TWI669399B - A method for in-vitro expansion of nature killer cells (nk cells) and nature killer t cells (nkt cell) and the pharmaceutical composition thereof. - Google Patents

Abstract

The invention relates to a method for amplifying natural killer cells and natural killer T cells in vitro, which is cultured at an initial cell density of 0.5×10 ⁶ to 5×10 ⁶ cells/mL, and the cell density is adjusted again at a specific time after culture. 0.5x10 ⁶ ~ 5x10 ⁶ cells / mL, in order to maintain a better cell growth environment, thereby increasing the amplification efficiency of the cells, according to which the method of the present invention can be used in a shorter time than the prior art. The total number of cells was obtained, and the cell survival rate and the ratio of NK/NKT cells were high, and the proportion of NK/NKT cells with cytotoxicity was also high in the activated state.

Description

Method for amplifying natural killer cells and natural killer T cells in vitro and pharmaceutical composition thereof

The invention relates to a method for amplifying natural killer cells (NK cells) and natural killer T cells (NKT cells) in vitro, in particular to a method for expanding three to seven thousand times NK/NKT cells in vitro, and the method The expanded NK/NKT cells have a cell survival rate of more than 85%, active NK/NKT cells of more than 50%, and a killing effect on cancer cells of more than 76%.

Immunotherapy, which refers to the way to prevent and treat diseases by strengthening the body's own immune system or by externally giving immunity to the organism. It has high specificity, high efficiency and durability. In the past years, immune cell therapy derived from the concept of immunotherapy has also been widely explored.

In 2011, immune cell therapy was first proposed in the international journal Nature , which is considered a possible treatment for cancer; immune cell therapy focuses on stimulating the immune system and can destroy surgery (such as radiation therapy or chemotherapy) After the cancer cells remain, the therapeutic effect is improved, and the toxic effects of surgery or chemotherapy and radiotherapy are reduced to improve the quality of life and prognosis of the patient; for example, immunotherapy is used to treat cancer, which is separated from the blood of the patient. The immune cells, after proper in vitro culture, amplification and activation, are again returned to the patient to produce a specific immune response to tumors of specific tissues in the body, and use their own immune cells to return for treatment and avoid immunity. Rejection; in the immune cells, since the natural killer cells (NK cells) can directly kill the target cells without presenting the antigen through the antigen presenting cells, it is predicted to have a comparison with the T cells. Higher anti-tumor efficiency.

Natural killer cells are the first line of defense against foreign substances in the human immune system. Because their cell surface lacks a specific antigen receptor (TCR), the immune response caused by it is a non-specific defense, natural killer cells. It is located in the peripheral blood circulation, can attack the virus-infected cells, protect the human body from viral infection, and can also cause rejection of tumor cells and bone marrow transplantation to attack these foreign cells, when natural killer cells and these heterogeneous cells Upon exposure, it drives its activating receptor, thereby activating itself and causing natural killer cells to release factors such as perforin and granzyme; perforin will form pores in the labeled heterogeneous cell membrane. Then, after the granzyme enters the target heterogeneous cell, the permeability of the mitochondrial membrane is changed, and the apoptosis-related protein is activated to initiate an apoptosis reaction, thereby fragmenting the DNA in the labeled heterogeneous cell. And cause cell breakdown; in addition, a group of natural killer T cell lines simultaneously express T cell surface antigens (eg, CD3, TCRαβ) and A natural killer cell surface antigen (eg, CD56) heterogeneous immune cell, so it can simultaneously induce a specific immune response like T cells, and a non-specific immune response like NK cells, is also considered to develop immune cell therapy A big focus.

However, natural killer cells account for only 5-10% of lymphocytes in the peripheral blood of the human body, and natural killer T cells account for less than 1%. Therefore, it is possible to efficiently amplify natural killer cells and natural killer T cell lines in vitro. A major issue, until now, researchers are still trying to improve natural killer cells and The in vitro expansion efficiency of natural killer T cells, its cell survival rate, cell purity and cytotoxicity, further enhance its applicability in medicine and medical applications.

In one aspect, the invention provides a method for expanding natural killer cells (NK cells) and natural killer T cells (NKT cells) in vitro, wherein the method is performed at an initial cell density of 0.5×10 ⁶ to 5×10 ⁶ cells/mL. Culture to provide a better growth environment for cells, and when the total number of cells reaches 2.5x10 ⁶ ~ 5x10 ⁶ cells / mL, the cell culture medium is first updated and the cell density is adjusted to 0.5x10 ⁶ ~ 5x10 ⁶ cells / mL, that is, when After the cells are in a stable state, the cell density is readjusted to enhance the growth efficiency of the cells.

In another aspect, the present invention provides a method for expanding natural killer cells and natural killer T cells in vitro, wherein the cell culture medium is renewed every 2 to 3 days, and the cell density is re-adjusted to 0.5×10 ⁶ ~ 5x10 ⁶ cells/mL, which can continue to provide a better growth environment for cells.

In another aspect, the present invention provides a method for amplifying natural killer cells and natural killer T cells in vitro, wherein the cell culture solution used comprises human recombinant interleukin-2 (rhIL-2) for activation. NK/NKT cells, in turn, promote the secretion of perforin by NK cells to bind to the target cells and cause cytotoxic effects, while also contributing to immune cell growth to increase the total number of NK/NKT cells after expansion.

In another aspect, the present invention provides a method for amplifying natural killer cells and natural killer T cells in vitro, wherein the cell culture solution used in the method further comprises an anti-CD3 monoclonal antibody. To promote the activation of NK/NKT cells to enhance the cytotoxicity of NK/NKT cells to cancer cells without affecting the growth of NK/NKT cells.

Accordingly, one aspect of the present invention relates to a method for expanding natural killer cells and natural killer T cells in vitro, comprising the steps of: suspending a peripheral mononuclear cells (PBMCs) in a cell culture solution. to obtain a cell mixture; adjusting the cell density of the cell is a mixture of ^{0.5x10 6 ~ 5x10 6 cells / mL} , as an initial cell density, the cell culture medium using the cell mixture was cultured in a cell In the culture tray; when the total number of cells in the cell culture plate reaches 2.5 x 10 ⁶ ~ 5 x ^{10 6} cells / mL, the cell culture medium is renewed, and the cell density is re-adjusted to 0.5 x 10 ⁶ ~ 5 x 10 ⁶ cells / mL; 2-3 days update the cell culture medium, and re-adjust the cell density of ^{0.5x10 6 ~ 5x10 6 cells / mL} , incubated for at least 10 to 16 days; and washing the cells and obtains the cell mixture is a cell mixture In the community, the cell population is a mixture of natural killer cells and natural killer T cells that have been expanded.

In some embodiments of the present invention, before the step of suspending the peripheral blood mononuclear cells, the method further comprises the step of collecting the peripheral blood mononuclear cells, comprising: centrifuging a peripheral blood sample to make the periphery The blood sample is layered into a plasma layer, a buffy coat and a red blood cell layer; the plasma layer is removed and the white blood cell layer is obtained, and the white blood cell layer is mixed with Hanks balanced salt solution (HBSS) and a single nuclear ball. The cell separation solution is used to obtain a leukocyte mixture; centrifugation to separate the leukocyte mixture; and the supernatant is removed, and an intermediate layer of white liquid is obtained to obtain the peripheral mononuclear cells.

In another specific embodiment of the present invention, the cell culture fluid system comprises: a stem cell growth medium (SCGM); a human interleukin-2 recombinant protein (rhIL-2), the concentration range thereof is Between 250 and 1,000 IU/mL; one serum, the volume percentage is between 1 and 10%; and the anti-CD3 monoclonal antibody, which ranges from 10 to 500 ng/mL.

In another embodiment of the present invention, in the step of adjusting the initial cell density, the cell density of the cell mixture is adjusted to 0.5×10 ⁶ to 2.5×10 ⁶ cells/mL.

In another embodiment of the present invention, in the step of renewing the cell culture medium and re-adjusting the cell density, the cell density of the cell mixture is adjusted to 0.5 x 10 ⁶ to 2.5 x 10 ⁶ cells/mL.

In another embodiment of the present invention, in the step of culturing the cell mixture, the cell mixture is cultured in an environment of 37 ° C and 5% carbon dioxide.

In another embodiment of the present invention, before the step of obtaining the cell population, the method further comprises: analyzing the ratio of the natural killer cells and the natural killer T cells in the cell mixture, and the ratio is between 30 and 90%. The cell surface antigen of the natural killer cell line is a cell population of CD16 ⁺ CD56 ⁺ CD3 ^- , and the cell surface antigen of the natural killer T cell line is a cell population of CD16 ⁺ CD56 ⁺ CD3 ⁺ .

In another embodiment of the present invention, after the step of washing the cell mixture and obtaining the cell population, the method further comprises the step of suspending the cell population with a preservation solution, the preservation solution being a monophosphate buffer solution or an extracellular cell. Liquid (Plasma-Lyte A).

In another embodiment of the present invention, the amplified natural killer cell and natural killer T cell mixture has a cell viability of at least 80%, and the natural killer cell and the natural killer T cell mixture have The natural killer cell and natural killer T cell line of cytotoxicity reaches at least 50%.

Another aspect of the invention relates to a pharmaceutical composition of natural killer cells and natural killer T cells, comprising the amplified natural killer cell and natural killer T cell mixture prepared by the method described above; and medicine An acceptable solvent.

S10~S18‧‧‧Steps

S101~S104‧‧‧Steps

1 is a schematic flow chart of a first embodiment of the present invention; FIG. 2 is a schematic flow chart of a second embodiment of the present invention; and FIG. 3A-3B is an amplified natural killer cell (NK cell)/natural killer T cell ( Proportion analysis of NKT cells; Figure 4A-4B is a ratio analysis of amplified natural killer cells (NK cells) / natural killer T cells (NKT cells); Figure 5 is an amplified natural killer cell (NK cells) //Analysis of the cytotoxic ability of natural killer T cells (NKT cells).

In view of the prior art, there are still many areas for improvement in the expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells) in vitro, since NK cells only account for about 5-10% of human immune cells, NKT cells are more Less than 1%, in order to make it more suitable for medical applications, it is necessary to obtain a larger number of cells in a shorter culture time, and to be able to amplify NK/NKT cells in the cell population to enhance NK/NKT cells. The ratio, which in turn increases the purity of the cells, in addition, the cell survival rate of the amplification must be maintained, and the NK/NKT cells are effectively stimulated and activated during cell expansion to enhance their immunity. According to the present invention, a method for amplifying natural killer cells (NK cells)/natural killer T cells (NKT cells) in vitro is proposed to improve the deficiencies of the prior art; hereinafter, the technology of the present invention will be The means and characteristics are explained.

definition

As used in this specification, the term "Nature killer cell" (NK cell) means a cytotoxic lymphocyte that is part of the innate immune system and can be induced to perform non-specificity. Cell killing effect.

As used in this specification, the term "Nature killer T cell" (NKT cell) means an immune cell that simultaneously expresses an αβ T cell receptor (TCR) and a molecular marker associated with a natural killer cell, which is simultaneously specific. Cellular immunotoxicity and non-specific cellular immunotoxicity.

As used herein, the term "peripheral mononuclear cells" (PBMCs) means a cell having a single cell nucleus and having a nuclear morphology in a circular shape, which comprises lymphocytes such as T cells, B cells, and NK cells. Mononuclear cells and dendritic cells, and mononuclear cells in peripheral blood can also be called peripheral blood mononuclear cells (PBMC).

Materials and Methods

Reagent preparation

Cell culture medium (medium)

Based packaging line one embodiment of the invention using ^{Clonetics TM CellGro® SCGM BulletKit TM (CC} -3205) as the basic medium, 500 ml capacity (mL) of cell basal medium comprising a substrate (Stromal Cell Basal Medium), 0.5mL Human fibroblast growth factor (h-FGF-B), 0.5 mL of insulin (Insulin), 0.5 mL of fetal bovine serum (FBS), and 0.5 mL of gentamicin/amplemycin (Gentamicin sulfate/Amphotericin; GA-1000).

Recombinant human interleukin-2 (rhIL-2)

In a sterile working environment, take 1 ml (mL) of sterile water from a sterile syringe, inject into a container containing 1.1 mg (mg) of rhIL-2 powder (Proleukin ^® ), and dissolve evenly to obtain a concentration of 1.1 mg / ml (mg). /mL) of rhIL-2 stock solution, then take the rhIL-2 stock solution in a sterile syringe, and place it in a 50mL centrifuge tube, then add 43mL of cell culture medium to adjust the concentration of rhIL-2 solution to 500 units / microliter (U/μl) to obtain a diluted solution of rhIL-2; the diluted solution of rhIL-2 was dispensed into a micro-sterilized centrifuge tube and stored in a refrigerator at -20 ° C for use.

Anti-cluster of differentiation-3 monoclonal antibody (Anti-CD3 mAb)

The anti-CD3 monoclonal antibody stock solution (1 mg of Anti-CD3 monoclonal antibody in 1 mL of solvent) was taken out from the original packaging (Takara) under sterile working conditions, and then the Anti-CD3 monoclonal antibody stock solution was mixed. To 4 mL of the cell culture medium, the concentration of the Anti-CD3 monoclonal antibody solution was diluted to 20 μg/mL; the anti-CD3 monoclonal antibody solution diluted solution was dispensed into a micro-sterilized centrifuge tube, and stored in a refrigerator at -20 ° C for use. .

Amplification of cell culture medium of natural killer cell (NK cell) / natural killer T cell (NKT cell)

Take the aforementioned rhIL-2 diluted solution, Anti-CD3 monoclonal antibody dilution solution and serum into the cell culture medium, and mix them evenly to configure to amplify natural killer cells/natural killer T cells. a cell culture solution comprising rhIL-2 at a final concentration of 250 to 1,000 IU/mL, an anti-CD3 monoclonal antibody at a final concentration of 10 to 500 ng/mL, and a serum having a final concentration of 1 to 10%. .

Peripheral blood sample

The peripheral blood samples of the subjects are collected by the hospital physician's clinic and stored in a sterile blood collection tube, and the samples are transported to the Good Tissue Practice (GTP) core laboratory according to the specifications for subsequent processing.

Cell

Preparation of peripheral blood mononuclear cells (PBMC)

The peripheral blood was transferred from the blood collection tube to a 50 mL centrifuge tube for gradient centrifugation. The centrifugal force of the centrifuge (AUBOCA, 4000/4200) was set to 600 x g, and the centrifuge function of the centrifuge was turned off, and the mixture was centrifuged at room temperature for 10 minutes to be The peripheral blood sample is divided into three layers, from top to bottom, respectively, plasma, Buffy coat and Erythrocytes; then the plasma layer is removed and the white blood cell layer is transferred to a new sterile centrifuge tube. The Hanks balanced salt solution (HBSS) was used to slowly mix the leukocytes to obtain a leukocyte dilution solution; on the other hand, 1 to 3 mL of mononuclear cell fraction (Ficoll-Paque Plus) was placed in a single-nuclear sphere separation centrifuge tube. After centrifugation at 600xg for 30 seconds at room temperature, it was set aside; then, the above-mentioned leukocyte dilution solution was added to a mononuclear sphere separation centrifuge tube containing a mononuclear cell separation solution, and centrifuged at 600 x g for 10 minutes at room temperature. Layer, successively remove the supernatant, transfer the white intermediate layer (ie, peripheral blood mononuclear cells) to a new sterile centrifuge tube, add HBSS to mix evenly to wash the cells, centrifuge at room temperature for 10 minutes, then remove the supernatant. , Then cells were resuspended again HBSS, centrifuged for 10 minutes at room temperature, each supernatant was collected and peripheral blood mononuclear cells to spare ball.

Cell test

Cell surface antigen analysis

Take 1×10 ⁶ cells in a 15 mL centrifuge tube, centrifuge at 500 g for 5 minutes, remove the supernatant, resuspend the cells in 4 mL of Dulbeccos Phosphate Buffered Saline (DPBS), and repeat this step twice. Then, 5 μl of the cell fluorescent labeling reagent was added, and the reaction was incubated at 4 ° C for 15 minutes in the dark. Then, the cells were washed with 4 mL of DPBS, centrifuged at 500 g for 5 minutes, and then the supernatant was removed. Finally, 0.5 mL of DPBS was added to resuspend the cells, followed by The cell surface fluorescent label was analyzed and quantified by flow cytometry (SONY; SH800Z); the aforementioned cell fluorescent labeling reagent contained the fluorescent label Anti-CD3 mAb associated with T cells (Invitrogen; Cat# 11-0038-42) ), and fluorescent markers associated with NK cells, Anti-CD16 mAb (Invitrogen; Cat# 17-0168-42), Anti-CD56 mAb (Invitrogen; Cat#12-0567-41), and Anti-CD314 (NKG2D) mAb (BD; Cat# 562365).

cell counts

A cell sample from which the cell survival rate is to be analyzed is obtained from the cell culture tray, and the cell sample is uniformly suspended by a micropipette to obtain a cell suspension, and then 20 μl of the cell suspension and 20 μl of 0.4% trypan blue dye (Trypan blue; Gibco; Cat# 15250-061) uniformly mixed to obtain a cell mixture, and then 10 to 20 μl of the cell mixture was injected into the groove between the coverslip and the cell counting plate (Marienfeld; Cat# AP-0650030) using a micropipette. Then, the cell counter disk was observed under a microscope, and the cells were counted after the cells were at rest.

The four corners of the cell counting disk respectively include a 4*4 area, and the counting range includes the four areas; the bright cells in the four areas are counted, the number of living cells is obtained, and the four areas are counted. The dark cells and dark blue cells obtain the number of dead cells, and then calculate the cell density, cell total and cell survival rate by using these values; among them, cell density (cells/ml) = (four regions live cells / 4) x (dilution factor 2) x 10 ⁴ /mL; total cells (cells) = cell density (cells / ml) x cell culture volume (mL); cell viability % = viable cells (cells) / (live Number of cells + number of dead cells) (cells) x%.

Cytotoxicity test

In the experimental system, the amplified NK/NKT cells were used as effector cells, and the K562 cell line was used as the target cells. The effector cells and the labeled cells were mixed and cultured in a ratio of 1:1. After 4 hours, the cells were stained with the staining agent 7-AAD. Since 7-AAD could not penetrate the cell membrane of normal cells, it could only penetrate cells that are in the process of apoptosis or dead, so it can be detected by cells. The signal of 7-AAD is used as the basis for apoptosis.

Next, please refer to FIG. 1 , which is a schematic flow chart of a first embodiment of the present invention; as shown in the figure, the in vitro expanded natural killer cells (NK cells) and natural killer T cells (NKT cells) described in this embodiment. The method comprises the following steps: Step S10: suspending a peripheral mononuclear cell (PBMCs) in a cell culture solution to obtain a cell mixture; and step S12: cells of the cell mixture density was adjusted to ^{0.5x10 6 ~ 5x10 6 cells / mL} , as an initial cell density, the cell culture medium using the cell mixture was cultured in a cell culture dish; step S14: when the total of the cell culture plate The cell culture medium is renewed when the number of cells reaches 2.5 x 10 ⁶ to 5 x ^{10 6} cells/mL, and the cell density is re-adjusted to 0.5×10 ⁶ to 5×10 ⁶ cells/mL; Step S16: the cell culture medium is updated every 2 to 3 days. and re-adjust the cell density of ^{0.5x10 6 ~ 5x10 6 cells / mL} , incubated for at least 10 to 16 days; and step S18: the cell mixture is washed to obtain a population of cells and the cells of the mixture, i.e., the cell population Once amplified Natural killer cells and natural killer cell mixtures T.

Before the step S10, the flow chart of the second embodiment shown in FIG. 2 is further included. As shown in the figure, the step of collecting the peripheral blood mononuclear cell line in the embodiment includes the following steps: Step S101: centrifuging one a peripheral blood sample, the peripheral blood sample is layered into a plasma layer, a buffy coat and a red blood cell layer; step S102: removing the plasma layer and obtaining the white blood cell layer, and mixing the white blood cell layer with Hanks Balanced salt solution (HBSS) and a mononuclear cell fraction to obtain a leukocyte mixture; step S103: centrifugation to layer the leukocyte mixture; and step S104: removing the supernatant and obtaining an intermediate layer of white liquid, That is, the peripheral mononuclear cells are obtained.

This embodiment is a process for preparing peripheral blood mononuclear cells as described in the materials and methods; wherein, in step S11, a layered peripheral blood sample is obtained, and then in step S12, a 10 mL sterile pipette is used together with an auxiliary device to remove the blood sample. Plasma, in the process of aspiration, stops when the liquid surface is close to 0.4 to 0.5 cm from the white blood cell layer to avoid consumption of white blood cells, and also avoids disrupting the interface of peripheral blood stratification, and then the white blood cells are mixed and diluted with HBSS to It is suitable for the subsequent separation of the mononuclear sphere; and in the step S14, in the process of sucking up the supernatant, when the liquid surface is close to 0.5-1 cm from the middle layer white liquid, the suspension is stopped, also to avoid consumption of white blood cells. And avoiding disrupting the layered interface; by the method of the present embodiment, peripheral blood mononuclear cells can be obtained, which is the initial cell source for amplifying NK/NKT cells in the first embodiment.

Next, in step S10, the peripheral blood mononuclear cells obtained in steps S11 to S14 are suspended in the cell culture solution, and the cell culture solution needs to provide sufficient nutrients during cell growth, and the cells are expanded. While providing appropriate factors to stimulate and activate NK/NKT cells, for the above purposes, the cell culture solution of the present embodiment uses a stem cell growth medium (SCGM) as a basal medium, and the concentration range is from 150 to 1,500 IU. One of the /mL human interleukin-2 recombinant protein (rhIL-2), and an anti-CD3 monoclonal antibody (anti-CD3 mAb) at a concentration ranging from 10 to 800 ng/mL as activated NK / NKT cell stimulating factor, in the present embodiment, rhIL-2 can also promote the growth of NK / NKT cells to increase the total number of cells of NK / NKT cells, in addition, this example also added volume percentage between 1 ~ 10% of the serum; the above additive, in a preferred embodiment, the final concentration of 250-1,000 IU/mL of rhIL-2, 50-500 ng/mL of anti-CD3 mAb and 1~ 5% serum, which can be the source of the peripheral blood sample Serum, or serum from other animal sources.

In step S12, the cell density of the cell mixture obtained in step S11 is adjusted to 0.5×10 ⁶ to 5×10 ⁶ cells/mL, and the initial cell density is placed on the cell culture plate carrying the cell culture solution. Then, the cell culture plate was placed in a cell culture incubator, and mononuclear cells were cultured at 37 ° C and 5% carbon dioxide, and this step was defined as the day 0 of culture, and the initial cell density provided cells. Proper contact with the cells and good growth space to promote the cells to a stable state for subsequent growth and division; in a preferred embodiment, 0.5 x 10 ⁶ to 2.5 x 10 ⁶ cells/mL The number of cells was used as the initial cell density, and the cells were cultured in a thin neck culture dish (flask).

In the step S14, when the total number of cells in the cell culture tray is increased to about 2.5× ^{10 6} to 5× ^{10 6} cells/mL, the cell culture medium in the cell culture tray is renewed, and the cell density is adjusted again to 0.5×10. ⁶ ~ 5x10 ⁶ cells / mL, in order to maintain a good growth environment in the cell culture tray and sufficient growth nutrients; in a preferred embodiment, the total cell number is grown to 2.5x10 ⁶ ~ 3.5x10 ⁶ cells / mL, Or until the fifth day of culture, at this time, the cells are in a stable state and have better cell growth and division efficiency, that is, the cell density is again adjusted to 0.5 x 10 ⁶ to 5 x 10 ⁶ cells/mL, and the amplification is continued. NK/NKT cells.

Subsequently, after step S14, the cell culture medium is updated every 2 to 3 days, and the cell density is re-adjusted to 0.5×10 ⁶ to 5×10 ⁶ cells/mL to continuously maintain a good growth environment in the cell culture tray and sufficient. The nutrient is grown and cultured until day 10 to day 16; in a preferred embodiment, in the process of amplifying NK/NKT cells, on day 5, day 7, day 9, and day after culture. The cell density was readjusted on days 12, 14, and 16, and the cell density was adjusted to 0.5 x 10 ⁶ to 5 x 10 ⁶ cells/mL.

Continuing, before step S16, a part of the cell mixture in the cell culture tray is taken as a test sample for cell counting and cell characterization; the cell trait analysis is performed by analyzing the cells as described in the materials and methods. The surface antigens CD16, CD56 and CD3 define the NK/NKT cells contained in the expanded cell population to analyze the proportion of NK cells and NKT cells, wherein the NK cell line cell surface antigen CD16 ⁺ CD56 ⁺ CD3 ^- The cell population, while the NKT cells are the cell surface antigen CD16 ⁺ CD56 ⁺ CD3 ⁺ cell population, and the cell population amplified by the procedure of the present embodiment has a NK/NKT cell ratio of up to 90%.

Finally, in step S18, after washing the cell population, the amplified natural killer cell and natural killer T cell mixture is obtained, which is suspended in a physiological grade sterile phosphate buffer solution. The cell population was floated, centrifuged for 10 minutes to remove the supernatant, and this step was repeated at least three times to ensure removal of the cell culture fluid and the added substances it contained.

The amplified natural killer cell and natural killer T cell mixture obtained by the foregoing steps may be mixed and stored in a suitable solvent, or further prepared as a pharmaceutical composition, and the solvent may be a monophosphate buffer solution or a cell. External liquid (Plasma-Lyte A)

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example

Example 1. Optimization of initial cell number

The purpose of this example is to establish a preferred initial cell density for use in the method of in vitro expansion of natural killer cells (NK cells)/natural killer T cells (NKT cells) of the present invention, which is obtained by the method described above for obtaining peripheral blood mononuclear cells. The cells were cultured with autologous serum of the peripheral blood sample source (containing 250 IU/ml of rhIL-2 and 50 ng/ml of Anti-CD3 monoclonal antibody), and cell density assessment was performed by the aforementioned cell counting method; As shown in Table 1, the cells were cultured at initial cell densities of 1.00 x 10 ⁶ , 2.00 x 10 ⁶ , 3.00 x 10 ⁶ and 5.00 x 10 ⁶ cells/ml, respectively, and ⁵ , ⁷ , ⁹ , 12, ¹⁴ , 16 after culture. On the 18th and 20th day, the cells were taken out from the culture plate for counting, and the cell density was readjusted to 1.00 x 10 ⁶ cells/ml. From the results, it was found that when the NK/NKT cells were expanded in vitro at these initial cell densities, The cell survival rate can be maintained above 90%. Among them, when cultured at an initial cell density of 1.00 x 10 ⁶ cells/ml, the cell expansion efficiency is the best, and the number of cells that can be obtained after amplification is also the highest; Therefore, in a preferred embodiment of the present invention, optional ^{1.00 x10 6, 2.00x10 6, 3.00} x10 6 and 5.00 x10 ⁶ cells / ml initial cell density of the culture.

Example 2: Optimization of cell culture days

In order to achieve better cell growth efficiency, the present embodiment continues to explore the required number of cell culture days and the time interval for re-adjusting the cell density; in this embodiment, the peripheral blood mononuclear cells are obtained. A preferred embodiment of 1.00 x 10 ⁶ cells/mL is the initial cell density, using autologous serum of the peripheral blood sample source (containing 250 IU/ml of rhIL-2 and 50 ng/ml of Anti-CD3 monoclonal antibody). The cells were counted in the culture time indicated in each group in Table 2. After the cell count, the cell density in the culture plate was readjusted to 1.00 x 10 ⁶ cells/mL to continue the culture.

As shown in Table 2, the system is divided into three test groups. The first group performs the first cell density adjustment on the 5th day after the cells are placed on the culture plate, and then re-adjusts the cell density to 1.00 every 2 days. X10 ⁶ cells/mL, followed by continuous culture for 3 days on the 9th day, and then re-adjusted the cell density to 1.00 x 10 ⁶ cells/mL every 2 days from the 12th day of culture, from the results, the second group Compared with the third group, the total number of cells in the first group continued to rise steadily after the 12th day of culture, and the total number of cells reached on the 20th day was 2 to 20 times higher than that of the other groups. And the cell survival rate is also maintained above 95%; accordingly, in a preferred embodiment of the present invention, the cell density adjustment interval as described in the first group of Table 2 is used as a culture condition for amplifying NK/NKT cells. In order to achieve better cell growth efficiency.

Example 3: Optimization of Human Recombinant Interleukin-2 (rhIL-2) Concentration in Cell Culture Medium

Since the amount of the additive in the cell culture fluid also affects the cell growth efficiency during cell expansion and culture, the present embodiment continues to investigate the cell density and the cell density interval at the beginning of the establishment of Examples 1 and 2. Under the culture condition, the concentration of rhIL-2 can further increase the cell growth efficiency; the peripheral blood mononuclear cells are obtained, and the peripheral cell density is obtained by using a peripheral blood test with a preferred embodiment of 1.00 x 10 ⁶ cells/mL. The autologous serum of the body source (containing rhIL-2 and 50 ng/ml of Anti-CD3 monoclonal antibody) was cultured, and the test group was respectively containing 250, 500, 750 or 1,000 IU/ml of rhIL-2.

As shown in Table 3, the cell expansion trend was the most stable in the group cultured with 500 IU/ml rhIL-2, and the total number of cells that could be obtained on the 20th day of culture was 1 to 3 times higher than that of the other groups; Accordingly, a preferred embodiment of the present invention can be cultured with 250 to 1,000 IU/ml of rhIL-2. Further, from the results of the present example, the cell culture medium for amplifying cells of the present invention can also be known. The immune cell growth can be further aided, i.e., the concentration of rhIL-2 selected in one embodiment of the invention increases the total number of NK/NKT cells in the expanded cell population.

Example 4: Optimization of antibody concentration and number of additions of anti-differentiation cluster-3 (Anti CD-3) in cell culture medium

The in vitro expanded immune cell line is applied to the human body to enhance immunity, thereby killing the tumor cells, in order to improve the immunotoxicity produced by the in vitro expanded immune cells entering the human body, in addition to the proliferation of a large number of cells The obtained cells also need to have cytotoxicity. Here, the present embodiment further explores the anti-CD-3 monoclonal antibody for activating NK/NKT cells in vitro, which is preferably used in the process of expanding the cultured cells. The obtained peripheral mononuclear cells are obtained, and a preferred embodiment 1.00 x 10 ⁶ cells/mL is used as the initial cell density, and the autologous serum of the peripheral blood sample source (including 500 IU/ml of rhIL-2) is used. The cells were cultured with Anti-CD3 monoclonal antibody, and the test group was an anti-CD3 monoclonal antibody containing 50, 100, 250 or 500 ng/ml, respectively.

As shown in Table 4, the group cultured with 50 ng/ml and 500 ng/ml of Anti-CD3 monoclonal antibody reached a higher total number of cells on the 20th day of culture than the other groups. In order to reduce the preparation cost and reduce the residue of foreign substances in the prepared cell population, thereby reducing the immune response induced by the foreign substance, thereby reducing the risk of side effects when applied to the human body, a preferred embodiment of the present invention In the example, 50 to 500 ng/ml of Anti-CD3 monoclonal antibody was used for culture, and further, as shown in Table 5, the condition of adding 50 ng/ml of Anti-CD3 monoclonal antibody to the cell culture solution was used. After the cells were cultured, they were added once on days 0, 5, and 7, respectively, and the cells were more stably expanded than the other groups, except that the total number of cells after amplification in Example 3 was maintained, and the amplification was improved. The proportion of NK/NKT cells with virulence activity in the cell population.

Example 5: Proportion of natural killer cells (NK cells) and natural killer T cells (NKT cells) obtained by amplification

According to the data of the above examples, the method for amplifying natural killer cells and natural killer T cells in vitro can obtain 3,000 to 7,000 times of cells in the peripheral mononuclear cells within 18 days of culture. And the cell viability is higher than 85%; then, this example further establishes that the method of the present invention can indeed amplify NK/NKT cells in the primary cells (i.e., peripheral mononuclear cells).

In the present embodiment, the expanded cell population is obtained by the foregoing examples, and then the flow cytometry is used to analyze the effective cells (ie, NK/NKT cells) in the expanded cell population. The ratio of NK/NKT cells in the expanded cell population is quantified by flow cytometry according to the cell marker of NK cells and NKT cells, respectively. The NK cell specificity marker is CD56. And NK cell activation receptors CD16 and NKG2D, in addition, CD3 was used as a T cell-specific marker to analyze NKT cells; the assay was performed by cell population analysis using the cell surface antigen analysis protocol described in Materials and Methods.

3A and FIG. 3B, FIG. 3A shows the results of analysis of cell surface granularity and cell particle size. As shown by the results, most of the cell samples analyzed in this example are concentrated in the same community, indicating that their cell types are consistent. Moreover, the cell samples were not clearly distinguished into multiple communities, indicating the consistency of cell survival rate; Figure 3B is the analysis result of NK/NKT cells in the cell population, as shown in the figure, the cell population in the upper left corner As shown in Fig. 3A, the cell population that does not express CD3 but exhibits CD56 (CD3 ^- CD56 ⁺ ) is NK cells, which accounts for 5.64% of the total cells. In addition, the CD3 is also expressed in the upper right corner. The cell population of CD56 (CD3 ⁺ CD56 ⁺ ), which is NKT cells, and the cell ratio thereof is 55.82%, from which it can be seen that the proportion of NK/NKT cells in the cell population amplified by the method of the present invention reaches 62.5. % (NK cells 5.64 + NKT cells 55.82).

Please refer to 4A and 4B. Figure 4A shows the cells in the cell population showing CD16 and CD56 at the same time. The ratio is 23.03% of the total cells. After selecting the CD16 ⁺ CD56 ⁺ cell population from Figure 4A, analyze CD16 ⁺ In the cell population of CD56 ⁺ , the distribution of cells of NKG2D and CD3 was expressed. The result is shown in Fig. 4B. The cell population of CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ^{- in} the upper left corner is NK cells, and CD16 ^{+ is in the} upper right corner ^. The cell population of CD56 ⁺ NKG2D ⁺ CD3 ⁺ is NKT cells. According to the quantitative results of flow cytometry, the proportion of NK/NKT cells expressing NK cell-specific markers CD16, CD56 and NKG2D is 16.9% ( 23.03%*(15.37+58.3)%=16.9%).

Example 6. Cytotoxicity of natural killer cells (NK cells) and natural killer T cells obtained by amplification

Finally, in order to establish the method for in vitro expansion of NK/NKT cells of the present invention, the prepared NK/NKT cells are required to meet the requirements for preparation as a pharmaceutical composition, and the present embodiment utilizes the cytotoxicity described in the materials and methods. The test further analyzes the poisoning effect of the amplified NK/NKT cells on cancer cells; see Figures 5A and 5B, Figure 5A shows that the cell samples exhibit consistent cell type and cell survival rate, as can be clearly seen in Figure 5B. The K562 cells co-cultured with the NK/NKT cells obtained by amplification have a cell state of about 76.30% in the early apoptosis or apoptosis process, that is, the toxic effect of the amplified NK/NKT cells on cancer cells. Up to 76.30%.

In summary of the above examples, the method for in vitro expansion of natural killer cells and natural killer T cells disclosed in the present invention is confirmed by experiments, the initial cell density used in the step, and the cell culture is updated at a specific time point. The cell density in the plate can indeed give the cells a better growth environment, thereby increasing the amplification efficiency of the cells. In addition, the cell culture medium containing the additives rhIL-2 and anti-CD3 monoclonal antibodies can be more In addition to improving cell growth efficiency, the amplified NK/NKT cells can also be activated, and thus, the cells obtained by the method of the present invention can obtain not only a higher total number of cells but also a higher level than conventional techniques. Good cell viability, as well as higher NK/NKT cell ratio, and even more, the expanded NK/NKT cells also have better cytotoxicity, that is, the method of the present invention can indeed target cell populations. The NK/NKT cells are efficiently expanded and activated, and can be further applied to the preparation of pharmaceutical compositions.

While the foregoing specification has been described by the embodiments of the embodiments It will be appreciated, however, that the present invention may be modified and modified from the foregoing description to make it more suitable for various uses and applications without departing from the spirit and scope of the invention. Pieces. Accordingly, the scope of the present invention and its claims are intended to be illustrative, rather than limiting the scope of the invention

Claims (8)

A method for in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells), comprising the steps of: suspending a peripheral mononuclear cell (PBMCs) in a cell culture medium; to obtain a cell mixture; adjusting the cell density of the cell is a mixture of ^{0.5x10 6 ~ 5x10 6 cells / mL} , as an initial cell density, the cell culture medium using the cell mixture was cultured in a cell culture In the tray; when the total number of cells in the cell culture plate reaches 2.5× ^{10 6} 5× ^{10 6} cells/mL, the cell culture medium is renewed, and the cell density is re-adjusted to 0.5×10 ⁶ to 5×10 ⁶ cells/mL; every 2 3 days to update the cell culture medium, and re-adjust the cell density of ^{0.5x10 6 ~ 5x10 6 cells / mL} , incubated for at least 10 to 16 days; and the cells were washed and obtain a mixture of the cell population of the cell mixture The cell population is a mixture of the natural killer cell and the natural killer T cell; wherein the cell culture system comprises: a stem cell growth medium (SCGM); a human interleukin-2 recombinant protein (rhIL-2) , its concentration The range is between 250 and 1000 IU/mL; one serum is between 1 and 10% by volume; and the anti-CD3 monoclonal antibody is in the range of 10 to 500 ng/mL. Wherein the natural killer cell line cell surface antigen is a cell population of CD16 ⁺ CD56 ⁺ CD3 ^- and the cell surface antigen of the natural killer T cell line is a cell population of CD16 ⁺ CD56 ⁺ CD3 ⁺ . The method of claim 1, wherein the step of suspending the peripheral blood mononuclear cells further comprises the step of collecting the peripheral blood mononuclear cells, the method comprising: Centrifuging a peripheral blood sample to stratify the peripheral blood sample into a plasma layer, a buffy coat and a red blood cell layer; removing the plasma layer and obtaining the white blood cell layer, and mixing the white blood cell layer with Hanks balance Salt solution (HBSS) and a mononuclear cell separation solution to obtain a leukocyte mixture; centrifugation to layer the leukocyte mixture; and removing the supernatant, and obtaining an intermediate layer of white liquid, thereby obtaining the peripheral mononuclear ball cell. The method according to claim 1, wherein in the step of adjusting the initial cell density, the cell density of the cell mixture is adjusted to 0.5 x 10 ⁶ to 2.5 x 10 ⁶ cells/mL. The method according to claim 1, wherein in the step of renewing the cell culture medium and re-adjusting the cell density, the cell density of the cell mixture is adjusted to 0.5×10 ⁶ to 2.5×10 ⁶ cells/mL. The method of claim 1, wherein in the step of culturing the cell mixture, the cell mixture is cultured in an environment of 37 ° C and 5% carbon dioxide. The method of claim 1, wherein after the step of washing the cell mixture and obtaining the cell population, the method further comprises the step of suspending the cell population with a preservation solution, the preservation solution being a monophosphate buffer solution or An extracellular fluid (Plasma-Lyte A). The method of claim 1, wherein the amplified natural killer cell and natural killer T cell mixture has a cell viability of at least 60%, and the amplified natural killer cell and natural The natural killer cell and natural killer T cell line with cytotoxicity in the killer T cell mixture reaches at least 50%. A pharmaceutical composition of natural killer cells and natural killer T cells, comprising the amplified natural killer cell and natural killer T cell mixture prepared by the method described in claim 1; and a medicine An acceptable solvent.