A method for culturing peripheral blood NK cells in vitro

Abstract

This invention relates to the field of immune cell culture technology, and particularly to a method for in vitro culture of peripheral blood NK cells. The invention involves sequentially performing a first culture and a second culture on in vitro cultured peripheral blood NK cells. In the first culture stage, star anise extract and IL-2 are used. The active components in the star anise extract synergistically interact with IL-2 to induce an increase in NK cell activity. Furthermore, in the second culture stage, epimedium extract is used to culture the NK cells, which improves the NK cell environment and promotes rapid NK cell proliferation. The entire process requires no additional treatment and is simple. Therefore, the technical solution of this invention not only enhances the proliferation capacity of NK cells but also prolongs their original cytotoxic activity.

Description

Technical Field

[0001] This invention relates to the field of immune cell culture technology, and in particular to a method for in vitro culture of peripheral blood NK cells. Background Technology

[0002] Natural killer (NK) cells are part of the innate immune system. Unlike T cells and B cells, they do not require antigen-specific receptors to recognize pathogens or tumor cells, offering new ideas and methods for the treatment of various diseases, such as tumors, inflammatory diseases, and drug hypersensitivity reactions. However, during in vitro culture of NK cells, environmental damage reduces their proliferative capacity and makes it difficult to maintain their original viability, severely limiting their use.

[0003] Existing research has found that certain traditional Chinese medicines (TCMs) can enhance the resilience of NK cells. For example, Chinese patent CN118995603 B describes a TCM culture medium and a method for in vitro expansion of NK cells. This method involves a specific ratio and extraction of five herbs: Sanghuang (Phellinus linteus), Echinacea purpurea, Astragalus membranaceus, Ganoderma lucidum (Reishi mushroom), and Polygonum multiflorum (He Shou Wu). The resulting TCM extract can improve the NK cell environment, enhance NK cell activity, promote NK cell proliferation, and kill tumor cells. However, this method requires a large amount of TCM raw materials and necessitates cyclical irradiation with 433MHz microwaves for 280 seconds and 2450MHz microwaves for 70 seconds during cell culture, with a 6-hour interval between the two irradiations, to further enhance the efficacy of the TCM extract. This presents a certain obstacle to practical application. Summary of the Invention

[0004] In view of this, the present invention proposes a method for in vitro culture of peripheral blood NK cells, which uses fewer types of Chinese herbal raw materials and has a simple culture process.

[0005] This invention provides a method for in vitro culture of peripheral blood NK cells, comprising the following steps: NK cells were seeded into the first culture medium for the first culture to obtain induced NK cells; The induced NK cells were seeded into a second culture medium for a second culture to obtain in vitro cultured NK cells; The first culture medium includes: star anise extract and IL-2; The second culture medium includes: Epimedium extract.

[0006] Preferably, during the first culture, the seeding density of the NK cells is ≥1×10⁻⁶. 6 cells / mL.

[0007] Preferably, the first culture medium is based on RPMI-1640 medium, and 260-300 IU / mL of IL-2 and 0.1-0.5 g / mL of star anise extract are added to the basic culture medium. The preparation method of the star anise extract includes: crushing star anise, mixing and soaking it with water, heating and extracting it, concentrating it under reduced pressure, and freeze-drying it to obtain the star anise extract.

[0008] Preferably, the ratio of star anise to water is 1g:20-30mL; The heating extraction temperature is 90-95℃, and the heating extraction time is 30-40 minutes.

[0009] Preferably, the second culture medium is based on RPMI-1640 medium, and 1.5-2.5 g / mL of Epimedium extract is added to the basic medium; The preparation method of the Epimedium extract includes: pulverizing Epimedium and mixing it with an ethanol solution for extraction, concentrating under reduced pressure and freeze-drying to obtain the Epimedium extract; The volume concentration of the ethanol solution is 45%-55%.

[0010] Preferably, during the mixed extraction, the ratio of Epimedium to ethanol solution is 1g:8-10mL; The temperature for the mixed extraction is 58-62℃, and the extraction time is 1.5-2 hours.

[0011] Preferably, the conditions for the first culture include: a temperature of 36-38℃ and a time of 30-48h.

[0012] Preferably, the conditions for the second culture include: a temperature of 36-38℃ and a time of 10-13 days.

[0013] Preferably, during the second culture period, the nutrient solution is replenished once every 2 days; The nutrient solution uses water as a solvent and contains 1-3 mg / mL of glucose and 0.5-0.8 mg / mL of Epimedium extract.

[0014] This invention provides the application of the method described in the above technical solution in improving the proliferation capacity and / or prolonging the killing activity of NK cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for in vitro culture of peripheral blood NK cells, comprising the following steps: seeding NK cells in a first culture medium for a first culture to obtain induced NK cells; seeding the induced NK cells in a second culture medium for a second culture to obtain in vitro cultured NK cells; the first culture medium includes: star anise extract and IL-2; the second culture medium includes: epimedium extract.

[0016] This invention involves sequentially culturing peripheral blood NK cells in vitro using a first culture and a second culture. In the first culture stage, star anise extract and IL-2 are used. The active components in the star anise extract synergistically interact with IL-2 to induce increased NK cell activity. Furthermore, the second culture stage uses epimedium extract to culture NK cells, which improves the NK cell environment and promotes rapid NK cell proliferation. The entire process requires no additional treatment and is simple. Therefore, the technical solution of this invention not only enhances NK cell proliferation but also prolongs the original cytotoxic activity of NK cells. Detailed Implementation

[0017] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0018] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0019] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0020] The Epimedium mentioned in this invention is Epimedium sagittatum (Sagittaria sagittatum). Epimedium sagittatum (Sieb. and Zucc.) Maximus The star anise mentioned is white-flowered star anise (.); Illicium philippinense Merr. ).

[0021] All of the reagents can be replaced by other commercially available products of equivalent specifications and functions known in the art.

[0022] Example 1 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium After crushing the star anise, pass it through a 50-mesh sieve to obtain star anise powder. The star anise powder was mixed with water at a ratio of 1g:20mL and soaked at 28℃ for 2h. Then, it was heated at 95℃ for 40min for extraction. The heated mixture was separated into solid and liquid components, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain star anise extract. First culture medium: RPMI-1640 medium was used as the basal medium, with 260 IU / mL of IL-2 and 0.1 g / mL of star anise extract added to the basal medium.

[0023] (2) Preparation of the second culture medium The leaves of Epimedium are crushed and passed through a 50-mesh sieve to obtain Epimedium powder. The powdered Epimedium was mixed with a 50% ethanol solution at a ratio of 1 g: 10 mL, and then extracted at 58 °C for 1.5 h. After solid-liquid separation, the filtrate was concentrated under reduced pressure and freeze-dried to obtain Epimedium extract. Second culture medium: RPMI-1640 medium was used as the basal medium, and 1.5 g / mL of Epimedium extract was added to the basal medium. (3) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6 NK cells were cultured at 37°C and 5% CO2 for 40 h to obtain induced NK cells. The induced NK cells were then seeded in a second culture medium (ensuring a seeding density of 1×10⁻⁶ cells / mL). 6 (cells / mL) were cultured at 37°C and 5% CO2 for 12 days. During the second culture, nutrient solution was added every 2 days.

[0024] The nutrient solution uses water as a solvent and contains 1 mg / mL of glucose and 0.5 mg / mL of epimedium extract. The preparation method of epimedium extract is the same as that of epimedium extract in step (2) above.

[0025] Comparative Example 1 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium After crushing the star anise, pass it through a 50-mesh sieve to obtain star anise powder. The star anise powder was mixed with water at a ratio of 1g:20mL and soaked at 28℃ for 2h. Then, it was heated at 95℃ for 40min for extraction. The heated mixture was separated into solid and liquid components, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain star anise extract. First culture medium: RPMI-1640 medium was used as the basal medium, with 260 IU / mL of IL-2 and 0.1 g / mL of star anise extract added to the basal medium.

[0026] (2) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6(cells / mL) were cultured at 37°C and 5% CO2 for 14 days, with nutrient solution added every 2 days during the culture process.

[0027] The nutrient solution uses water as a solvent and contains 1 mg / mL of glucose and 0.1 mg / mL of star anise extract; the preparation method of the star anise extract is the same as that in step (1) above.

[0028] Comparative Example 2 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the second culture medium The leaves of Epimedium are crushed and passed through a 50-mesh sieve to obtain Epimedium powder. The powdered Epimedium was mixed with a 50% ethanol solution at a ratio of 1 g: 10 mL, and then extracted at 58 °C for 1.5 h. After solid-liquid separation, the filtrate was concentrated under reduced pressure and freeze-dried to obtain Epimedium extract. Second culture medium: RPMI-1640 medium was used as the basal medium, and 1.5 g / mL of Epimedium extract was added to the basal medium. (2) In vitro culture NK cells were seeded in the second culture medium (ensuring a seeding density of 1×10⁶ NK cells). 6 (cells / mL) were cultured at 37°C and 5% CO2 for 14 days, with nutrient solution added every 2 days during the culture process.

[0029] The nutrient solution uses water as a solvent and contains 1 mg / mL of glucose and 0.1 mg / mL of star anise extract. The preparation method of the epimedium extract is the same as that of the epimedium extract in step (2) above.

[0030] Comparative Example 3 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium The same as step (1) in Example 1.

[0031] (2) Preparation of the second culture medium The same as step (2) in Example 1.

[0032] (3) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6 NK cells were cultured at 37°C and 5% CO2 for 40 h to obtain induced NK cells. The induced NK cells were then seeded in a second culture medium (ensuring a seeding density of 1×10⁻⁶ cells / mL).6 The culture medium (cells / mL) was cultured at 37°C and 5% CO2 for 12 days. During the second culture, fresh second culture medium was added every 2 days.

[0033] Comparative Example 4 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium After crushing the star anise, pass it through a 50-mesh sieve to obtain star anise powder. The star anise powder was mixed with water at a ratio of 1g:20mL and soaked at 28℃ for 2h. Then, it was heated at 95℃ for 40min for extraction. The heated mixture was separated into solid and liquid components, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain star anise extract. First culture medium: RPMI-1640 medium was used as the basal medium, with 260 IU / mL of IL-2 and 0.1 g / mL of star anise extract added to the basal medium.

[0034] (2) Preparation of the second culture medium Second culture medium: RPMI-1640 medium was used as the basal medium, and 1.5 μg / mL of icariin was added to the basal medium.

[0035] (3) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6 NK cells were cultured at 37°C and 5% CO2 for 40 h to obtain induced NK cells. The induced NK cells were then seeded in a second culture medium (ensuring a seeding density of 1×10⁻⁶ cells / mL). 6 The culture medium (cells / mL) was cultured at 37°C and 5% CO2 for 12 days. During the second culture, fresh second culture medium was added every 2 days.

[0036] Comparative Example 5 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium Ganoderma lucidum ( Ganoderma lucidum (Curtis) P. Karst. By replacing the star anise in step (1) of Example 1, Ganoderma lucidum extract was prepared; The leaves of Epimedium are crushed and passed through a 50-mesh sieve to obtain Epimedium powder. The powdered Epimedium was mixed with a 50% ethanol solution at a ratio of 1 g: 10 mL, and then extracted at 58 °C for 1.5 h. After solid-liquid separation, the filtrate was concentrated under reduced pressure and freeze-dried to obtain Epimedium extract. First culture medium: RPMI-1640 medium was used as the basal medium, with 260 IU / mL IL-2, 0.1 g / mL Ganoderma lucidum extract and 1.5 g / mL Epimedium extract added to the basal medium. (2) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6 The culture medium (cells / mL) was cultured at 37°C and 5% CO2 for 14 days. During the second culture, fresh first culture medium was added every 2 days.

[0037] Comparative Example 6 A method for in vitro culture of peripheral blood NK cells, comprising the following steps: (1) Preparation of the first culture medium First culture medium: RPMI-1640 medium was used as the basal medium, with 260 IU / mL of IL-2 added to the basal medium.

[0038] (2) Preparation of the second culture medium Second culture medium: RPMI-1640 medium; (3) In vitro culture NK cells were seeded in the first culture medium (ensuring a seeding density of 1 × 10⁶ cells / mL). 6 NK cells were cultured at 37°C and 5% CO2 for 40 h to obtain induced NK cells. The induced NK cells were then seeded in a second culture medium (ensuring a seeding density of 1×10⁻⁶ cells / mL). 6 The culture medium (cells / mL) was cultured at 37°C and 5% CO2 for 12 days. During the second culture, fresh second culture medium was added every 2 days.

[0039] Application Example 1 1. Amplification fold detection After the culture was completed, the culture flasks of Example 1 and Comparative Examples 1-5 were gently shaken to ensure that the substances in the culture flasks were mixed evenly. The cell suspension in each culture flask was aspirated and counted. The cells were counted using trypan blue staining and the live cells were counted using a hemocytometer (excluding dead cells stained by blue). Each treatment was set up with 3 replicates and the average value was taken to calculate the amplification factor (the result was only retained to the integer part). Fold of expansion = Nt / N0 (Initial NK cell seeding number is N0, total number of cells collected after culture is N) t The results are shown in Table 1.

[0040] Table 1. Fold increase of NK cells in different treatments

[0041] As can be seen from the data in Table 1, the expansion fold of NK cells was significantly increased after the first and second cultures using star anise extract and epimedium extract. Compared with Comparative Examples 1-6, the expansion fold of NK cells in Example 1 was 450-fold, while the highest expansion fold in the comparative examples was only 187-fold.

[0042] Application Example 2 Preparation before the experiment: Take K562 cells in the logarithmic growth phase (K562 cell concentration is 1×10⁻⁶). 5 NK cells (NK cell concentration of 1×10⁶ / mL) were used as target cells in Example 1 and Comparative Examples 1-6, respectively. 6 (cells / mL) were used as effector cells, ensuring that the effector cells and target cells were seeded at an effector-to-target ratio of 10:1 into 96-well plates and mixed thoroughly to form the test group. The test group consisted of 100 μL each of effector cells and target cells; The effector group consisted of 100 μL each of effector cells and RPMI 1640 medium. The target cell group consisted of 100 μL each of target cells and RPMI 1640 medium. The final volume of each well was 200 μL, and each group had 3 replicates. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. 10 μL of LTT solution was added to each well and incubated for 4 h. At the end of the incubation, the OD value at 570 nm was measured using a microplate reader, and the killing rate was calculated to evaluate the cell killing rate (%), accurate to the integer place. Kill rate = [OD value of target cell group - (OD value of test group - OD value of effector group) / OD value of target cell group] × 100%, the results are shown in Table 2.

[0043] Table 2. Kill rate of NK cells in different treatments

[0044] As can be seen from the data in Table 2, the killing rate of NK cells in Example 1 was as high as 89%.

[0045] After cryopreserving the NK cells cultured in vitro in Example 1 and Comparative Example 3 for 4 days, the NK cells were revived in a water bath at 37°C to obtain reviving NK cells. The reviving NK cells were used as the test object, and the above-mentioned killing rate detection method was used to evaluate the killing rate (%) of the reviving NK cells, accurate to the integer place. The results are shown in Table 3.

[0046] Table 3 Kill rate of NK cells after resuscitation in different treatments

[0047] As can be seen from the data in Table 3, after NK cells are cultured in vitro using the technical solution provided by this invention, even after being frozen for 4 days and then thawed, the cells can maintain high cytotoxic activity. In other words, the technical solution of this application is beneficial to prolonging the cytotoxic rate (i.e., cytotoxic activity) of NK cells.

[0048] In summary, this invention involves sequentially performing a first culture and a second culture on peripheral blood NK cells cultured in vitro. During the first culture stage, star anise extract and IL-2 are used. The active components in the star anise extract synergistically interact with IL-2 to induce an increase in NK cell activity. Furthermore, the second culture stage uses epimedium extract to culture NK cells, which not only improves the NK cell environment but also promotes rapid NK cell proliferation. The entire process requires no additional treatment and is simple. Therefore, the technical solution of this invention not only enhances NK cell proliferation but also prolongs the original cytotoxic activity of NK cells.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro culture of peripheral blood NK cells, characterized in that, Includes the following steps: NK cells were seeded into the first culture medium for the first culture to obtain induced NK cells; The induced NK cells were seeded into a second culture medium for a second culture to obtain in vitro cultured NK cells; The first culture medium includes: star anise extract and IL-2; The second culture medium includes: Epimedium extract.

2. The method according to claim 1, characterized in that, During the first culture, the seeding density of the NK cells was ≥1×10⁻⁶. 6 cells / mL.

3. The method according to claim 1, characterized in that, The first culture medium was based on RPMI-1640 medium, with 260-300 IU / mL of IL-2 and 0.1-0.5 g / mL of star anise extract added to the basic medium; The preparation method of the star anise extract includes: crushing star anise, mixing and soaking it with water, heating and extracting it, concentrating it under reduced pressure, and freeze-drying it to obtain the star anise extract.

4. The method according to claim 3, characterized in that, The ratio of star anise to water is 1g:20-30mL; The heating extraction temperature is 90-95℃, and the heating extraction time is 30-40 minutes.

5. The method according to claim 1, characterized in that, The second culture medium was based on RPMI-1640 medium, with 1.5-2.5 g / mL of Epimedium extract added to the medium; The preparation method of the Epimedium extract includes: pulverizing Epimedium and mixing it with an ethanol solution for extraction, concentrating under reduced pressure and freeze-drying to obtain the Epimedium extract; The volume concentration of the ethanol solution is 45%-55%.

6. The method according to claim 5, characterized in that, When performing mixed extraction, the ratio of Epimedium to ethanol solution is 1g:8-10mL. The temperature for the mixed extraction is 58-62℃, and the extraction time is 1.5-2 hours.

7. The method according to claim 1, characterized in that, The conditions for the first culture include: a temperature of 36-38℃ and a time of 30-48h.

8. The method according to claim 1, characterized in that, The conditions for the second culture include a temperature of 36-38℃ and a time of 10-13 days.

9. The method according to claim 1 or 8, characterized in that, During the second culture period, nutrient solution was added once every 2 days; The nutrient solution uses water as a solvent and contains 1-3 mg / mL of glucose and 0.5-0.8 mg / mL of Epimedium extract.

10. The use of the method according to any one of claims 1-9 in enhancing NK cell proliferation and / or prolonging cytotoxic activity.

Images