TWI778302B - A culture medium for in vitro expansion and activation of natural killer cells - Google Patents

Abstract

A cell culture medium for in vitro expansion and activation of natural killer cells which contains nattokinase. Such medium provides an optimized environment specifically for natural killer cells. Without using any cancer feeder cell, using such medium could efficiently increase expansion fold, and yield high purity natural killer cell with high cytotoxicity activity.

Description

Cell culture medium for in vitro expansion and activation of natural killer cells

The present invention relates to a cell culture medium for in vitro expansion and activation of natural killer cells.

Natural killer cells (Natural Killer Cells; NK cells) are a group of cells with CD3 ^- CD56 ⁺ phenotype, which usually exist in lymph nodes, various organs and peripheral blood. NK cells account for about 5-20% of lymphocytes in the peripheral blood of the human body, so peripheral blood is one of the convenient sources of NK cells. NK cells were discovered in the 1970s to have cancer-killing properties without prior education or priming. Later studies found that NK cells can not only kill cancer cells, but also kill virus-infected cells, cancerous cells, and aged stressed cells.

Past studies have shown that NK cells have the potential to treat tumors. The practice of autologous NK cells in the treatment of cancer is to separate the NK cells from the patient's peripheral blood, culture them for about 14 days by in vitro cell expansion and activation, and then infuse them into the patient's body to achieve the effect of cancer treatment. In the past, in the implementation of in vitro cell expansion and activation methods, NK cells were mostly co-cultured with cancer feeder cells to achieve the purpose of greatly increasing the number of NK cells. However, the use of cancer feeder cell expansion methods still cannot solve the problem of safety. Infusion of expanded natural killer cells It is difficult to determine whether the cancerous feeder cells have been completely removed before entering the patient, raising concerns about safety. Furthermore, even if the number of NK cells after in vitro culture is greatly increased, if the activity of the NK cells in killing cancer cells is not good, the effect of subsequent application in autologous cell therapy is still very limited. Accordingly, it is necessary to develop a medium that can effectively increase the NK cell expansion multiple and generate NK cells with high purity and high cancer cell cytotoxicity without co-culture with cancer feeder cells.

Nattokinase is an extracellular enzyme secreted by Bacillus subtilis natto. It is known to have the activity of dissolving thrombus and the function of decomposing fibrin. It can be used in the prevention and treatment of cardiovascular diseases. Nattokinase is currently administered orally in the form of nutritional supplements. Although some studies have discussed that individual consumption of natto or natto extract can help regulate individual immunity (Takeda et al., Traditional & Kampo Medicine, Vol.3 Iss.2 100-106, 2016), only the active ingredients of its effect and related mechanisms are still unclear. In addition, there is no relevant research on the method and effect of nattokinase applied to in vitro immune cell culture.

In one aspect, the present invention provides a medium for in vitro expansion and activation of natural killer cells, the medium comprising nattokinase in a concentration range of 5FU/ml to 20FU/ml.

In another aspect of the present invention, there is provided a medium for in vitro expansion and activation of natural killer cells, the medium further comprises coenzyme Q10.

In some embodiments of the present invention, the medium comprises CoQ10 at a concentration of 100 nM to 1000 nM.

In another aspect of the present invention, it provides a medium for in vitro expansion and activation of natural killer cells, the medium further comprises interleukin-2 (IL-2), interleukin-12 (IL-12) and interleukin-12 (IL-12). Interleukin-18 (IL-18); wherein the concentration of the interleukin-2 (IL-2) in the medium ranges from 200 to 1000 IU/ml; wherein the interleukin-12 (IL-12) is present in the medium The concentration in the medium is between 10-50 ng/ml; wherein the concentration of the interleukin-18 (IL-18) in the medium is between 100-200 ng/ml.

In another aspect, the present invention provides a medium for in vitro expansion and activation of natural killer cells, the medium further comprises a basal medium; wherein the basal medium is AIM V, X-VIV015, CellGro SCGM, KBM 501, DMEM or RPMI-1640 medium.

In some embodiments of the present invention, among the cells cultured in the aforementioned medium, 21% to 41% are NKG2A ⁺ CD3 ^- CD56 ⁺ cells.

In some embodiments of the present invention, among the cells cultured in the aforementioned medium, 41% to 71% are TRAIL ⁺ CD3 ^- CD56 ⁺ cells.

In some embodiments of the present invention, 92% to 97% of the cells cultured in the aforementioned medium are natural killer cells.

In some embodiments of the present invention, when the cells are cultured in the aforementioned medium, when the cells are co-cultured with K562 cells at a ratio of 5:1 in vitro, the cells have 63% to 81% cancer cell killing. active.

Figure 1: Steps S11~S14‧‧‧

FIG. 1 is a flow chart of a method for in vitro expansion and activation of NK cells according to Embodiment 1 of the present invention;

Figure 2 is an analysis of cell expansion folds produced by the present invention;

Figure 3 shows the test results of the ability of the NK cells produced by the present invention to kill cancer cells. A549 as a cancer cell line poisoned by NK cells;

Figure 4 shows the results of mouse experiments in which NK cells produced by the present invention inhibit the growth of cancer cells.

In view of the above-mentioned problems to be solved, the present invention proposes a culture medium for in vitro expansion and activation of NK cells, which contains nattokinase without using cancer feeder cells for co-cultivation, which can provide a good culture of NK cells. environment, effectively increase the NK cell expansion multiple, and generate NK cells with high purity and high cancer cell cytotoxicity.

definition

For the purpose of this specification, "natural killer cells" (NK cells for short) refer to cells that present CD3 ^- CD56 ⁺ on their cell surface antigens.

For the purpose of this specification, "cancer feeder cell" refers to in vitro immune cell culture, adding live cancer cells to co-culture to achieve the effect of enhancing immune cell expansion, and other live cancer cells added are called Cancer feeder cells.

For the purpose of this specification, "cell expansion fold" is determined as follows: "number of cells after 14 days of in vitro culture" divided by "number of initial NK cells isolated from peripheral blood mononuclear cells".

For the purpose of this specification, "the activity of killing cancer cells" refers to the K562 cell line or other cancer cell lines as the target cell (target cell), and the NK cell as the effector cell (effector cell). When the (E:T ratio) was 5:1, the ratio of target cell death was taken as the poisoning efficiency.

Materials and Methods

The peripheral blood sample of the present invention is based on the plan passed by the ethics committee. Whole blood is collected from the subject's arm, placed in a sterile blood collection tube, and stored at room temperature for subsequent processing.

The basal medium used in the present invention can be selected from commercially available products such as: CellGro SCGM (CellGenix Company), KBM 501 (Kohjin Bio Company), AIM-V (Thermo Fisher Company), X-VIV015 (Lonza Company), DMEM and RPMI 1640, etc. culture medium.

The culture medium of the present invention may contain appropriate components such as proteins, cytokines, antibodies, serum, and compounds. The cytokines are sometimes interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-12 (IL-12) , Interleukin-15 (IL-15), Interleukin-18 (IL-18) or Interleukin-21 (IL-21).

Method for isolating NK cells from PBMC

Including but not limited to the use of Dynabeads (Invitrogen Company), CliniMACS magnetic beads (Miltenyi Biotec Company) or other commercially available immunomagnetic beads to separate and remove cells expressing cell surface antigens CD3 and/or CD34 to achieve the effect of purifying NK cells.

Analysis of cell surface antigens by flow cytometry

The expanded and activated cells were placed in a 96-well plate at 2×10 ⁵ cells/50 μl, and 3 μl of fluorescently labeled antibodies were added to react at 4°C for 15 minutes, followed by phosphate buffered saline (PBS for short) ) After washing 3 times, 400 μl PBS was added to suspend the cells, and the fluorescent markers on the cell surface were analyzed by flow cytometer (Beckman Company). The above fluorescently labeled antibodies include anti-CD3 antibody (Invitrogen company), anti-CD56 antibody (Invitrogen company), anti-TRAIL antibody (BD Bioscience company) and anti-NKG2A antibody (R&D system company).

Test method for the ability of NK cells to kill cancer cells

The expanded and activated cells were used as effector cells, and K562 cell line (chronic myeloid blood cancer cell line), HepG2 cell line (liver cancer cell line), HT-29 cell line (colorectal cancer cell line) ) or A549 cell line (lung cancer cell line) as the target cell. The effected cells and target cells were mixed and cultured at 0.25:1, 0.5:1, 1:1 or 5:1, reacted for 4 hours, and then stained with 7-AAD to determine the number of apoptotic cells.

Method for expanding and activating NK cells in vitro

As shown in Figure 1, step 11 is to separate peripheral blood mononuclear cells (PBMC) from the blood sample. The unrefrigerated or frozen peripheral blood samples were placed in a 50ml centrifuge tube and centrifuged. After the centrifugation, the plasma in the upper layer was removed, and an appropriate amount of PBS was added to dilute, and then 3 ml of Ficoll-paque Plus (GE Healthcare) was added. After centrifugation at room temperature for 20 minutes, the cells in the PBMC layer were aspirated and washed three times with PBS to obtain PBMCs.

Next, purify NK cells from PBMCs (step 12 in Figure 1): Suspend PBMCs in 80 μl of PBS containing 0.5% BSA, 2 mM EDTA per 1×10 ⁷ PBMCs. Add 30 μl of CD3 magnetic beads (MACS CD3 MicroBeads human, Miltenyi Biotec Company) to the PBMC suspension under the condition of every 1×10 ⁷ cells, stir the PBMC suspension containing magnetic beads evenly, and place it at 4° C. next 15 minutes. After that, the PBMC suspension was centrifuged at 300 x g for 10 minutes and the supernatant was removed, PBMC was suspended in 500 μl of PBS containing 0.5% BSA and 2 mM EDTA, and the PBMC suspension was passed through an LD Column chromatography tube (Miltenyi Biotec Company), the cells expressing CD3 antigen on the cell surface were separated from the suspension by magnetic force, the chromatography tube was washed with 2 x 1 ml of PBS containing 0.5% BSA, 2 mM EDTA, and the effluent cells were collected to obtain the purified NK cells.

Next, carry out NK cell expansion and activation: NK cells are uniformly prepared into a cell suspension with a concentration of 2~10× ^{10 5} /ml in medium, and then transferred to a culture plate, at 37°C and 5% CO ₂ environment NK cells were expanded under the following conditions (step 13 in Figure 1). The NK cell culture medium was based on AIM-V medium (Thermo Fisher Scientific), and was supplemented with interleukin-2 (IL-2) (Thermo Fisher Scientific) at a concentration ranging from 200 to 1000 IU/ml. Interleukin-12 (IL-12) (Sigma-Aldrich) at concentrations ranging from 10 to 50 ng/mL, and interleukin-18 (IL-18) (Thermo Fisher) at concentrations ranging from 100 to 200 ng/mL Scientific Corporation), and nattokinase (NSK-SD; Japan Bio Science Laboratory Corporation) at concentrations ranging from 5 to 20 FU/ml. The culture medium may be supplemented with coenzyme Q10 (Sigma-Aldrich) at a concentration ranging from 100 to 1000 nM. In a preferred embodiment of the present invention, 100 nM of coenzyme Q10 (Sigma-Aldrich) is additionally added to the medium.

After that, observe the growth status of the cells every two to three days, and replace the fresh NK cell medium according to the cell growth status until 14±2 days (step 14 in Figure 1). The NK cell culture medium, which is changed every two to three days, is formulated in the manner described in paragraph 0036. Finally, transfer the mixture of cells and medium in the culture plate to a centrifuge tube, collect the cells in the mixture by centrifugation, and then wash with PBS. After repeating this step three times, the cells are evenly dispersed with PBS. That is, the expanded and activated NK cells are obtained.

The expanded and activated NK cells obtained by the aforementioned method can be stored in a suitable excipient, which can be a monophosphate buffer, and finally prepared into a pharmaceutical composition.

Example

Example 1, NK cell expansion fold test

In order to test and optimize the NK cell expansion and activation method, this example discusses the supplements and the amount of the NK cell culture medium. The experimental group uses culture medium A and medium B respectively. Or medium C, and the control group used medium D. Both the control group and the experimental group were tested with PBMC isolated from the peripheral blood of the same subject. The medium A, medium B, medium C and medium D are all based on AIM-V medium (Thermo Fisher Scientific company) as the basal medium, and the interleukin-2 (IL -2) (Thermo Fisher Scientific), interleukin-12 (IL-12) (Sigma-Aldrich) in the range of 10~50ng/ml, and interleukin in the range of 100~200ng/mL IL-18 (Thermo Fisher Scientific). In order to test the effect of adding nattokinase on the in vitro expansion and activation of NK cells, the experimental groups (medium A, medium B and medium C) were supplemented with 20FU/ml (FU means fibrin degradation unit) or 5FU /ml of nattokinase. In order to test the effect of adding nattokinase and coenzyme Q10 at the same time on the in vitro expansion and activation of NK cells, 20FU/ml of nattokinase and 100nM of coenzyme Q10 were added to the medium A at the same time. The components of medium A, medium B, medium C and medium D are summarized in Table 1 below.

Table 1. Culture medium ingredients

The experimental results of FIG. 2 show that after culturing for 14 days with medium A, medium B or medium C containing nattokinase, the NK cell expansion multiples all reach more than 1000 times, specifically, the expansion multiples are 1135 to 1750 times. In contrast, after culturing for 14 days in medium D without nattokinase, the NK cells expanded only 498 times. It can be seen that adding 5~20FU/ml of nattokinase can effectively increase the expansion ratio of NK cells. Comparing the experimental results of medium A and medium B, it was shown that when coenzyme Q10 was added to the medium containing nattokinase, the expansion ratio of NK cells increased from 1326 times to 1750 times.

Example 3. Test of the cytotoxic ability of NK cells to cancer cells

In order to further explore the activity of NK cells expanded by the present invention, this example tests the ability of NK cells to kill cancer cells after expansion in different media. First, medium A, medium B or medium C was used in the experimental group, and medium D was used in the control group, and the composition of the medium was shown in Table 1. The control group and the experimental group were both expanded and activated with PBMC isolated from the peripheral blood of the same subject. The experimental results show (Fig. 3A), after culturing for 14 days with medium A, medium B or medium C containing nattokinase, when the ratio of E:T is 5:1 and K562 is used as the cancer cell line poisoned by NK cells, poisoning The efficiency is above 60%. Specifically, the poisoning efficiency is 63.1%~81.2%. In contrast, after 14 days of culture in medium D without nattokinase, the poisoning efficiency was only 39.8%. It can be seen that adding 5~20FU/ml of nattokinase to the medium can not only increase the expansion multiple of NK cells, but also have a higher cytotoxic activity against cancer cells after the expanded NK cells. Comparing the experimental results of medium A and medium B, it is shown that adding coenzyme Q10 to the medium containing nattokinase can further improve the cytotoxicity of NK cells to cancer cells, from 63.1% to 81.2%.

Continuing from the above, the NK cells amplified and activated in a preferred embodiment medium A were used to test the cytotoxicity of the NK cells to different cancer cell lines. The experimental results showed (Fig. 3B) that the NK cells (namely, the medium group A) after the expansion and activation of the medium containing nattokinase and coenzyme Q10 can effectively kill the HepG2 cell line (hepatoma cell line) and the HT-29 cell line ( colorectal cancer cell lines) and A549 cell line (lung cancer cell line), and compared with the use of the medium without nattokinase (ie, medium D group), it has significantly higher toxicity to cancer cells.

Example 4. Analysis of cell surface antigens

In order to test the proportion of NK cells (that is, the purity) and the activation state of the NK cells in the cells produced by the present invention, anti-CD3, anti-CD56, anti-TRAIL and anti-NKG2A antibodies were used to label cell surface antigens , and then analyzed by flow cytometry, and the results are shown in Table 2. The surface of activated NK cells expresses TRAIL (TNF-related apoptosis-inducing ligand) molecule, which has better cancer cell killing activity. NKG2A is an inhibitory receptor located on the surface of NK cells. If NKG2A is expressed on the surface of NK cells, it shows that the activity of the NK cells is in a state of being inhibited. In other words, when the surface of NK cells does not express NKG2A antigen, the NK cells have better cancer cell killing activity.

Table 2. Results of cell surface antigen analysis

Table 2 shows that 92% to 97% of the cells produced by using medium A, medium B or medium C are NK cells, indicating that the present invention can produce extremely high-purity NK cells. Furthermore, 41% to 71% of cells produced using medium A, medium B or medium C were TRAIL ⁺ CD3 ^- CD56 ⁺ cells, indicating that most of the cells were NK cells in a highly activated state; Of the cells produced in medium D containing nattokinase, only 37% to 54% were TRAIL ⁺ CD3 ^- CD56 ⁺ cells. In addition, only 21% to 41% of the cells produced using Medium A, Medium B or Medium C were NKG2A ⁺ CD3 ^- CD56 ⁺ cells, showing that most cells had better cancer cell killing activity; Up to 41% to 53% of cells produced in medium D without nattokinase were NKG2A+CD3-CD56+ cells.

Example 5. Mouse experiment in which NK cells inhibit tumor cell proliferation

All animal experiments were performed in accordance with appropriate animal care and using ethical experimental procedures and guidelines approved by the Laboratory Animal Care and Use Committee or Panel (IACUC). In order to test the ability of the expanded NK cells of the present invention to inhibit the proliferation of cancer cells, female NOD/SCID mice (average body weight of 20 grams) were assigned to different groups, a control group (n=5), a group of Experimental group (n=5). 100 μl of K562 cells (tumor cells) containing 1×10 ⁷ were subcutaneously injected into the mice of the control group and the experimental group, which was called the 0th day of the experiment. On the 0th, 4th, 8th, 12th and 14th days of the experiment, 100 μl containing 1×10 ⁷ cells produced in a preferred embodiment medium A were administered to the experimental group by intravenous injection; The same volume of 100 μl of normal saline was administered to the control group by intravenous injection. The activity, body weight, coat color and subcutaneous tumor growth of the mice were continuously recorded until the 18th day. The experimental results showed that on the 18th day, the tumor volume of the mice in the experimental group was 97.6 mm ^{3 , which was significantly smaller than the tumor volume of 364.8 mm 3 in} the control group. Accordingly, in this example, NK cells after expansion and activation in the medium containing nattokinase and coenzyme Q10 can effectively inhibit the growth of tumor cells in vivo, indicating that the activity of NK cells after expansion is excellent, and has application in clinical treatment .

Claims (10)

A kind of substratum for in vitro amplification and activation of natural killer cells, it comprises nattokinase, basal medium, interleukin-2 (IL-2), interleukin-12 between 5FU/ml and 20FU/ml in a concentration range (IL-12) and interleukin-18 (IL-18). Such as the medium of claim 1, which further comprises coenzyme Q10. The medium of claim 2, wherein the concentration of the coenzyme Q10 ranges from 100 nM to 1000 nM. The medium of claim 1, wherein the concentration of the interleukin-2 (IL-2) in the medium is between 200 and 1000 IU/ml; wherein the interleukin-12 (IL-12) is present in the medium. The concentration in the medium is between 10-50 ng/ml; wherein the concentration of the interleukin-18 (IL-18) in the medium is between 100-200 ng/ml. The medium of claim 1, wherein the basal medium is AIM V, X-VIV015, CellGro SCGM, KBM 501, DMEM or RPMI 1640 medium. The medium according to any one of claims 1 to 5 of the claimed scope, wherein the cells cultured in the medium do not need to be co-cultured with cancer feeder cells, and can achieve a cell expansion multiple of between 1135-fold and 1750-fold. According to the medium according to any one of items 1 to 5 of the claimed scope, 21% to 41% of the cultured cells are NKG2A ⁺ CD3 ^- CD56 ⁺ cells. According to the medium according to any one of items 1 to 5 of the claimed scope, 41% to 71% of the cultured cells are TRAIL ⁺ CD3 ^- CD56 ⁺ cells. According to the medium according to any one of items 1 to 5 of the claimed scope, the cultured cells contain 92% to 97% of natural killer cells. According to the medium according to any one of the claims 1 to 5 of the claimed scope, when the natural killer cells and K562 cells are co-cultured in vitro at a ratio of 5:1, the natural killer cells have a ratio of 63% to 81%. Cancer cell cytotoxicity.

Images