized water at a ratio of 1 g: 30 mL. Heat at 90°C for 2 h. Filter and collect the filtrate. Add deionized water to the filter residue. Repeat the above operation twice. Combine the filtrates and concentrate them to 20% of the original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol. Let stand at 4°C for 12-15 h. Centrifuge at 5000 rpm for 15-20 min. Wash the precipitate with anhydrous ethanol. Redissolve in distilled water. Deproteinize 5 times using the Sevage method. Remove the Sevage solution under reduced pressure. Dialyze in distilled water for 3 days, changing the water every 8 h. Freeze dry to obtain honeysuckle extract. (2) Dissolve the honeysuckle extract obtained in step (1) in deionized water, add sodium periodate, stir at room temperature in the dark for 10-12 h, add ethylene glycol and stir for 30 min, dialyze with deionized water for 3 days, change the water every 8 h, freeze dry to obtain oxidized honeysuckle extract. (3) ε-polylysine (EPL) was added to dimethyl sulfoxide (DMSO) and stirred to dissolve. Succinic anhydride was added and stirred at 50°C for 1 h. The mixture was dialyzed in deionized water for 4 days, with the water changed every 4 h. The mixture was then freeze-dried to obtain modified polylysine. (4) Add the modified polylysine obtained in step (3) to DMSO, add CDI, stir at 60℃ for 4 h, cool to room temperature, add DMAP and resveratrol (RSV) in the dark, stir at room temperature for 24 h, dialyze in deionized water for 4 days, change the water every 4 h, freeze dry to obtain EPL-RSV. (5) Mix paraffin with Span 80 to obtain an oil phase. Add the honeysuckle extract obtained in step (2), EPL-RSV obtained in step (4), and polyethylene glycol 4000 to a PBS buffer at pH 6.5 and mix to obtain an aqueous phase. Add the aqueous phase dropwise to the oil phase with stirring. Stir at 800-1000 rpm for 30 min, stir at 300 rpm for 4-6 h, centrifuge at 5000 rpm for 8-12 min, wash the precipitate with petroleum ether and deionized water, vacuum dry, resuspend in PBS, filter through a 150 μm sieve, and freeze dry to obtain porous microspheres.

[0009] Further, in step (2), the honeysuckle extract has a mass concentration of 20-30 mg / mL in deionized water.

[0010] Further, in step (2), the mass ratio of sodium periodate to honeysuckle extract is 1:2-3.

[0011] Furthermore, in step (2), the volume ratio of ethylene glycol to deionized water is 1:50.

[0012] Furthermore, in step (3), the mass concentration of polylysine in DMSO is 0.1 g / mL. Further, in step (3), the mass ratio of polylysine to succinic anhydride is 8-10:1.

[0013] Further, in step (4), the mass concentration of the modified polylysine in DMSO is 10-20 mg / mL.

[0014] Further, in step (4), the mass ratio of the modified polylysine, CDI, DMAP and resveratrol is 10:0.8:1:3.

[0015] Furthermore, in step (5), the volume ratio of the paraffin wax to the Span 80 is 49:1.

[0016] Further, in step (5), the mass ratio of the oxidized honeysuckle extract, EPL-RSV and polyethylene glycol 4000 is 2-4:3-5:1-2.

[0017] Further, in step (5), the mass-to-volume ratio of the oxidized honeysuckle extract in PBS buffer is 1% w / v.

[0018] Furthermore, in step (5), the volume ratio of the aqueous phase to the oil phase is 1:3-5.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing human placental NK cells. By adding a specific combination of cytokines—IL-2, IL-15, IL-12, IL-18, and IL-21—to the basal culture medium and introducing porous microspheres, a 3D culture system is constructed, significantly improving the in vitro expansion efficiency and cytotoxic activity of placental-derived NK cells. This invention uses postpartum placental tissue as the source of NK cells. The placenta is medical waste, making the sourcing non-invasive and widely available, eliminating the need to draw large amounts of peripheral blood from the patient and avoiding physical harm to the donor / patient. This invention optimizes the scientific ratio of the five cytokines, constructing a synergistic expansion system. IL-2 is the core proliferation factor, maintaining the rapid division and proliferation of NK cells; IL-15 promotes NK cell survival and self-renewal, reducing apoptosis during culture; IL-12 and IL-18 synergistically activate the cytotoxic effect of NK cells, promoting the secretion of effector cytokines such as IFN-γ, continuously enhancing cell killing ability during expansion; IL-21 effectively delays cell function exhaustion during long-term expansion. This invention prepares porous microspheres. Polysaccharides are extracted from honeysuckle. Sodium periodate is used to oxidize the o-dihydroxy groups in the honeysuckle extract to aldehyde groups, yielding oxidized honeysuckle extract. Succinic anhydride is used to introduce carboxyl groups onto the polylysine molecular chain. The carboxyl groups then undergo esterification based on the hydroxyl groups of resveratrol, covalently binding resveratrol and polylysine to obtain EPL-RSV. The aldehyde groups in the oxidized honeysuckle extract and the amino groups in EPL-RSV undergo a Schiff base reaction, cross-linking to form a porous structure, thus producing porous microspheres. The porous microspheres of this invention possess a rich, interconnected porous structure, providing cells with a three-dimensional growth space mimicking in vivo lymph nodes, improving the space utilization and cell carrying capacity of the culture system. The microspheres can be uniformly suspended in the culture medium, allowing for sufficient contact between cells, cytokines, and nutrients, significantly improving the uniformity of the culture environment. The porous microspheres of this invention are cross-linked and cured by a Schiff base reaction between the aldehyde groups of oxidized honeysuckle extract and the free amino groups of EPL-RSV, eliminating the need for toxic chemical cross-linking agents such as glutaraldehyde. All raw materials are natural bioactive materials with high biocompatibility. The functional components of this invention synergistically enhance cell activity and cytotoxicity. The honeysuckle extract in the porous microspheres exhibits significant anti-inflammatory and antioxidant activities, scavenging reactive oxygen species accumulated in the culture system, reducing oxidative stress damage to NK cells, maintaining cell physiological activity, and delaying cell aging. The ε-polylysine in EPL-RSV has broad-spectrum antibacterial activity, effectively inhibiting bacterial and fungal contamination in the culture system and improving the stability of long-term culture. Grafted resveratrol further enhances antioxidant activity, strengthens NK cell activity, and reduces functional exhaustion during long-term cell expansion. Attached Figure Description

[0020] Figure 1 This is a scanning electron microscope image of the porous microspheres described in Embodiment 1 of the present invention; Figure 2 This refers to the fold increase in NK cells described in Examples 1-3 and Comparative Examples 1-4 of the present invention; Figure 3 The killing activity of NK cells described in Examples 1-3 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0022] Example 1: A method for preparing human placental NK cells, comprising the following steps: S1. Placental tissue isolation: Under aseptic conditions, fresh placental tissue was collected and rinsed repeatedly 5 times with PBS buffer containing 1% penicillin-streptomycin. The amnion was peeled off, and the placental chorionic villus tissue was collected and cut into 3 mm pieces. 3 The fragments were washed three times with PBS buffer, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded to obtain placental tissue homogenate. S2. Placental tissue digestion: The placental tissue homogenate obtained in step S1 was digested with a compound enzyme solution. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum was immediately added to terminate the digestion. The mixture was filtered through a 200-mesh sieve, and the filtrate was collected. The cells were washed twice with PBS buffer, resuspended in PBS buffer, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 Cells were collected at a concentration of 100 cells / mL to obtain a cell suspension. The specific digestion process was as follows: placental tissue homogenate was mixed with compound enzyme solution at a volume ratio of 1:5, and placed in a constant temperature incubator at 37℃ and 5% CO2 for 40 min with shaking. The mixture was gently blown and mixed every 10 min. The compound enzyme solution was serum-free DMEM medium containing type I collagenase, type IV collagenase, and DNase I. The final concentration of type I collagenase was 1.2 mg / mL, the final concentration of type IV collagenase was 0.7 mg / mL, and the final concentration of DNase I was 25 μg / mL. S3. Mononuclear cell isolation: The cell suspension obtained in step S2 is slowly stacked along the tube wall above the surface of an equal volume of human lymphocyte separation solution. Centrifuge at 4°C and 500 g for 20 min, aspirate the mononuclear cells in the middle white membrane layer, wash twice with PBS buffer, centrifuge at 4°C and 600 g for 8 min, discard the supernatant, and obtain placental mononuclear cells. S3. NK cell sorting: Placental mononuclear cells obtained in step S3 were collected and sorted using an NK cell negative selection kit and an immunomagnetic bead negative selection method to obtain CD56 cells. + CD3 - Placental NK cells; S4. NK cell expansion culture: Resuspend the NK cells obtained in step S3 in complete culture medium and adjust the cell concentration to 1×10⁻⁶. 6 Cells were seeded at a density of 1.5 × 10⁶ cells / mL into culture flasks pre-coated with 10 μg / mL CD16 antibody and incubated at 37°C in a 5% CO₂ incubator. Complete culture medium was added every 3 days to maintain a cell concentration of 1.5 × 10⁶ cells / mL. 6 Cells were cultured at a density of 10 cells / mL for 14 days and then collected. The complete culture medium consisted of Lonza X-VIVO 15 serum-free medium supplemented with IL-2 800 IU / mL, IL-15 40 ng / mL, IL-12 10 ng / mL, IL-18 20 ng / mL, IL-21 10 ng / mL, and porous microspheres 3 mg / mL.

[0023] The method for preparing the porous microspheres includes the following steps: (1) Wash and dry the honeysuckle, pulverize it through a 100-mesh sieve to obtain honeysuckle powder. Add 5 g of honeysuckle powder to 150 mL of deionized water at a material-liquid ratio of 1 g: 30 mL. Heat at 90℃ for 2 h, filter and collect the filtrate. Add deionized water to the filter residue. Repeat the above operation twice. Combine the filtrates and concentrate them to 20% of the original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol, let stand at 4℃ for 15 h, centrifuge at 5000 rpm for 20 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, deproteinize 5 times using the Sevage method, remove the Sevage solution under reduced pressure, dialyze in distilled water for 3 days, change the water every 8 h, freeze dry to obtain honeysuckle extract. (2) Dissolve 3 g of honeysuckle extract obtained in step (1) in 100 mL of deionized water, add 1 g of sodium periodate, stir at room temperature in the dark for 12 h, add 2 mL of ethylene glycol and stir for 30 min, dialyze with deionized water for 3 days, change the water every 8 h, freeze dry to obtain oxidized honeysuckle extract. (3) Add 12 g of ε-polylysine (EPL) to 120 mL of dimethyl sulfoxide (DMSO) and stir to dissolve. Add 1.2 g of succinic anhydride and stir at 50 °C for 1 h. Dialyze in deionized water for 4 days, changing the water every 4 h. Freeze dry to obtain modified polylysine. (4) Add 10 g of the modified polylysine obtained in step (3) to 500 mL of DMSO, add 0.8 g of CDI, stir at 60 °C for 4 h, cool to room temperature, add 1 g of DMAP and 3 g of resveratrol (RSV) in the dark, stir at room temperature for 24 h, dialyze in deionized water for 4 days, change the water every 4 h, freeze dry to obtain EPL-RSV; (5) Mix paraffin and Span 80 at a volume ratio of 49:1 to obtain an oil phase. Add 1 g of oxidized honeysuckle extract obtained in step (2), 1.25 g of EPL-RSV obtained in step (4), and 0.4 g of polyethylene glycol 4000 to 100 mL of PBS buffer at pH 6.5 and mix to obtain an aqueous phase. Add the aqueous phase dropwise to the oil phase with stirring. The volume ratio of the aqueous phase to the oil phase is 1:5. Stir at 1000 rpm for 30 min, stir at 300 rpm for 6 h, centrifuge at 5000 rpm for 12 min, wash the precipitate with petroleum ether and deionized water, vacuum dry, resuspend in PBS, filter through a 200 μm sieve, freeze dry, and obtain porous microspheres.

[0024] Example 2: A method for preparing human placental NK cells, comprising the following steps: S1. Placental tissue isolation: Under aseptic conditions, fresh placental tissue was collected, rinsed three times repeatedly with PBS buffer containing 1% penicillin-streptomycin, the amnion was peeled off, and the placental chorionic villus tissue was collected and cut into 1 mm pieces. 3 The fragments were washed twice with PBS buffer, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded to obtain placental tissue homogenate. S2. Placental tissue digestion: The placental tissue homogenate obtained in step S1 was digested with a compound enzyme solution. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum was immediately added to terminate the digestion. The mixture was filtered through a 200-mesh sieve, and the filtrate was collected. The cells were washed twice with PBS buffer, resuspended in PBS buffer, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 Cells were collected at a concentration of 100 cells / mL to obtain a cell suspension. The specific digestion process was as follows: placental tissue homogenate was mixed with compound enzyme solution at a volume ratio of 1:3, and placed in a constant temperature incubator at 37℃ and 5% CO2 for 30 min with shaking and mixing, with gentle pipetting every 10 min. The compound enzyme solution was serum-free DMEM medium containing type I collagenase, type IV collagenase, and DNase I. The final concentration of type I collagenase was 0.8 mg / mL, the final concentration of type IV collagenase was 0.3 mg / mL, and the final concentration of DNase I was 15 μg / mL. S3. Mononuclear cell isolation: The cell suspension obtained in step S2 is slowly stacked along the tube wall above the surface of an equal volume of human lymphocyte separation solution. Centrifuge at 4°C and 500 g for 20 min, aspirate the mononuclear cells in the middle white membrane layer, wash twice with PBS buffer, centrifuge at 4°C and 600 g for 8 min, discard the supernatant, and obtain placental mononuclear cells. S3. NK cell sorting: Placental mononuclear cells obtained in step S3 were collected and sorted using an NK cell negative selection kit and an immunomagnetic bead negative selection method to obtain CD56 cells. + CD3 - Placental NK cells; S4. NK cell expansion culture: Resuspend the NK cells obtained in step S3 in complete culture medium and adjust the cell concentration to 1×10⁻⁶. 6 Cells were seeded at a density of 1 × 10⁶ cells / mL into culture flasks pre-coated with 5 μg / mL CD16 antibody and incubated at 37°C in a 5% CO₂ incubator. Complete culture medium was added every 2 days to maintain a cell concentration of 1 × 10⁶ cells / mL. 6 Cells were cultured for 14 days and then collected. The complete culture medium consisted of Lonza X-VIVO 15 serum-free medium supplemented with IL-2 500 IU / mL, IL-15 20 ng / mL, IL-12 5 ng / mL, IL-18 10 ng / mL, IL-21 5 ng / mL, and porous microspheres 2 mg / mL.

[0025] The method for preparing the porous microspheres includes the following steps: (1) Wash and dry the honeysuckle, pulverize it through an 80-mesh sieve to obtain honeysuckle powder. Add 5 g of honeysuckle powder to 150 mL of deionized water at a material-to-liquid ratio of 1 g: 30 mL. Heat at 90°C for 2 h, filter and collect the filtrate. Add deionized water to the filter residue. Repeat the above operation twice. Combine the filtrates and concentrate them to 20% of the original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol, let stand at 4°C for 12 h, centrifuge at 5000 rpm for 15 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, deproteinize 5 times using the Sevage method, remove the Sevage solution under reduced pressure, dialyze in distilled water for 3 days, change the water every 8 h, freeze dry to obtain honeysuckle extract. (2) Dissolve 2 g of honeysuckle extract obtained in step (1) in 100 mL of deionized water, add 1 g of sodium periodate, stir at room temperature in the dark for 10 h, add 2 mL of ethylene glycol and stir for 30 min, dialyze with deionized water for 3 days, change the water every 8 h, freeze dry to obtain oxidized honeysuckle extract. (3) Add 12 g of ε-polylysine (EPL) to 120 mL of dimethyl sulfoxide (DMSO) and stir to dissolve. Add 1.5 g of succinic anhydride and stir at 50 °C for 1 h. Dialyze in deionized water for 4 days, changing the water every 4 h. Freeze dry to obtain modified polylysine. (4) Add 10 g of the modified polylysine obtained in step (3) to 1000 mL of DMSO, add 0.8 g of CDI, stir at 60℃ for 4 h, cool to room temperature, add 1 g of DMAP and 3 g of resveratrol (RSV) in the dark, stir at room temperature for 24 h, dialyze in deionized water for 4 days, change the water every 4 h, freeze dry to obtain EPL-RSV; (5) Mix paraffin and Span 80 at a volume ratio of 49:1 to obtain an oil phase. Add 1 g of oxidized honeysuckle extract obtained in step (2), 1.5 g of EPL-RSV obtained in step (4), and 0.5 g of polyethylene glycol 4000 to 100 mL of PBS buffer at pH 6.5 and mix to obtain an aqueous phase. Add the aqueous phase dropwise to the oil phase with stirring. The volume ratio of the aqueous phase to the oil phase is 1:3. Stir at 800 rpm for 30 min, stir at 300 rpm for 4 h, centrifuge at 5000 rpm for 8 min, wash the precipitate with petroleum ether and deionized water, vacuum dry, resuspend in PBS, filter through a 200 μm sieve, and freeze dry to obtain porous microspheres.

[0026] Example 3: A method for preparing human placental NK cells, comprising the following steps: S1. Placental tissue isolation: Under aseptic conditions, fresh placental tissue was collected and rinsed four times repeatedly with PBS buffer containing 1% penicillin-streptomycin. The amnion was then detached, and the placental chorionic villus tissue was collected and cut into 2 mm pieces. 3 The fragments were washed three times with PBS buffer, centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded to obtain placental tissue homogenate. S2. Placental tissue digestion: The placental tissue homogenate obtained in step S1 was digested with a compound enzyme solution. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum was immediately added to terminate the digestion. The mixture was filtered through a 200-mesh sieve, and the filtrate was collected. The cells were washed twice with PBS buffer, resuspended in PBS buffer, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7Cells were collected at a concentration of 1 / mL to obtain a cell suspension. The specific digestion process was as follows: placental tissue homogenate was mixed with compound enzyme solution at a volume ratio of 1:4, and placed in a constant temperature incubator at 37℃ and 5% CO2 for 35 min with shaking. The mixture was gently blown and mixed once every 10 min. The compound enzyme solution was serum-free DMEM medium containing type I collagenase, type IV collagenase, and DNase I. The final concentration of type I collagenase was 1 mg / mL, the final concentration of type IV collagenase was 0.5 mg / mL, and the final concentration of DNase I was 20 μg / mL. S3. Mononuclear cell isolation: The cell suspension obtained in step S2 is slowly stacked along the tube wall above the surface of an equal volume of human lymphocyte separation solution. Centrifuge at 4°C and 500 g for 20 min, aspirate the mononuclear cells in the middle white membrane layer, wash twice with PBS buffer, centrifuge at 4°C and 600 g for 8 min, discard the supernatant, and obtain placental mononuclear cells. S3. NK cell sorting: Placental mononuclear cells obtained in step S3 were collected and sorted using an NK cell negative selection kit and an immunomagnetic bead negative selection method to obtain CD56 cells. + CD3 - Placental NK cells; S4. NK cell expansion culture: Resuspend the NK cells obtained in step S3 in complete culture medium and adjust the cell concentration to 1×10⁻⁶. 6 Cells were seeded at a density of 1.2 × 10⁶ cells / mL into culture flasks pre-coated with 8 μg / mL CD16 antibody and incubated at 37°C in a 5% CO₂ incubator. Complete culture medium was added every 3 days to maintain a cell concentration of 1.2 × 10⁶ cells / mL. 6 Cells were cultured for 14 days and then collected. The complete culture medium consisted of Lonza X-VIVO 15 serum-free medium supplemented with IL-2 600 IU / mL, IL-15 30 ng / mL, IL-12 7 ng / mL, IL-18 15 ng / mL, IL-21 7 ng / mL, and porous microspheres 2.5 mg / mL.

[0027] The method for preparing the porous microspheres includes the following steps: (1) Wash and dry the honeysuckle, pulverize it through a 90-mesh sieve to obtain honeysuckle powder. Add 5 g of honeysuckle powder to 150 mL of deionized water at a material-to-liquid ratio of 1 g: 30 mL. Heat at 90°C for 2 h, filter and collect the filtrate. Add deionized water to the filter residue. Repeat the above operation twice. Combine the filtrates and concentrate them to 20% of the original volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol, let stand at 4°C for 14 h, centrifuge at 5000 rpm for 18 min, wash the precipitate with anhydrous ethanol, reconstitute with distilled water, deproteinize 5 times using the Sevage method, remove the Sevage solution under reduced pressure, dialyze in distilled water for 3 days, change the water every 8 h, freeze dry to obtain honeysuckle extract. (2) Dissolve 2.5 g of honeysuckle extract obtained in step (1) in 100 mL of deionized water, add 1 g of sodium periodate, stir at room temperature in the dark for 11 h, add 2 mL of ethylene glycol and stir for 30 min, dialyze with deionized water for 3 days, change the water every 8 h, freeze dry to obtain oxidized honeysuckle extract. (3) 12 g of ε-polylysine (EPL) was added to 120 mL of dimethyl sulfoxide (DMSO) and stirred to dissolve. 1.4 g of succinic anhydride was added and stirred at 50 °C for 1 h. The mixture was dialyzed in deionized water for 4 days, with the water changed every 4 h. The mixture was then freeze-dried to obtain modified polylysine. (4) Add 10 g of the modified polylysine obtained in step (3) to 750 mL of DMSO, add 0.8 g of CDI, stir at 60 °C for 4 h, cool to room temperature, add 1 g of DMAP and 3 g of resveratrol (RSV) in the dark, stir at room temperature for 24 h, dialyze in deionized water for 4 days, change the water every 4 h, freeze dry to obtain EPL-RSV; (5) Mix paraffin and Span 80 at a volume ratio of 49:1 to obtain an oil phase. Add 1 g of oxidized honeysuckle extract obtained in step (2), 1.5 g of EPL-RSV obtained in step (4), and 0.5 g of polyethylene glycol 4000 to 100 mL of PBS buffer at pH 6.5 and mix to obtain an aqueous phase. Add the aqueous phase dropwise to the oil phase with stirring. The volume ratio of the aqueous phase to the oil phase is 1:4. Stir at 900 rpm for 30 min, stir at 300 rpm for 5 h, centrifuge at 5000 rpm for 10 min, wash the precipitate with petroleum ether and deionized water, vacuum dry, resuspend in PBS, filter through a 200 μm sieve, and freeze dry to obtain porous microspheres.

[0028] The only difference between Comparative Example 1 and Example 1 is that IL-12, IL-18 and IL-21 are not added to the complete culture medium; all other operations are the same as in Example 1.

[0029] The only difference between Comparative Example 2 and Example 1 is that polylysine is used instead of EPL-RSV; all other operations are the same as in Example 1.

[0030] The only difference between Comparative Example 3 and Example 1 is that dextran was used instead of honeysuckle extract; all other operations were the same as in Example 1.

[0031] The only difference between Comparative Example 4 and Example 1 is that porous microspheres are not added; all other operations are the same as in Example 1.

[0032] Experimental Example 1: The porous microspheres prepared in Example 1 were observed and photographed using a scanning electron microscope. The results are as follows: Figure 1 As shown.

[0033] Figure 1 The results showed that the porous microspheres prepared by this invention have a rich pore structure, which can ensure the space for normal cell proliferation and provide NK cells with a 3D growth microenvironment that mimics the lymph nodes in vivo, thereby effectively improving cell activity.

[0034] Experimental Example 2: After amplification in Examples 1-3 and Comparative Examples 1-4, cells were collected and counted. The fold increase after 14 days of culture was statistically analyzed. The results are as follows: Figure 2 As shown.

[0035] Figure 2 The results showed that the NK cell expansion fold in Examples 1-3 was significantly better than that in Comparative Examples 1-4. In Comparative Example 1, the absence of cytokines IL-12, IL-18, and IL-21 in the culture medium resulted in decreased cell growth rate. In Comparative Example 2, the use of polylysine instead of EPL-RSV, the lack of carboxyl groups on the polylysine surface, and the absence of resveratrol grafting all contributed to decreased cell growth rate. In Comparative Example 3, the substitution of honeysuckle extract with dextran resulted in decreased cell expansion rate. In Comparative Example 4, the absence of porous microspheres reduced the cell expansion fold. These findings indicate that the placental-derived NK cell preparation method of the present invention can effectively promote NK cell expansion and improve cell yield.

[0036] Experimental Example 3: Using K562 cells (human chronic myeloid leukemia cell line) as target cells, and NK cells expanded from Examples 1-3 and Comparative Examples 1-4 as effector cells, the target cells were arranged at a density of 5 × 10⁻⁶ cells / cells. 3 100 μL of effector cells were seeded per well in a 96-well plate at a ratio of 10:1 to target cells. CCK-8 was added simultaneously. The plates were incubated at 37°C with 5% CO2 for 4 hours. Cell killing efficiency was calculated by reading the values ​​at 450 nm. Results are shown below. Figure 3 As shown.

[0037] Figure 3The results showed that the NK cell killing efficiency of Examples 1-3 was significantly better than that of Comparative Examples 1-4. Comparative Example 1 did not add cytokines IL-12, IL-18, and IL-21 to the culture medium, resulting in decreased killing activity. Comparative Example 2 used polylysine instead of EPL-RSV, did not introduce carboxyl groups onto the polylysine surface, and did not graft resveratrol, leading to decreased cell killing activity. Comparative Example 3 used dextran instead of honeysuckle extract, resulting in a decreased killing rate. Comparative Example 4 did not add porous microspheres, resulting in reduced cell killing activity. These findings indicate that the NK cells prepared according to this invention have high activity.

[0038] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for preparing human placental NK cells, characterized in that, Includes the following steps: S1. Placental tissue separation: Take placental tissue, cut it into small pieces, centrifuge it, discard the supernatant, and obtain placental tissue homogenate; S2. Placental tissue digestion: The placental tissue homogenate obtained in step S1 is digested and resuspended to obtain a cell suspension; S3. Mononuclear cell isolation: The cell suspension obtained in step S2 is separated using lymphocyte separation solution to obtain placental mononuclear cells; S3. NK cell sorting: Placental mononuclear cells obtained in step S3 are sorted for NK cell selection to obtain CD56. + CD3 - Placental NK cells; S4. NK cell expansion culture: The NK cells obtained in step S3 were resuspended in complete culture medium, seeded into CD16 antibody-coated culture flasks, and complete culture medium was added. The complete culture medium is prepared by adding IL-2 500-800 IU / mL, IL-15 20-40 ng / mL, IL-12 5-10 ng / mL, IL-18 10-20 ng / mL, IL-21 5-10 ng / mL, and porous microspheres 2-3 mg / mL to the basal medium Lonza X-VIVO 15 serum-free medium. The method for preparing the porous microspheres includes the following steps: (1) Wash, dry and pulverize honeysuckle, add it to deionized water, heat to extract, concentrate, precipitate with alcohol, deproteinize by Sevage method, dialyze, freeze dry to obtain honeysuckle extract. (2) Dissolve the honeysuckle extract obtained in step (1) in deionized water, add sodium periodate, stir, add ethylene glycol, dialyze, freeze dry to obtain oxidized honeysuckle extract. (3) Add ε-polylysine to DMSO, add succinic anhydride, stir, dialyze, freeze dry to obtain modified polylysine; (4) Add the modified polylysine obtained in step (3) to DMSO, add CDI, stir, add DMAP and resveratrol, stir, dialyze, freeze dry to obtain EPL-RSV; (5) Mix paraffin with Span 80 to obtain an oil phase. Add the honeysuckle extract obtained in step (2), EPL-RSV obtained in step (4), and polyethylene glycol to PBS buffer and mix to obtain an aqueous phase. Add the aqueous phase to the oil phase, stir, centrifuge, precipitate, wash, dry, filter, and freeze dry to obtain porous microspheres.

2. The method for preparing human placental-derived NK cells according to claim 1, characterized in that, In step S2, the composite enzyme solution is a serum-free DMEM medium containing type I collagenase, type IV collagenase, and DNase I.

3. The method for preparing human placental-derived NK cells according to claim 2, characterized in that, In step (2), the mass ratio of sodium periodate to honeysuckle extract is 1:2-3.

4. The method for preparing human placental-derived NK cells according to claim 3, characterized in that, In step (3), the mass ratio of polylysine to succinic anhydride is 8-10:

1.

5. The method for preparing human placental-derived NK cells according to claim 4, characterized in that, In step (4), the mass ratio of the modified polylysine, CDI, DMAP and resveratrol is 10:0.8:1:

3.

6. The method for preparing human placental-derived NK cells according to claim 5, characterized in that, In step (5), the mass ratio of the oxidized honeysuckle extract, EPL-RSV and polyethylene glycol 4000 is 2-4:3-5:1-2.

7. The method for preparing human placental-derived NK cells according to claim 6, characterized in that, In step (5), the volume ratio of the aqueous phase to the oil phase is 1:3-5.

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