Method and culture medium for in-vitro culture and amplification of NK cells

Abstract

The invention discloses a method for culturing and amplifying NK (Natural Killer) cells in vitro, which comprises the following steps: adding mononuclear cells into an activation culture medium, then adding the mononuclear cells into a culture flask which is coated with a coating solution in advance, putting the culture flask into an incubator, culturing for 16-22 days under the conditions of 37 DEG C and 5% CO2, and harvesting the NK cells, wherein the culture medium is supplemented and activated on the fifth day of culture; and from the sixth day of culture, supplementing the amplification culture medium once every two days. The invention further discloses a culture medium for culturing the NK cells. The culture medium comprises an activation culture medium and a supplementary amplification culture medium. According to the method for culturing and amplifying the NK cells in vitro, amplification of more than 3000 times can be achieved, the purity of the NK cells is larger than 90% during harvesting, the cell viability is larger than 90%, and the NK cells have good in-vitro killing activity after being co-incubated with the K562 cells. According to the culture method, large-scale amplification of the NK cells can be realized, and a low-cost and high-safety technical solution is provided for the field of immune cell therapy.

Description

TECHNICAL FIELD

[0001] The application relates to a method for culturing and expanding NK cells in vitro and a culture medium, and belongs to the technical field of NK cell culture. BACKGROUND

[0002] Natural killer cells (NK cells) as the core effector cells of the innate immune system play an important role in tumor immune surveillance and clearance, and the in vitro expansion technology of NK cells is a research hotspot in the field of immune cell therapy. At present, the in vitro culture of NK cells by factor method is one of the mainstream technologies in this field, and the core is to simulate the in vivo microenvironment by adding various cytokines (such as IL-2, IL-15, IL-18, IL-21, etc.) in the culture medium to induce NK cell proliferation and maintain its killing activity.

[0003] The cytokines (such as IL-2, IL-15) used in the factor method are naturally occurring immunomodulatory molecules in the human body, and do not need to introduce heterologous components (such as tumor cell lines or viral vectors), so the safety is significantly better than that of the feeder layer cell culture method. For example, after peripheral blood or umbilical cord blood-derived NK cells are cultured by pure factors, the product is more likely to pass the regulatory review when clinically declared, and there will be no safety controversy caused by the potential tumorigenicity of the feeder layer cells. In addition, NK cells cultured by the factor method rarely cause graft-versus-host disease (GVHD) and cytokine release syndrome (CRS) when used in vivo, and its safety has been verified in clinical trials of liver cancer, colon cancer and other solid tumors. Reasonable cytokine combination can precisely regulate the proliferation and activation of NK cells. The culture process of the factor method can be highly standardized, and through optimization of the culture medium formula (such as serum-free culture medium) and automation equipment, the batch-to-batch stability can be realized.

[0004] Although the factor method has made certain progress in the in vitro culture of NK cells, its technical bottlenecks are still prominent, and there are the following deficiencies:

[0005] (1) High culture cost: the preparation process of recombinant cytokines (especially NKp44, IL-15, etc.) is complex and expensive, and the cost increases significantly in large-scale culture, which limits the economic efficiency of clinical application.

[0006] (2) High dependence on exogenous components: the traditional culture system needs to add various cytokines and serum and other complex components, which not only increases the heterogeneity risk (such as immunogenicity reaction) of the culture system, but also may introduce potential biological safety hazards (such as pathogenic microorganism contamination).

[0007] (3) Expansion efficiency and function maintenance conflict: In the existing method, feeder cells (such as irradiated K562 cell line) are often used for co-culture to provide auxiliary signals when pursuing cell expansion times, but the introduction of feeder cells leads to the complication of the culture system, and there is a risk of residual contamination of heterologous cells, and the killing activity of NK cells in long-term culture is prone to decline.

[0008] (4) Complicated operation process: The preparation, irradiation treatment and co-culture process of feeder cells and NK cells increase the difficulty of quality control in vitro culture, and it is difficult to meet the needs of standardized production.

[0009] In summary, the existing factor method for in vitro culture of NK cells has problems such as high cost, complex exogenous components, and dependence on feeder cell co-culture, and it is urgent to develop a low-cost, low-exogenous intervention and feeder cell-free auxiliary culture system without autologous plasma to break through the bottleneck of existing technology in NK cell large-scale expansion and clinical application. SUMMARY

[0010] In view of the above existing technology, the present application provides a method for in vitro culture and expansion of NK cells and a culture medium.

[0011] The present application is realized by the following technical solutions:

[0012] A method for in vitro culture and expansion of NK cells, comprising the following steps: adding mononuclear cells (MNCs) into an activation culture medium, controlling the cell density at 1.5x10 6 ~2.0x10 6 cells / mL, then adding into a culture flask coated with a coating solution, and placing in a culture box for culture at 37℃ and 5% CO2 for 16-22 days to harvest NK cells; wherein, the activation culture medium is supplemented on the 5th day of culture; and the expansion culture medium is supplemented every two days to maintain the cell density at 1.0x10 6 ~1.5x10 6 cells / mL from the 6th day of culture;

[0013] The activation culture medium is prepared by the following method: adding 1% (volume percentage) GlutaMAX TM , an appropriate amount of OK432 to make the final concentration 50ng / mL, an appropriate amount of IL-2 to make the final concentration 300ng / mL, an appropriate amount of IL-18 to make the final concentration 25ng / mL, and an appropriate amount of IL-21 to make the final concentration 10ng / mL into the NK cell serum-free culture medium, and mixing well to obtain the activation culture medium.

[0014] The supplement expansion culture medium is prepared by the following method: adding 1% (volume percentage) GlutaMAXTM and an appropriate amount of IL-2 to make the final concentration 200 ng / mL, and mix well.

[0015] Further, the additional activation medium is equal to the initial activation medium.

[0016] Further, the NK cell serum-free medium is selected from KBM 581 medium, which is a commercially available medium.

[0017] Further, the coating solution is a 5 μg / mL CD16 antibody solution.

[0018] A medium for culturing NK cells, comprising an activation medium and a supplementary expansion medium; the activation medium is prepared by adding 1% (volume percentage) GlutaMAX TM , an appropriate amount of OK432 to make the final concentration 50 ng / mL, an appropriate amount of IL-2 to make the final concentration 300 ng / mL, an appropriate amount of IL-18 to make the final concentration 25 ng / mL, and an appropriate amount of IL-21 to make the final concentration 10 ng / mL, and mix well; and the supplementary expansion medium is prepared by adding 1% (volume percentage) GlutaMAX TM and an appropriate amount of IL-2 to make the final concentration 200 ng / mL, and mix well.

[0019] The medium for culturing NK cells is used for in-vitro culturing and expanding NK cells.

[0020] The GlutaMAX TM is a commercially available cell culture additive, which is a stable dipeptide form of L-glutamine, and can be purchased on the market; the OK432 is a bacterial preparation made from hemolytic streptococcus Su strain treated with penicillin and freeze-dried, which is a commercially available drug and can be purchased on the market.

[0021] The method for in-vitro culture and expansion of NK cells of the present application is suitable for in-vitro expansion of NK cells from human mononuclear cells (MNCs), and the NK cells can be expanded by more than 3000 times in a large system culture (cell culture bag) for 16-22 days; and the purity of the NK cells is greater than 90% at the time of harvesting, the cell viability is greater than 90%, and the in-vitro killing activity is good when co-incubated with K562 cells (E:T = 1:1 > 60%, E:T = 2:1 > 80%). The method for in-vitro culture and expansion of NK cells of the present application does not need to rely on autologous plasma and feeder cells, widens the range of in-vitro culture of NK cells, enhances the expansion and in-vitro killing performance of NK cells, reduces the culture cost, simplifies the culture operation, and reduces the risk of cell contamination in the culture process. The culture cost is greatly reduced.

[0022] The present application constructs a culture microenvironment with low exogenous component intervention through the optimized combination of specific antibodies, cytokines, inactivated bacterins and basic additives. Compared with the existing factor culture system, the present application significantly reduces the culture cost and the use of exogenous biological components while ensuring the high expansion capacity and tumor killing activity of NK cells. The matching in-vitro culture method discards the traditional feeder cell co-culture mode, and effectively solves the technical problems of low in-vitro expansion efficiency of NK cells, dependence on feeder cell assisted culture and detection of residual feeder cells in the prior art by precisely regulating the concentration ratio of each component in the culture system and the culture conditions. The culture method of the present application can realize the large-scale expansion of NK cells, and provides a low-cost and high-safety technical solution for the field of immune cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Purity flow cytometry detection results of NK cells (Day 0), wherein the detection results of Example 1, Example 2, Example 3 and Example 4 are sequentially arranged from left to right and from top to bottom.

[0024] Figure 2 : Purity flow cytometry detection results of NK cells (Day 18), wherein the detection results of Example 1, Example 2, Example 3 and Example 4 are sequentially arranged from left to right and from top to bottom.

[0025] Figure 3 : Viability detection results of NK cells.

[0026] Figure 4 : Expansion fold of NK cells.

[0027] Figure 5 : Killing experiment results of NK cells. DETAILED DESCRIPTION

[0028] The application will be further described in connection with the following examples. However, the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0029] The instruments, reagents, and materials involved in the following examples are conventional instruments, reagents, and materials available in the prior art, and can be obtained through regular commercial channels, unless otherwise specified. The experimental methods and detection methods involved in the following examples are conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0030] GlutaMAX of the application TM OK432, IL-2, IL-18, IL-21, CD16 antibody, etc., are all conventionally purchased, wherein GlutaMAX TM GlutaMAX is purchased from Promocell / PB180419, OK432 is purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd. / GMP-TL107-0100, IL-2 is purchased from QuanGang Pharmaceutical Co., Ltd. / 125SER, IL-18 is purchased from Fein Biological / P1642, IL-21 is purchased from Novoprotein / GMP-CC45, and CD16 is purchased from Gene Baygene 4Abio / FHU016-03-100.

[0031] The single nucleated cells of the application are obtained by conventional methods from the umbilical cord blood of a puerpera. The umbilical cord blood of 4 puerperas is collected, and 4 single nucleated cells are obtained by separation, which are used as the single nucleated cells of Examples 1, 2, 3, and 4, respectively, for experiments.

[0032] Example 1: In vitro culture and expansion of NK cells

[0033] (I) Preparation of reagents

[0034] Coating solution: dilute the CD16 antibody solution to a concentration of 5 μg / mL with D-PBS buffer (i.e., Dulbecco's phosphate buffered saline) to obtain.

[0035] Activation medium: add 0.6 mL of GlutaMAX TM , an appropriate amount of OK432 to a final concentration of 50 ng / mL, an appropriate amount of IL-2 to a final concentration of 300 ng / mL, an appropriate amount of IL-18 to a final concentration of 25 ng / mL, and an appropriate amount of IL-21 to a final concentration of 10 ng / mL to 60 mL of KBM 581 medium, and mix well to obtain.

[0036] Expansion medium: add 10 mL of GlutaMAX TMand an appropriate amount of IL-2 to make the final concentration of 200 ng / mL, and mix well.

[0037] (II) Culture method

[0038] As follows:

[0039] (1) On Day -1, 8 mL of coating solution was added to a T75 culture flask, and incubated at 4°C for 12 h; the coated T75 culture flask was gently washed twice with D-PBS buffer;

[0040] (2) On Day 0 (Note: this is the actual first day of culture, recorded as Day 0), the mononuclear cells were resuspended with 30 mL of activation medium, and the cell density was controlled at 1.5 x 10 6 cells / mL, and transferred to a T75 culture flask, and mixed by cross shaking, and placed in an incubator for culture at 37°C, 5% CO2;

[0041] (3) On Day 1-3, the cells were cultured at rest;

[0042] (4) On Day 4, 30 mL of activation medium was added;

[0043] (5) On Day 5-21, the medium was counted and supplemented every two days: 30 mL of expansion medium was added to maintain the cell density at 1.0 x 10 6 -1.5 x 10 6 cells / mL;

[0044] (6) On Day 18 (Note: at this time, the cells were actually cultured for 19 days), the NK cells were harvested.

[0045] The mononuclear cell samples of Examples 2, 3, and 4 were different from Example 1, and the steps were the same as Example 1.

[0046] Detection of experimental NK cells

[0047] The cells cultured in Examples 1, 2, 3, and 4 were subjected to flow target detection and in vitro killing experiments.

[0048] (1) Flow target detection

[0049] On Day 0, flow target detection was performed to detect the purity of NK cells, and the results are shown in Figure 1 Table 1. The purity of NK cells (i.e. CD56+CD3-) in Examples 1, 2, 3, and 4 was 7.78%, 4.38%, 10.11%, and 21.74%, respectively. It can be seen that the purity of NK cells in mononuclear cells isolated from umbilical cord blood of different individuals differs greatly, which also indicates that the method of the present application has high compatibility for the purity of initial state NK cells.

[0050] On Day 18, flow target point detection was performed to detect the purity of the NK cells, and the results are shown in Table 1. Figure 2 As shown in Table 1, the purity of the NK cells in Examples 1, 2, 3 and 4 was 96.03%, 92.45%, 96.54% and 97.46%, respectively. It can be seen that the purity of the NK cells can be increased to more than 90% at the time of harvest by the method of the present application for umbilical cord mononuclear cells from different sample sources.

[0051] (2) Viability detection of NK cells

[0052] On Day 0-21, samples were taken regularly for viability detection. The detection method was as follows: the cultured NK cells were mixed, samples were taken in the culture bottle with a pipette and in the culture bag with a syringe, the sample was reserved in a 1.5 ml sterile centrifuge tube, and the viability of the NK cells was detected by AO / PI (acridine orange / propidium iodide) fluorescence staining method. The results are shown in Table 2. Figure 3 As shown in Table 2, on Day 18, the viability of the NK cells in Examples 1, 2, 3 and 4 was 92.31%, 92.23%, 96.23% and 93.36%, respectively, all of which were more than 90%.

[0053] (3) On Day 0-18, samples were taken regularly for detection of expansion fold. The detection method was as follows: the cultured NK cells were mixed, samples were taken in the culture bottle with a pipette and in the culture bag with a syringe, the sample was reserved in a 1.5 ml sterile centrifuge tube, and the concentration of the NK cells was detected by AO / PI fluorescence staining method. The measured value was multiplied by the purity of the flow target point detection, and the ratio compared with Day 0 was the expansion fold. The results are shown in Table 3. Figure 4 As shown in Table 3, on Day 18, the expansion fold of the NK cells in Examples 1, 2, 3 and 4 was 3369, 3177, 3563 and 4177, respectively. It can be seen that the method of the present application can have a good proliferation effect on NK cells, and the proliferation effect is particularly obvious after Day 12.

[0054] (4) On Day 18, in vitro killing experiment was performed on the harvested NK cells as follows:

[0055] ① The NK cells in good growth state were resuspended with culture medium and counted, and the cell density was adjusted to 2.0 x 10 5 cells / mL, 1.0 x 10 5 cells / mL and 0.5 x 10 5 cells / mL, respectively, then 50 μL per well was seeded, and the sample was added in a four-replicate well manner.

[0056] ②Take K562-FFluc-EGFP cells, centrifuge at 1000 rpm / min for 5 min, adjust the cell density to 1.0 x 10 5 cells / mL, then add 50 μL per well to different NK cell plates, co-culture at 37℃, 5% CO2 for 4 h, and detect luciferase expression level.

[0057] ③Detect luciferase expression level; transfer the contents in the Bright-Glo TM Buffer into the corresponding Bright-Glo TM Substrate, invert and mix, form Bright-Glo TM reagent, add 50 μL of Bright-Glo TM reagent per well, after 5 min, measure with a microplate reader (Molecular Decices SpectraMax i3x).

[0058] The results of the killing experiment of NK cells are shown in Table 1. Figure 5 As can be seen from Table 1, when the effector-target ratio is 2:1, the killing rate of NK cells of each example is generally high, and the overall killing rate is more than 80%; when the effector-target ratio is 1:1, the killing rate decreases, but is still more than 60%; when the effector-target ratio is 1:2, the killing rate can also be maintained at more than 50%. This shows that the NK cells obtained by the culture of the application have good tumor killing activity.

[0059] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure disclosed herein. Modifications obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for in vitro culture and expansion of NK cells, characterized in that: Mononuclear cells were added to the activation medium, and the cell density was controlled at 1.5 × 10⁻⁶. 6 ~2.0×10 6 Cells / mL were added to pre-coated culture flasks and incubated at 37°C with 5% CO2 for 16–22 days. NK cells were then harvested. Activation medium was added on day 5 of culture. From day 6 onwards, expansion medium was added every two days to maintain a cell density of 1.0 × 10⁶ cells / mL. 6 ~1.5×10 6 cells / mL; The activation medium was prepared by adding 1% GlutaMAX to a serum-free NK cell culture medium. TM Mix an appropriate amount of OK432 to achieve a final concentration of 50 ng / mL, an appropriate amount of IL-2 to achieve a final concentration of 300 ng / mL, an appropriate amount of IL-18 to achieve a final concentration of 25 ng / mL, and an appropriate amount of IL-21 to achieve a final concentration of 10 ng / mL. The supplemental amplification medium was prepared by adding 1% GlutaMAX to the serum-free NK cell culture medium. TM Add an appropriate amount of IL-2 to achieve a final concentration of 200 ng / mL, mix well, and the product is ready.

2. The method for in vitro culture and expansion of NK cells according to claim 1, characterized in that: The serum-free NK cell culture medium was selected from KBM 581 medium.

3. The method for in vitro culture and expansion of NK cells according to claim 1, characterized in that: The coating solution is a 5 μg / mL CD16 antibody solution.

4. A culture medium for culturing NK cells, characterized in that: This includes activation medium and supplemental amplification medium; The activation medium was prepared by adding 1% GlutaMAX to a serum-free NK cell culture medium. TM Mix an appropriate amount of OK432 to achieve a final concentration of 50 ng / mL, an appropriate amount of IL-2 to achieve a final concentration of 300 ng / mL, an appropriate amount of IL-18 to achieve a final concentration of 25 ng / mL, and an appropriate amount of IL-21 to achieve a final concentration of 10 ng / mL. The supplemental amplification medium was prepared by adding 1% GlutaMAX to the serum-free NK cell culture medium. TM Add an appropriate amount of IL-2 to achieve a final concentration of 200 ng / mL, mix well, and the product is ready.

5. The application of the culture medium for culturing NK cells as described in claim 4 in the in vitro culture and expansion of NK cells.

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