CN110628714B

CN110628714B - Serum-free cell culture solution for in vitro amplification of natural killer cells and natural killer T cells

Info

Publication number: CN110628714B
Application number: CN201810644666.9A
Authority: CN
Inventors: 叶明功
Original assignee: Precision Biotechnology Co ltd
Current assignee: Precision Biotechnology Co ltd
Priority date: 2018-06-21
Filing date: 2018-06-21
Publication date: 2023-03-28
Anticipated expiration: 2038-06-21
Also published as: CN110628714A

Abstract

The invention relates to a serum-free cell culture solution for in vitro amplification of natural killer cells and natural killer T cells, which comprises a basic culture medium, a human interleukin-2 recombinant protein (rhIL-2), heparin and an anti-CD 3monoclonal antibody (anti-CD 3monoclonal antibody).

Description

Serum-free cell culture solution for in vitro amplification of natural killer cells and natural killer T cells

Technical Field

The invention relates to a cell culture solution for in vitro amplification of natural killer cells (NK cells) and natural killer T cells (NKT cells), in particular to a cell culture solution which can be used for in vitro amplification of three to seven thousand times of NK/NKT cells without adding serum, and the cell survival rate of the amplified NK/NKT cells is higher than 85%, the active NK/NKT cells reach more than 50%, and the poisoning effect on cancer cells reaches more than 76%.

Background

Immunotherapy (Immunotherapy), which broadly refers to a method for preventing and treating diseases by enhancing the autoimmune system of an organism or additionally endowing the organism with immune ability, has the characteristics of high specificity, high efficiency, durability and the like; in recent decades, immune cell therapy (immunotherapy) derived from the concept of immunotherapy has been widely studied.

In 2011 immune cell therapy was first proposed as a potential comment in the international journal, nature, which is considered as a possible treatment for cancer; the immune cell therapy focuses on stimulating the immune system and can destroy the residual cancer cells after operations (such as radiotherapy or chemotherapy), thereby improving the curative effect, and reducing the toxic effect of operations or chemotherapy and radiotherapy so as to improve the life quality and prognosis of patients; for example, cancer is treated by immunocyte therapy, in which immunocytes are separated from blood of a patient, cultured, amplified and activated in vitro, and then returned to the patient to generate specific immune response to tumors of specific tissues in the body, and immune rejection is avoided by using the returned immunocytes; among the immune cells, natural killer cells (NK cells) are expected to have higher antitumor efficiency than T cells because they can directly poison target cells without presenting antigens through antigen presenting cells.

The natural killer cells serve as a first line of defense against foreign substances in the immune system of a human body, the immune response initiated by the natural killer cells is nonspecific defense due to the lack of specific antigen receptors (TCR) on the cell surfaces of the natural killer cells, the natural killer cells are positioned in peripheral blood circulation and can attack virus-infected cells to prevent the human body from being infected by viruses, and rejection reaction can be generated for tumor cells and bone marrow transplantation to attack the foreign cells; perforin forms a hole in a target heterogeneous cell membrane, and then after the granzyme enters the target heterogeneous cell, the permeability of a mitochondrial membrane is changed, apoptosis (apoptosis) -related protein is activated to start an apoptosis (apoptosis) reaction, so that DNA in the target heterogeneous cell is fragmented and the cell is decomposed; in addition, natural killer T cells are a heterogeneous population of immune cells that express both T cell surface antigens (e.g., CD3, TCR. Alpha. Beta.) and natural killer cell surface antigens (e.g., CD 56), and thus can induce both specific immune responses, such as T cells, and non-specific immune responses, such as NK cells, and are considered to be a major focus in the development of immune cell therapy.

However, the present invention is directed to solving the above-mentioned problems by providing a cell-growth-promoting agent, which is capable of promoting cell growth, by using the natural killer cell in vitro amplification technology, and by adding other animal-derived serum substances during the preparation process, which may cause rejection of the cell recipient, and by adding host plasma or serum during the preparation process, which only limits the usage of the amplified cells in the host, which is a major bottleneck in the clinical development of immune cell therapy.

Disclosure of Invention

In one aspect, the present invention provides a serum-free cell culture solution for in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells), which does not require the addition of plasma or serum for the expansion of NK/NKT cells, thereby solving the problem of rejection of heterogeneous plasma or serum by organisms.

Accordingly, one aspect of the present invention relates to a cell culture solution for in vitro expansion of natural killer cells (NK cells) and natural killer T cells (NKT cells), comprising: a basal culture medium; a human interleukin-2 recombinant protein (rhIL-2) at a concentration ranging from 50 to 1,500IU/mL; heparin at a concentration ranging from 1 to 15U/mL; and an anti-CD 3monoclonal antibody (anti-CD 3monoclonal antibody) at a concentration ranging from 30 to 600ng/mL.

In some embodiments of the invention, the concentration of the human interleukin-2 recombinant protein ranges from 100 to 1,000IU/mL.

In some embodiments of the invention, the heparin concentration ranges from 2 to 10U/mL.

In some embodiments of the invention, the concentration of the anti-CD 3monoclonal antibody ranges from 50 to 500ng/mL.

In some embodiments of the invention, the NK cell is CD16 ⁺ CD56 ⁺ CD3 ^- And the natural killer T cells are CD16 ⁺ CD56 ⁺ CD3 ⁺ The cell population of (1).

In some embodiments of the invention, the natural killer cell is a cell surface antigen CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ^- The cell population of (1).

In some embodiments of the invention, the natural killer T cell is CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ⁺ The cell population of (1).

Drawings

FIGS. 1A-1B are a ratio analysis of expanded natural killer cells (NK cells)/natural killer T cells (NKT cells);

FIGS. 2A-2B are a ratio analysis of expanded natural killer cells (NK cells)/natural killer T cells (NKT cells);

FIGS. 3A-3B are cytocidal potency assays of expanded natural killer (NK cells)/natural killer T cells (NKT cells).

Detailed Description

Other features and advantages of the present invention will be further illustrated by the following examples, which are intended to be illustrative only and not limiting.

In view of the many needs to be improved in the prior art for in vitro amplification of natural killer cells (NK cells) and natural killer T cells (NKT cells), it is still necessary to rely on host plasma or serum, or other animal-derived serum, to provide nutrients necessary for cell growth, however, the addition of other animal-derived serum substances during the preparation process may cause rejection of cell recipients, and the addition of host plasma or serum during the preparation process limits the use of the amplified cells to the host, which is a major bottleneck in the clinical development of immune cell therapy; accordingly, the present invention provides a serum-free cell culture solution for in vitro amplification of natural killer cells (NK cells) and natural killer T cells (NKT cells), and a specific preparation method is used to effectively amplify NK/NKT cells, so as to improve the deficiencies of the prior art; hereinafter, the technical means and features of the present invention will be described.

Definition of

As used herein, the term "natural killer cell" (NK cell) means a cytotoxic lymphocyte cell (lymphocyte) belonging to the innate immune system (innate immune system) that can be induced to perform a non-specific cytotoxic effect.

As used herein, the term "NK cell" means an immune cell that expresses both α β T Cell Receptor (TCR) and a molecular marker associated with NK cells, and has both specific cellular immunotoxicity and non-specific cellular immunotoxicity.

For the purposes of the present specification, the term "mononuclear cells" (PBMCs) means cells having a single nucleus and a round shape, which include lymphocytes (lymphocytes), monocytes (monocytes) and dendritic cells (dendritic cells) such as T cells, B cells and NK cells, and the mononuclear cells located in peripheral blood may also be referred to as Peripheral Blood Mononuclear Cells (PBMCs).

Materials and methods

Preparation of reagents

Cell basic culture medium (medium)

One embodiment of the present invention uses SILAC Advanced DMEM/F-12 (Gibco) ^TM )、UltraGRO ^TM Or RPMI-1640medium (Sigma) as basic medium, which contains growth factor (growth factor) and cytokine (cytokine) for cell growth and division, and the original package is liquid medium, and is stored at appropriate temperature for use after being subpackaged.

Human recombinant interleukin-2 (recombiant human interleukin-2, rhIL-2)

Under aseptic operation environment, 1mL of sterile water is taken from a sterile syringe, and 1.1 mg of rhIL-2 powder is injected

After being uniformly dissolved, the rhIL-2 stock solution with the concentration of 1.1 milligram per milliliter (mg/mL) is obtained, then the rhIL-2 stock solution is taken out by a sterile syringe and is placed in a 50mL centrifuge tube, and 43mL of cell culture medium is added to adjust the concentration of the rhIL-2 solution to be 500 units per microliter (U/microliter) so as to obtain a rhIL-2 diluted solution; and subpackaging the rhIL-2 diluted solution into a micro sterile centrifuge tube, and storing in a refrigerator at the temperature of-20 ℃ for later use.

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

Taking an Anti-CD 3monoclonal antibody stock solution (Anti-CD 3monoclonal antibody with the concentration of 1mg is dissolved in 1mL of solvent) out of an original package (Takara) under an aseptic working environment, and mixing the Anti-CD 3monoclonal antibody stock solution into 4mL of cell culture medium to dilute the Anti-CD 3monoclonal antibody solution to the concentration of 20 mu g/mL; and subpackaging the Anti-CD 3monoclonal antibody solution diluted solution into a micro-aseptic centrifuge tube, and storing in a refrigerator at the temperature of-20 ℃ for later use.

Heparin (Heparin)

And (3) under the aseptic operation environment, subpackaging the heparin stock solution into 1mL centrifuge tubes, and storing in a refrigerator at the temperature of-20 ℃ for later use.

Serum-free cell culture solution for amplifying natural killer cells (NK cells)/natural killer T cells (NKT cells)

The rhIL-2 diluted solution, the Anti-CD 3monoclonal antibody diluted solution and heparin are respectively added into the three different cell basal culture media and are uniformly mixed to prepare three serum-free cell culture solutions for amplifying natural killer cells/natural killer T cells, wherein the serum-free cell culture solutions comprise rhIL-2 with the final concentration of 100-1,000IU/mL, anti-CD 3monoclonal antibody with the final concentration of 50-500 ng/mL and heparin with the final concentration of 2-10U/mL in a preferred embodiment.

Peripheral blood test body

Peripheral blood samples of the subjects are collected and stored in sterile blood collection tubes by outpatient clinics of hospital physicians, and the samples are transported to a human body Tissue excellent Practice (GTP) core laboratory according to the specifications for subsequent processing.

Cell (cell)

Preparation of Peripheral Blood Mononuclear Cell (PBMC)

Transferring peripheral blood from a blood collection tube to a 50mL centrifuge tube for gradient centrifugation, setting the centrifugal force of a centrifuge (AUBOCA, 4000/4200) to be 600Xg, closing the braking function of the centrifuge, and centrifuging at room temperature for 10 minutes to divide the peripheral blood sample into three layers, namely Plasma (Plasma), a Buffy coat (Buffy coat) and red blood cells (Erythrocytes) from top to bottom; then removing the plasma layer and transferring the leukocyte layer to a new sterile centrifuge tube, and slowly mixing the leukocytes using Hanks Balanced Salt Solution (HBSS) to obtain a leukocyte diluted solution; on the other hand, 1-3 mL of mononuclear cell separation liquid (Ficoll-Paque Plus) is put into a mononuclear cell separation centrifuge tube and centrifuged for 30 seconds at the room temperature by the centrifugal force of 600Xg for later use; then, the above-mentioned leukocyte diluted solution is added into a mononuclear cell separation centrifuge tube containing mononuclear cell separation liquid, and centrifuged at 600xg for 10 minutes at room temperature to separate layers, and then the supernatant is removed, and the white intermediate layer (i.e. peripheral blood mononuclear cells) is transferred to a new sterile centrifuge tube, and then added with HBSS to be uniformly mixed to clean the cells, and centrifuged at room temperature for 10 minutes to remove the supernatant, and then the cells are resuspended with HBSS, and centrifuged at room temperature for 10 minutes to collect the supernatant and peripheral blood mononuclear cells respectively for use.

Cell assay

Cell surface antigen analysis

Take 1x10 ⁶ cells are put into a 15mL centrifuge tube, centrifuged for 5 minutes by 500g of centrifugal force, then supernatant is removed, 4mL of Dulbeccos Phosphate Buffered Saline (DPBS) is used for resuspending the cells, the step is repeated twice, then 5 mu l of cell fluorescence labeling reagent is added, the cells are reacted for 15 minutes in a dark place at 4 ℃, then 4mL of DPBS is added for washing the cells, centrifuged for 5 minutes by 500g of centrifugal force, then supernatant is removed, finally 0.5mL of DPBS is added for resuspending the cells, and then the fluorescence labeling on the surfaces of the cells is analyzed and quantified by a flow cytometer (SONY; SH 800Z); the aforementioned cell fluorescent labeling reagents include T cell-associated fluorescent labeled Anti-CD3 mAb (Invitrogen; cat # 11-0038-42), and NK cell-associated fluorescent labeled Anti-CD16 mAb (Invitrogen; cat # 17-0168-42), anti-CD56 mAb (Invitrogen; cat # 12-0567-41), and Anti-CD314 (NKG 2D) mAb (BD; cat # 562365).

Cytotoxicity assays

The experiment uses the amplified NK/NKT cells as effector cells (effector cells) and K562 cell line as target cells (target cells), and the effector cells and the target cells are expressed in a ratio of 1:1, culturing for 4 hours after mixing, and staining cells by using a stain 7-AAD, wherein 7-AAD can not permeate cell membranes of normal cells and can only permeate cells in an apoptosis process or apoptotic cells, so that the signal of 7-AAD in the cells can be detected to serve as the basis of apoptosis.

Cell counting

A cell sample to be analyzed for cell viability is obtained from a cell culture disc, the cell sample is evenly suspended by a micropipette to obtain a cell suspension, then 20 mu l of the cell suspension is evenly mixed with 20 mu l of 0.4% Trypan blue dye (Trypan blue; gibco; cat # 15250-061) to obtain a cell mixed solution, then 10-20 mu l of the cell mixed solution is taken by the micropipette to be injected into a groove between a cover glass and a cell counting disc (Marienfeld; cat # AP-0650030), then the cell counting disc is placed under a microscope for observation, and the cell counting is started after the cells are static.

The four corners of the cell counting plate respectively comprise a 4-by-4 area, and the counting range comprises the four areas; counting the bright cells in the four areas to obtain the number of live cells, counting the dark cells and the dark blue cells in the four areas to obtain the number of dead cells, and calculating the parameter values such as cell density, total cell number and cell survival rate according to the numerical values; wherein, cell density (cells/ml) = (number of living cells in four regions/4) x (dilution factor 2) x10 ⁴ Per mL; total number of cells (cells) = cell density (cells/mL) x volume of cell culture fluid (mL); cell survival% = viable cell number (cells)/(viable cell number + dead cell number) (cells) x%.

Other features and advantages of the present invention will be further illustrated and described in the following examples, which are given by way of illustration only and are not intended to limit the scope of the invention.

Examples

Example 1 testing of serum-free culture solution

The purpose of this embodiment is to establish a serum-free cell culture solution that can be used for in vitro amplification of natural killer cells (NK cells)/natural killer T cells (NKT cells), and the cell culture solution does not require additional autologous plasma or serum derived from peripheral blood sample, or serum derived from other animal sources; the peripheral blood mononuclear cells are obtained as described above, and autologous serum, SILAC Advanced DMEM/F-12, and ultraGRO of the peripheral blood specimen are used as the peripheral blood specimen-derived material ^TM Or RPMI-1640medium, wherein the autologous serum of the peripheral blood sample source contains 500IU/ml rhIL-2 and 50ng/ml Anti-CD 3monoclonal antibody, SILAC Advanced DMEM/F-12, ultraGRO ^TM And RPMI-1640medium respectively containing 500IU/ml rhIL-2, 50ng/ml Anti-CD 3monoclonal antibody and 2U/ml heparin, culturing, and then passing through the above-mentioned cell counting methodCarrying out cell density evaluation; as shown in Table I, the cells were expanded at least 3,000 times in number whether cultured in autologous serum or in a serum-free cell culture medium; accordingly, NK/NKT cells could be efficiently amplified by the serum-free cell culture solution of the present example.

In this example, since the host autologous plasma or serum is not added during the process of expanding NK/NKT cells, the NK/NKT cells obtained by expansion can be administered to the host itself or the non-host recipient, and thus, when the recipient to be treated with the cells is weak due to a disease, the risk that the number of autologous cells is insufficient for in vitro expansion is avoided.

A first table,

Example 2 optimization of initial cell number

In light of the above, the present example further discusses the preferred initial cell density for NK/NKT cell expansion using serum-free cell culture medium, peripheral blood mononuclear cells were obtained as described above, and UltraGRO was used ^TM As a basal medium, 500IU/ml rhIL-2, 50ng/ml Anti-CD 3monoclonal antibody and 2U/ml heparin were added for culture, and then cell density evaluation was performed by the aforementioned cell counting method.

As shown in Table two, the initial cell density was 1.00x10 ⁶ 、2.00x10 ⁶ 、3.00x10 ⁶ And 5.00x10 ⁶ cells/ml, and at 5, 7, 9, 12, 14, 16, 18 and 20 days after the culture, the cells were taken out from the culture dish, counted and the cell density was readjusted to 1.00x10 ⁶ From the results, it was found that the cell viability was maintained at 90% or higher in the in vitro expansion of NK/NKT cells at these initial cell densities, wherein the cell viability was 2.00x10 ⁶ ceWhen the initial cell density of lls/ml is used for culturing, the cell amplification efficiency is the best, and the number of cells obtained after amplification is the largest; accordingly, in an embodiment of the present invention, 1.00x10 may be selected ⁶ 、2.00x10 ⁶ 、3.00x10 ⁶ And 5.00x10 ⁶ cells/ml of initial cell density, in a preferred embodiment, 1.5x10 ⁶ ～2.5x10 ⁶ cells/ml served as the initial cell density for ex vivo expansion of NK/NK T cells.

A second table,

Example 3 optimization of Heparin (Heparin) concentration in serum-free cell culture media

In one embodiment of the present invention, heparin is used to replace part of the serum, so this embodiment will be discussed to further improve the cell growth efficiency of heparin concentration in the serum-free cell culture medium and the initial cell density culture conditions established in embodiments 1 and 2; it obtains the peripheral blood mononuclear cells as described above, in a preferred embodiment 2.00x10 ⁶ cells/mL as initial cell density, using UltraGRO ^TM The cells were cultured in culture medium (containing 500IU/ml rhIL-2 and 50ng/ml Anti-CD 3monoclonal antibody), and the test groups contained 2, 5 or 10U/ml heparin.

As shown in Table III, the group cultured with 2U/ml heparin has the most stable cell expansion trend, and the total number of cells obtained at the 20 th day of culture is 1.5-4 times more than that of other groups, and on the whole, the total number of cells after expansion is not negatively influenced after the heparin is added; accordingly, in one embodiment of the present invention, the cell culture medium for expanding cells can be cultured with 2 to 10U/ml of heparin, and the results of this embodiment also show that the cell culture medium for expanding cells can further help the growth of immunocytes, i.e., the concentration of heparin selected in one embodiment of the present invention can increase the ratio of NK/NKT cells in the expanded cell population.

A third table,

Example 4 optimization of Interleukin-2 (IL-2) concentration in serum-free cell culture media

Since the amount of additives in the cell culture medium also affects the cell growth efficiency during the cell expansion culture, the present example discusses the rhIL-2 concentration that can further improve the cell growth efficiency under the culture conditions established in examples 1 to 3; it obtains the peripheral blood mononuclear cells as described above, in a preferred embodiment 2.00x10 ⁶ cells/mL as initial cell density, using UltraGRO ^TM Culturing in cell culture solution (containing 2U/ml heparin and 50ng/ml Anti-CD 3monoclonal antibody), wherein the test groups respectively contain 100, 250, 500 or 1,000IU/ml rhIL-2; as shown in Table IV, the results demonstrate that the cells can maintain a stable growth trend after being cultured with 100-1,000IU/ml rhIL-2, and thus, one embodiment of the present invention can be cultured with 100-1,000IU/ml rhIL-2, and the results of this embodiment also show that the cell culture solution for amplifying cells used in the present invention can further help the growth of immune cells, i.e., the concentration of rhIL-2 selected in one embodiment of the present invention can increase the total number of NK/NKT cells in the amplified cell population.

The fourth part of the table,

Example 5 optimization of anti-Cluster-3 (CD-3) antibody concentration in serum-free cell culture solution

The immune cells expanded in vitro are applied to human bodies to improve immunity and further have a poisoning effect on tumor cells, and in order to improve the effect that the immune cells expanded in vitro enter the human bodiesIn addition to the proliferation of a large number of cells, the resulting cells need to be cytotoxic, and this example further discusses the amount of Anti CD-3 monoclonal antibody used to activate NK/NKT cells in vitro, preferably used in the cell expansion process; it obtains the peripheral mononuclear cells as described above, in a preferred embodiment 2.00x10 ⁶ cells/mL as initial cell density, using UltraGRO ^TM The cells were cultured in culture medium (containing 2U/ml heparin and 500IU/ml rhIL-2), and the test groups contained 50, 100, 250 or 500ng/ml Anti-CD 3monoclonal antibody.

As shown in table five, the groups cultured with 50, 100 and 500ng/ml Anti-CD 3monoclonal antibodies all achieved higher total cell numbers at day 20 than the other groups, and thus, in one embodiment of the present invention, anti-CD 3monoclonal antibodies of 50 to 500ng/ml were selected for culturing, and as shown in table six, anti-CD 3monoclonal antibodies of 50ng/ml were added to the cell culture medium once each at day 0, day 5 and day 7 after cell culture, so as to stably expand cells compared to the other groups, in addition to maintaining the total number of expanded cells achieved in the above-mentioned examples, and increasing the proportion of NK/NKT cells having cytotoxic activity in the expanded cell population.

Table five,

The sixth part of the table,

Example 6 expanded Natural killer cell (NK cell) and Natural killer T cell (NKT cell) ratios

The data of the above examples show that the cell culture solution of the present invention can be used in combination with the above method for in vitro amplification of natural killer cells and natural killer T cells without the need of adding extra serum or plasma, and the cell amplification amount can reach 3,000 to 7,000 times and the cell survival rate is higher than 85% within 18 days of obtaining peripheral mononuclear cells and culturing; next, it was confirmed that the method of the present invention can amplify NK/NKT cells in primary cells (i.e., peripheral monocyte cells).

In this embodiment, an amplified cell population is obtained through the previous embodiment, and then the proportion of effective cells (i.e., NK/NKT cells) in the amplified cell population is analyzed by using a Flow cytometry (Flow cytometry); quantifying the proportion of NK/NKT cells in the amplified cell population by a flow cytometer according to cell markers (cell markers) of the NK cells and the NKT cells respectively, wherein the NK cell markers are CD56 and NK cell activation receptors CD16 and NKG2D respectively, and in addition, CD3 is used as a T cell marker for analyzing the NKT cells; assays cell population analysis was performed using the cell surface antigen analysis protocol described in materials and methods.

Referring to fig. 1A and fig. 1B, fig. 1A shows the analysis results of the cell surface granularity and the cell particle size, as shown by the results, most of the cell samples analyzed in this embodiment are concentrated in the same colony, which shows that the cell types are consistent, and the cell samples are not clearly differentiated into multiple colonies, which shows the consistency of the cell survival rates; FIG. 1B shows the analysis of the ratio of NK/NKT cells to cell population, and as shown in the figure, the cell population in the upper left corner shows a cell population that does not express CD3 but expresses CD56 (CD 3) in the cell population collected in FIG. 1A ^- CD56 ⁺ ) NK cells, which account for 17.16% of total cells, and CD56 (CD 3) in the upper right corner of the cell population ⁺ CD56 ⁺ ) The ratio of NK/NKT cells in the cell population expanded by the serum-free cell culture solution of the invention is up to 62.28% (NK cells 17.6+ NKT cells 45.66).

Please refer to fig. 2A and 2B, wherein the upper right corner of fig. 2A is shownShowing that the proportion of cells expressing both CD16 and CD56 in the cell population was 31.59% of the total cells, and selecting CD16 from FIG. 2A ⁺ CD56 ⁺ After the cell population of (3), CD16 was analyzed ⁺ CD56 ⁺ The cell population (2) showed the distribution of NKG2D and CD3 cells, and the result is shown in FIG. 2B, with CD16 at the top left ⁺ CD56 ⁺ NKG2D ⁺ CD3 ^- The cell population of (1), which is NK cells, has a cell ratio of 33.95% and CD16 at the upper right corner ⁺ CD56 ⁺ NKG2D ⁺ CD3 ⁺ The cell population of (a) is NKT cells, and the cell ratio thereof is 44.55%, it can be seen from the quantitative results of flow cytometry that the NK/NKT cell ratio of CD16, CD56 and NKG2D, which represent the NK cell-specific markers, reaches 24.8% (31.59% ((33.95) +44.55) = 24.8%).

Example 7 cytotoxic Capacity of expanded Natural killer cells (NK cells) and Natural killer T cells

Finally, in order to establish the method for in vitro amplification of NK/NKT cells by using the cell culture solution provided by the present invention, the prepared NK/NKT cells can meet the requirements for preparing pharmaceutical compositions, and the cytotoxicity test described in the materials and the method is utilized in the present embodiment to further analyze the poisoning effect of the amplified NK/NKT cells on cancer cells; referring to fig. 3A and 3B, fig. 3A shows that the cell samples exhibited consistent cell morphology and cell survival rate, and fig. 3B clearly shows that the proportion of the cell status of K562 cells cultured with the expanded NK/NKT cells in the early apoptosis or apoptosis process was about 75.59%, i.e., the toxicity of the expanded NK/NKT cells against cancer cells was 75.59%.

In combination with the above description, the serum-free cell culture solution for in vitro expansion of natural killer cells and natural killer T cells disclosed in the present invention has been tested to confirm that it can achieve better cell expansion efficiency without adding serum and the ratio of NK/NKT cells expanded is higher than that of the prior art; the additive Heparin contained in the cell culture solution provides factors required by cell growth, and plays a role of replacing serum, and the rhIL-2 and anti-CD 3monoclonal antibodies contained in the cell culture solution can improve the cell growth efficiency and activate the expanded NK/NKT cells so that the cells have better cytotoxicity; accordingly, since the cell culture solution of the present invention does not require the addition of serum derived from other animals during amplification, when the obtained cells are prepared into a pharmaceutical composition, rejection by a cell recipient can be avoided, and since the cell culture solution does not require the addition of autologous plasma or serum derived from peripheral blood samples for culture, when the obtained cells are prepared into a pharmaceutical composition, the cells are not limited to be administered to a cell-derived host; thus, the present invention does address the bottleneck of clinical development of immune cell therapy.

While the foregoing specification illustrates and describes various embodiments of this invention, it will be appreciated that those skilled in the art can readily devise various arrangements that are not intended to limit the invention to the exact construction and operation illustrated and described. It is to be understood that the present invention may be otherwise adapted to various uses and conditions, and that certain changes and modifications may be made from the foregoing description without departing from the spirit and scope of the invention. Accordingly, the description and claims set forth herein are intended to be illustrative, but not limiting, of the scope of the present invention in any way.

Claims

1. A serum-free cell culture solution for in vitro amplification of NK cells and NK T cells, comprising:

a basal medium which is UltraGRO ^TM ；

A human interleukin-2 recombinant protein, the concentration of which ranges from 100 to 1000IU/mL;

heparin at a concentration ranging from 2 to 10U/mL; and

an anti-CD 3monoclonal antibody at a concentration ranging from 50 to 500ng/mL.

2. The cell culture solution of claim 1, wherein the human interleukin-2 recombinant protein is at a concentration of 500IU/mL.

3. The cell culture solution of claim 1, wherein the heparin concentration is 2U/mL.

4. The cell culture solution of claim 1, wherein the concentration of the anti-CD 3monoclonal antibody is 50ng/mL.

5. A method for in vitro expansion of NK cells comprising culturing peripheral blood mononuclear cells with the cell culture medium according to claim 1, wherein the NK cells are CD16 as a cell surface antigen ⁺ CD56 ⁺ CD3 ^- The cell population of (1).

6. The method of claim 5, wherein the NK cell is CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ^- The cell population of (1).

7. A method for in vitro expansion of NK T cells comprising culturing peripheral blood mononuclear cells with the cell culture medium of claim 1, wherein the NK T cells are CD16 as a cell surface antigen ⁺ CD56 ⁺ CD3 ⁺ The cell population of (1).

8. The method of claim 7, wherein the NK T cell is CD16 ⁺ CD56 ⁺ NKG2D ⁺ CD3 ⁺ The cell population of (1).