TW201809254A - Cell preparation method, and cell culture vessel - Google Patents

Abstract

In order to achieve the practical use of an antigen-specific immuno-cell therapy, a cell culture vessel 1 in which multiple depressed parts 4, which serve as cell culturing sections, are provided on one surface of the walls of the vessel is used, so that cells are settled in the depressed parts 4 to prepare desired cells. In this manner, it becomes possible to appropriately prepare antigen-specific immunocytes in a large quantity, simply and safely, at low cost, and with high efficiency.

Description

Cell preparation method and cell culture container

The present invention relates to a cell preparation method capable of efficiently formulating immune cells responsible for a specific immune response, particularly for viruses, bacteria or cancer and other immune diseases, and a cell culture container used in the method.

In recent years, with the advancement of research on the human immune system, there has been increasing interest in immunotherapy using immune response to treat and prevent conditions such as cancer or viral infectious diseases. In particular, since the discovery of virus-specific antigens and tumor-specific antigens, research into antigen-specific immunotherapy as a new antiviral and anticancer therapy has been carried out, and clinical trials have been conducted in various countries around the world. Among them, the use of autologous or allogeneic alloantigen-specific immune cells and antigen-specific genetically modified immune cells to continuously accumulate opportunistic infectious diseases, lymphoma, melanoma, nasopharyngeal carcinoma, and blood cancer after hematopoietic cell transplantation. And effective clinical data of autoimmune diseases.

As the fastest progressing medical technology, antigen-specific immune cell therapy has developed TIL therapy using tumor infiltrating cells, CTL therapy using cytotoxic T cells, and helper T cells in the past two decades. Cellular HTL therapy, CAR-T using genetically modified immune cells, CAR-NK therapy, and TCR-T therapy, etc., are undergoing clinical trials.

Different from the so-called non-specific immune cell therapy, which is traditionally led by LAK therapy, CIK therapy, etc., the core technology of antigen-specific immune cell therapy is to efficiently induce and transform only the immune cells responsible for specific immune responses in vitro. , Or grow to a sufficient amount.

For example, in CTL therapy and HTL therapy, peripheral blood mononuclear cells taken from the patient itself are used to induce cytotoxic T cells (CTL) or helper T cells (HTL) that are specific to the antigen, and then grow. Infusion to the patient. In many clinical trials using this therapy, the number of cells infused into a patient is preferably 1 × 10 ⁷ or more at a time. For this reason, for example, when the proportion of antigen-specific immune cells is 10%, the total number of cells at the end of the culture needs to be at least 1 × 10 ⁸ (hundreds of millions). Therefore, in order to reduce the burden on the blood collection subject, it is necessary to induce and grow more antigen-specific immune cells by one operation.

However, in peripheral blood, antigen-specific cytotoxic T cells (CTL) and helper T cells (HTL) exist only at a very low frequency of less than about one percent of the peripheral blood mononuclear cell portion. The cells are very difficult.

Moreover, it has been confirmed that the frequency of the presence of these antigen-specific immune cells varies from individual to individual, from type to type, and from allele (Allele). For example, the viral antigen HCMV, which is one of the therapeutic targets for opportunistic infectious diseases after hematopoietic stem cell transplantation, will be described as an example. HLA-A24 restricted HCMV-specific CTL more common in Asians The amount contained in peripheral blood is about one percent compared with the amount contained in peripheral blood of HLA-A2 restricted HCMV-specific CTL, which is more common in Europeans and Americans. This is considered to be one of the reasons why HLA-A24-restricted HCMV-specific CTLs are difficult to induce and grow in vitro. Therefore, there are few reports on in vitro formulation methods suitable for clinical applications regarding specific CTLs other than HLA-A2 restrictions.

As the average life expectancy in Asia is extended, it is expected that the number of patients infected with the virus and cancer will increase, and immunotherapy needs more HLA types as treatment targets than before. The inability to efficiently and universally formulate antigen-specific immune cells is a rate-limiting factor when transitioning to clinical trials or commercialization.

Although there have been many studies so far, the reported antigen-specific immune cell preparation method has the following problems, so it has not been used commercially.

(1) In order to induce antigen-specific immune cells, for example, in the case of antigen-specific CTLs, it is necessary to prepare antigen-presenting cells. Although dendritic cells are mostly used as antigen-presenting cells, the preparation of dendritic cells is complicated and expensive.

(2) When the induced antigen-specific CTL is further grown, antigen-presenting cells are used (see Non-Patent Document 1). Therefore, the same problem as the above (1) occurs. Furthermore, in order to prepare the antigen-presenting cells used for growth, blood must be collected again from the donor. In addition, more antigen-presenting cells are required for growth than for induction, so the burden on the donor is large.

(3) As another means for further growth of the induced antigen-specific CTL, non-specific stimulation of OKT3 or lectin is sometimes used. Of course However, this method is relatively easy to grow cells other than the antigen-specific CTL, so it lacks efficiency. In addition, the REM method is known as a more efficient growth method. The REM method requires a large number of peripheral blood mononuclear cells and EBV-LCL and has a significant problem of virus incorporation, so it is not a practical method in terms of commercialization.

Under such circumstances, in order to realize the practical application of antigen-specific immune cells, especially antigen-specific CTL cells, the present invention proposes a method for preparing peripheral blood mononuclear cells and antigen-specific cells without preparing antigen-presenting cells. A method for preparing peptides and stimulating substances to induce antigen-specific immune cells from peripheral blood mononuclear cells quickly and easily.

In addition, since complicated steps are required, conventional antigen-specific immune cell culture methods have had to rely on 24-well flat culture plates (see Non-Patent Document 2), flat flasks (see Non-Patent Document 3), and stirred culture tanks. (See Non-Patent Document 4) and the like.

When using an open culture container, there are many unresolved problems. For example, a culture method using EBV-LCL as a more efficient preparation method is given as an example. Specifically, in this culture method, EBV-LCL cells are pulsed with an antigen peptide, and then deactivated by X-ray (40 Gy) irradiation. Thereafter, it was mixed with the collected 1 × 10 ⁶ peripheral blood mononuclear cells at a ratio of 40: 1 and seeded on a 24-well flat culture plate. At this time, a maximum of 2 mL of medium was added to each well. During cell culture, whenever restimulation of EBV-LCL cells is required, or when the culture medium is depleted, the cells need to be recovered by centrifugation repeatedly, resuspended and seeded in fresh medium.

During the growth period of the antigen-specific immune cells, especially in the later stages of growth, the proportion of the antigen-specific immune cells in the total cells varies drastically compared with the pre-induction period, so it is not appropriate to control the necessary nutrients and cytokines in an appropriate amount.

Therefore, the cultivation of the open system is indispensable for frequent replacement of the culture medium. In order to prevent the incorporation of bacteria and viruses from the outside, it must be performed in a cell processing facility, which requires significant equipment and management costs.

Usually, the number of cells finally obtained in a 24-well flat culture plate is 1 × 10 ⁷ or less. It is not possible to obtain a sufficient number of cells for treatment without expanding the scale with a plurality of culture plates. Furthermore, the capacity of a culture vessel such as a flat culture plate or a flat flask is limited, and it is very unsuitable for mass preparation of cells.

In addition, non-breathable plastic culture vessels such as flat culture plates and flat flasks have problems that cannot be overcome at all. That is, it is impossible to adjust the efficient gas exchange and the maintenance of stable buffer energy at the same time. Research reports to date show that strict control of the gas exchange rate (O ₂ / CO ₂ ) of the medium and pH conditions is important for the culture of antigen-specific immune cells (see Non-Patent Document 5). In order to fully ensure gas exchange, an empirical volume of 1 mL of culture medium must have a surface area of 1 cm ² or more. That is, the shallower the medium covering the cells, the better the gas exchange rate. Conversely, the shallower the culture medium covering the cells, the poorer the nutrient supply capacity and the pH buffering capacity tend to become worse due to the accumulation of lactic acid, carbon dioxide, and the like by the cell's metabolic waste. After all, this dilemma cannot be overcome by non-breathable plastic culture vessels such as flat culture plates and flat flasks.

On the other hand, in order to improve the gas exchange rate, a stirred culture tank is designed. However, the shear stress generated by the stirring rod is likely to cause damage to the cells, and as a result, the dead cell rate increases, making it impossible to become a general-purpose culture container.

In addition, in order to solve several problems of the above-mentioned open-type culture container, a closed-type culture system using a flat culture bag has been used in the cultivation of non-specific immune cells such as LAK therapy and CIK therapy in recent years. However, the use of such flat culture bags for antigen-specific immune cells has problems of low induction efficiency and high dead cell rate.

The film material of the flat culture bag is very soft, and the cells in the container may be placed in a tilted state in the culture container, or due to the distortion of the film caused by standing, or even a little vibration from the surrounding environment and the incubator. Concentrated to a part of or near the edge of the container, the damage caused by the physical collision between the cells and the unevenness of the cell density in the liquid phase are the reasons for the decline in cell growth.

In addition, although the interval can be set to prevent concentration near the edges, the dispersion and concentration of cells in each region cannot be controlled, and as a result, it is difficult to effectively induce antigen-specific immune cells.

[Prior technical literature]

[Non-patent literature]

Non-Patent Document 1: Foster AE, Gottlieb DJ, Marangolo M, Bartlett A, Li YC, Barton GW, Romagnoli JA, Bradstock KF. Rapid, Large-scale generation of highly pure cytomegalovirus-specific cytotoxic T cells for adoptive immunotherapy., J Hematother Stem Cell Res. 2003 Feb; 12 (1): 93 -105.

Non-Patent Document 2: Leen AM, Christin A, Myers GD, Liu H, Cruz CR, Hanley PJ, Kennedy-Nasser AA, Leung KS, Gee AP, Krance RA, Brenner MK, Heslop HE, Rooney CM, Bollard CM. Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation., Blood. 2009 Nov 5; 114 (19): 4283-92.

Non-Patent Document 3: Hanley PJ, Lam S, Shpall EJ, Bollard CM. Expanding cytotoxic T lymphocytes from umbilical cord blood that target cytomegalovirus, Epstein-Barr virus, and adenovirus., J Vis Exp. 2012 May 7; (63): e3627.

Non-Patent Document 4: Bohnenkamp H, Hilbert U, Noll T. Bioprocess development for the cultivation of human T-lymphocytes in a clinical scale., Cytotechnology. 2002 Jan; 38 (1-3): 135-45.

Non-Patent Document 5: Nakagawa Y, Negishi Y, Shimizu M, Takahashi M, Ichikawa M, Takahashi H. Effects of extracellular pH and hypoxia on the function and development of antigen-specific cytotoxic T lymphocytes., Immunol Lett. 2015 Oct; 167 (2): 72-86.

In view of the above situation, in order to realize the practical application of the antigen-specific immune cell therapy, the present invention provides a cell preparation method that has both simplicity and safety, and can be used to mass-produce antigen-specific immune cells in large quantities at low cost and efficiency. And the cell culture container it uses.

The cell preparation method of the present invention is a cell preparation method for preparing antigen-specific immune cells through co-culture of peripheral blood mononuclear cells with an antigen-specific peptide or a transformation virus. This method is used on one side of the inner wall surface of a container. A method of setting a plurality of depressions as a concave part of the cell culture part, and culturing the cells to the concave part to prepare the target cells.

Furthermore, the cell culture container of the present invention is a cell culture container used in the above-mentioned cell preparation method, and is provided with a container body composed of a gas-permeable plastic film and an injection inlet / outlet port 3. The container body has a periphery. The portion of the container body has a convex shape, and the bottom surface of the container body is provided with a plurality of bowl-shaped concave portions having opening diameters of 1.5 mm or more as the cell culture portion.

With the cell preparation method of the present invention, a target cell can be prepared by a simple operation without requiring complicated operations. Moreover, the time required for preparation is greatly reduced.

Moreover, with the cell culture container of the present invention, all the culture steps can be performed in a closed system. This brings an increase in security. Since high security can be ensured, the level of necessary equipment can be reduced and the risk of contamination can be reduced.

1‧‧‧ culture container

2‧‧‧ container body

2b‧‧‧ underside

3‧‧‧ nozzle for injection

4‧‧‧ bowl-shaped recess

[Fig. 1] An explanatory diagram showing the outline of a cell culture container according to an embodiment of the present invention, (a) is a plan view, (b) is a side view, and (c) is a bottom view.

[Fig. 2] A microscope image showing a state of cell sedimentation in an evaluation example of the example.

Fig. 3 is a graph showing changes in the total number of viable cells in the induction step of the evaluation example of the example.

FIG. 4 is a graph showing a stained image of the HCMV-specific CTL positive rate and the number of CTL cells in the induction step of the evaluation example of the example.

Fig. 5 is a graph showing changes in the total number of viable cells in an induction step and a growth step in the evaluation example of the example.

FIG. 6 is a graph showing a stained image of the HCMV-specific CTL positive rate and the number of CTL cells in the growth step of the evaluation example of the example.

Hereinafter, a preferred embodiment of the present invention will be described with reference to the drawings.

[Culture container]

The cell culture container 1 shown in FIG. 1 is provided with a container body 2 composed of a gas-permeable plastic film, and an injection inlet / outlet port 3 composed of a tubular member such as a culture medium and cells that can be circulated.

In addition, the container body 2 has a pillow-shaped bulged convex shape in which the peripheral portion is sealed, and the bottom surface 2b of the container body 2 is provided with a plurality of bowl-shaped concave portions 4 having opening diameter r of 1.5 mm or more as a cell culture portion. On the other hand, the top surface 2a of the container body 2 is made flat.

The bowl-shaped recess 4 formed in the bottom surface 2b of the container body 2 is preferably formed in a U-shaped cross section such that cells are easily concentrated on the bottom thereof, but is not limited thereto. It is preferable to have a diameter-reducing structure with a bottom area smaller than the opening area such that cells tend to concentrate on the bottom. Specifically, in addition to a U-shaped cross section, a semicircular cross section, a V-shaped cross section, and a truncated inverse cone shape may be used.

In addition, in order to suppress the cell movement in the container body 2, the cells in culture stay in a bowl-shaped recess 4, and the depth d of the bowl-shaped recess 4 is the ratio d / r of the depth d of the bowl-shaped recess 4 to the diameter r as 0.05 ~ 1 is preferred.

In addition, the arrangement of the bowl-shaped recesses 4 is preferably a staggered shape as shown in the figure so that the area of the bowl-shaped recesses 4 occupying the bottom surface 2b is as large as possible, but may be arranged in a grid shape as required.

In addition, although the size of the container body 2 is not particularly limited, examples are For example, it is better to set a length of 50 ~ 500mm and a width of 50 ~ 500mm.

Such a cell culture container 1 can be manufactured as follows, for example.

First, a plastic film on the top surface side as the top surface 2 a of the container body 2 and a plastic film on the bottom surface side as the bottom surface 2 b of the container body 2 are prepared. The bowl-shaped recessed portion 4 is formed on the protruding portion while being formed while retaining the peripheral portion of the bottom side plastic film and protruding. These can be formed by general vacuum forming, air forming, and the like, and the convex shape and the bowl-shaped concave portion 4 can be formed into a desired shape by appropriately adjusting a mold or the like.

Next, while the bottom-side plastic film and the top-side plastic film formed as described above are overlapped, a tubular member forming the injection inlet / outlet nozzle 3 is sandwiched at a predetermined position, and the peripheral portion is sealed by thermal fusion, and Trim the periphery as needed. Thereby, the cell culture container 1 shown in FIG. 1 can be manufactured.

The gas permeability of the plastic film forming the container body 2 is preferably an oxygen permeability of 3000 ml / (m ² ‧day‧ atm) or more measured at a test temperature of 37 ° C according to a gas permeability test method according to JIS K 7126. It is better to be above 6000ml / (m ² ‧day‧atm).

In addition, it is preferable that part or all of the plastic film has transparency. By doing so, even if the cells are not removed from the container, the progress of the cell culture, the state of the cells, and the like can be confirmed at any time from the outside by visual observation or microscope observation.

The material used to form the plastic film of the container body 2 is not particularly limited as long as it has a desired gas permeability. For example Thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, tetrafluoroethylene-hexafluoropropylene copolymer (FEP). Alternatively, polysiloxane can be used. These materials may be used in a single layer or in the same or dissimilar materials. However, in consideration of the heat-adhesiveness at the time of sealing the peripheral portion, a layer having a function as a sealant layer is preferred.

In addition, in order to have flexibility while the bowl-shaped recess 4 still has moderate shape retention without deformation, the thickness of the plastic film used to form the container body 2 is preferably 30 to 200 μm.

In addition, as described above, the injection inlet / outlet port 3 is formed of a tubular member that can circulate, such as a culture medium and cells. As the member for forming the injection nozzle 3, for example, a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, a polystyrene elastomer, or FEP can be formed into a predetermined shape by injection molding, extrusion molding, or the like. The specific form of the injection inlet / outlet port 3 is not particularly limited. For example, it can be made into a nozzle with an injection inlet and outlet made of an elastomer such as rubber, or it can be made into a luer lock type nozzle so that it can be injected with an injection needle.

The injection port 3 can also be used for the injection of cells such as peripheral blood mononuclear cells, and can also be used for the injection of target cells such as prepared antigen-specific immune cells. Although the number of injection ports 3 is one in the example shown in the figure, a plurality of nozzles corresponding to an operation error may be provided. In this case, by providing caps at each nozzle to prevent contamination, and by setting the caps to different colors or shapes for identification, it is possible to more reliably prevent operation errors.

In such a cell culture container 1, for example, when an antigen-specific immune cell is induced from a peripheral blood mononuclear cell or transformation is performed to prepare a target cell, a closed system is maintained through a liquid delivery tube connected to the injection inlet / outlet port 3, The peripheral blood mononuclear cells or other cells isolated from the peripheral blood of the patient, the antigen-specific peptide prepared according to the patient's HLA type, a stimulating substance, and a predetermined culture medium are injected into the container body 2 at a predetermined blending ratio.

Peripheral blood monocytes and antigen-specific peptides injected into the container body 2 settle in the culture medium and are distributed to the bowl-shaped recesses 4 and are concentrated to the bottom of each bowl-shaped recess 4 to suppress the movement in the container. . In this way, the cells are in close contact with each other, and peripheral blood monocytes and antigen-specific peptides can be co-cultured. For antigen-specific immune cells with a very low frequency, effective antigen presentation can be achieved, which can effectively To induce antigen-specific immune cells.

Moreover, by making each bowl-shaped recessed part 4 into almost the same shape, the cell density of each bowl-shaped recessed part 4 can be made almost uniform. Therefore, even if the cells concentrated in the bowl-shaped recess 4 are placed in a tilted manner in the cell culture container 1, or due to the distortion of the film generated during standing, or even some vibrations from the surrounding environment and the incubator, It does not move to other bowl-shaped recesses 4 as a result, and a certain aggregation can be formed in each bowl-shaped recesses 4. Thereby, the cell density can be maintained in a state that can be effectively induced, and the cells can not be induced and grown because of the decrease in the growth rate due to the denseness or the decrease in the induction efficiency due to the dispersion. As a result, it is possible to achieve homogeneous cell growth, and it is effective in reducing dead cells and improving induction efficiency. fruit.

In addition, the top surface 2a of the container body 2 is flat. By pressing the top surface 2a downward and contacting the bottom of the bowl-shaped recesses 4 with flexibility, the cells settled in each bowl-shaped recesses 4 can be easily made. Suspend again. Thereby, even if the cell density distribution of each bowl-shaped recessed part 4 is uneven, the cells in each bowl-shaped recessed part 4 can be dispersed almost evenly.

In addition, when recovering the grown cells, for example, the contents can be easily discharged from the injection / injection nozzle 3 by reversing and tilting the container, so it can be omitted as in the case of using a 24-well flat culture plate plastic container. The laborious task of injecting cells one by one into each well and recovering them with a pipette. In addition, since the cells can be filled or the grown cells can be recovered while maintaining a closed system, contamination by microorganisms such as bacteria, mold mold, and viruses can be reduced. Therefore, it is possible to more suitably perform infusion or culturing of cells embedded in the body or adhered to the injured site.

Furthermore, since the container body 2 is composed of a gas-permeable plastic film, a sufficient amount of oxygen can be supplied to cells in culture even if a closed system is constructed.

As described above, with the cell culture container 1 of this embodiment, all the culture steps can be performed in a closed system. This brings an increase in security. Since high security can be ensured, the level of necessary equipment can be reduced and the risk of contamination can be reduced. In addition, since the container body 2 is formed by using a plastic film, the container body 2 is large in capacity and light in weight, and does not occupy space, which is suitable for a large number of cell cultures. A sufficient amount of culture medium can be injected into the container body 2 in advance.

[Cell preparation method]

Next, an embodiment of the cell preparation method of the present invention using the above-mentioned cell culture container 1 will be described.

The target cells in the present invention are cells that can induce antigen-specific immune cells through co-cultivation of antigen-specific peptides and stimulating substances, and can be transformed into antigen-specific genetically modified immune cells through co-culture with virus solutions. cell. Especially suitable for peripheral blood mononuclear cells.

First, a culture medium and cells and antigen-specific peptides or a transformation virus solution are injected into the cell culture container 1 from the injection inlet / outlet port 3, and the cells and the like settled in the culture medium are collected at the bottom of each bowl-shaped recess 4. At this time, the culture medium is injected not only into the bowl-shaped recessed portion 4 but also to the extent that the bottom surface 2 b of the container body 2 is covered.

As the medium, a liquid medium is used, and it is appropriately selected according to the type of the cell. It can be permeated in a medium suitable for antigen-specific immune cell culture (for example, RPMI1640, AIM-V, DULBECCO's Modified Eagle Medium), X-VIVO (manufactured by BioWhittaker Inc.), ALyS505N (Manufactured by Cell Science & Technology Institute, Inc.), KBM series lymphocyte culture medium (manufactured by Kohjin Bio Co., Ltd., etc.), IL-2, etc. are added to prepare a cytokine-containing medium. Serum, plasma, serum albumin, antibiotics, L-glutamine and the like can also be added. As IL-2, use recombinant human IL-2 (for example, provided by Chiron Corporation) Teeleeukin provided by PROLEUKIN or Shionogi & Co., Ltd. is preferred. The content of IL-2 is, for example, 10 to 1,000 IU / mL.

Examples of antigen-specific peptides include viruses (cytomegalovirus, Epstein-Barr Virus, adenovirus, etc.), tumor antigens (WT1, hTERT, MN / CA9, gp100, MART1, TRP1, TRP2, tyramine) Acidase, MAGE1, MAGE2, MAGE3, MAGE6, NY-ESO-1, MUM1, BAGE, GAGE1, GAGE2, CEA, PSA, etc.) HLA-binding peptides. Usually, only one antigen-specific peptide is used, but it is also possible to use two or more antigen-specific peptides.

As a transformation virus, retroviral vectors (including oncogenic retroviral vectors, lentiviral vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, monkey virus vectors, and poxviruses can be used. Vectors or viral vectors such as Sendai virus vector, Epstein Barr virus (EBV) vector, HSV vector and the like. As the above-mentioned viral vector, it is suitable to have a lack of replication ability which cannot autogenously reproduce in infected cells.

Although the number of cells seeded in each of the bowl-shaped recesses 4 is affected by the size of the bowl-shaped recesses, the cell density is preferably about 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL. This is because when the cell is less than 1 × 10 ⁴ cells / mL, the probability of the cells meeting each other is low, so the induction efficiency is low. On the other hand, if the cell death rate exceeds 1 × 10 ⁶ cells / mL, the cell death rate increases, and the cell growth efficiency decreases.

(1) Induction steps of antigen-specific immune cells

After the foregoing operations, the cell culture container 1 is moved to an incubator set with conditions (typically, 37 ° C., 5% CO ₂ ) suitable for the culture of the antigen-specific immune cells. After a predetermined time (for example, 1 to 4 days) has elapsed, the cell culture container 1 is taken out of the incubator, and a medium containing cytokines is injected from the injection inlet / outlet port 3. After returning to the incubator for a predetermined period of time (for example, 2 to 7 days), the cytokine-containing medium is again injected into the cell culture container 1 from the injection port 3. Thereafter, the culture in the incubator was continued. It is also possible to culture while adjusting the cell density while resuspending via the rotating cell culture container 1 in a state where fresh medium is added. This allows the medium to be added without changing the container.

(2) Growth steps of antigen-specific immune cells

A small number of cells were collected from the cell culture container 1 in the cell culture, and the frequency and number of the antigen-specific immune cells were measured by a quantitative method of the antigen-specific immune cells described later. After that, the process proceeds to a growth step.

In this step, the induced antigen-specific immune cells are grown, and a large number of antigen-specific immune cells are prepared. If the positive rate of antigen-specific immune cells can be fully obtained in the aforementioned induction step, continue to set the conditions suitable for the culture of antigen-specific immune cells (typically, 37 ° C, 5% CO ₂ ). to cultivate.

After a predetermined time (for example, 1 to 4 days) has elapsed, the cell culture container 1 is taken out of the incubator, and the growth medium is injected into the cell culture container 1 through the injection inlet / outlet nozzle 3. After returning to the incubator for a predetermined time (for example, 2 to 7 days), the growth medium is injected again from the injection inlet / outlet port 3. its After that, the culture in the incubator was continued. Thereafter, the process proceeds to a cell recovery step. The interval from when the growth medium injection operation is performed to the next growth medium injection operation is, for example, 1 to 6 days. The interval between the first injection operation and the second injection operation can also be set to 3 days, and the interval between the second injection operation and the third injection operation can be set to two days. The interval here is not necessary.

The growth medium contains cytokines such as IL-2, IL-15, or both. The content of IL-2 is, for example, 10 to 1,000 IU / mL. On the other hand, the content of IL-15 is, for example, 10 to 100 ng / mL.

If the positive rate of antigen-specific immune cells is relatively low in the aforementioned induction step, in order to promote the growth of antigen-specific immune cells, any of the following operations (a) to (e) may be performed first, or combined use More than two operations, then move to the growth step.

(a) Additional antigen-specific peptides.

By further adding antigen-specific peptides, the expression of cell surface stimulating factors is promoted. Thereby, in the subsequent growth step, the growth rate of the induced antigen-specific immune cells can be made faster, and the growth can be performed with high efficiency. The amount of the antigen-specific peptide in the additional medium is, for example, 0.01 to 100 μg / mL.

(b) Adding anti-CD3 antibodies.

By adding anti-CD3 antibodies, the expression of costimulatory factors is promoted on the cell surface. Thereby, in the subsequent growth step, the growth rate of the induced antigen-specific immune cells can be made faster, and the growth can be performed with high efficiency.

The anti-CD3 antibody used can utilize the CD3 molecule or a part of it. Prepared by immunological methods. Commercially available anti-CD3 antibodies can also be used. An example of a commercially available anti-CD3 antibody is OKT-3 antibody (manufactured by Jiansheng Pharmaceutical Co., Ltd.). From the viewpoint of safety, it is preferable to use OKT-3 antibody which has been approved by the pharmaceutical industry.

Furthermore, the anti-CD3 antibody may be a single antibody or a multiple antibody. However, from the viewpoint of specificity and efficiency, an anti-CD3 antibody using a monoclonal antibody is preferred. Two or more anti-CD3 antibodies may be used in combination. The amount of the anti-CD3 antibody in the culture medium is, for example, 0.01 to 10 μg / mL.

(c) Adding anti-PD-1 antibody.

By adding anti-PD-1 antibodies, the immune checkpoint PD-1 molecules that inhibit the growth of antigen-specific immune cells can be blocked, and the induced antigen-specific immune cells can grow efficiently.

The anti-PD-1 antibody to be used can be formulated using PD-1 molecules or a part of them by immunological methods. A commercially available anti-PD-1 antibody can also be used. An example of a commercially available anti-PD-1 antibody is Nivolumab (manufactured by Ono Pharmaceutical Industry Co., Ltd.). From a safety point of view, it is preferable to use an anti-PD-1 antibody that has been approved by the pharmaceutical industry.

Furthermore, the anti-PD-1 antibody may be a single antibody or a multiple antibody. However, from the viewpoint of specificity and efficiency, an anti-PD-1 antibody using a monoclonal antibody is preferred. Two or more anti-PD-1 antibodies may be used in combination. The amount of the anti-PD-1 antibody in the culture medium is, for example, 0.01 to 10 μg / mL.

In addition to PD-1, there are many immune checkpoint molecules such as PD-L1, CTLA-4, TIGHT, TIM-3, LAG-3, B7-H3, B7-H4, BTLA that inhibit the growth of antigen-specific immune cells. , VISTA, etc. Antibodies corresponding to these molecules can be similarly added to the culture medium. Can also be used in combination Two or more anti-immune checkpoint molecular antibodies.

(d) Adding a low-molecular inhibitor that suppresses immune checkpoint molecules or suppresses the performance of regulatory T cells (Treg), metabolic pathways, or enhances the performance of HLA molecules.

Examples of the substance that suppresses the expression of an immune checkpoint molecule include low-molecular compounds such as Nafamostat Mesylate, a serine protease inhibitor that inhibits the expression of PD-L1. Examples of substances that inhibit the expression of Treg include low-molecular-weight compounds that inhibit only lenalidomide in effector Tregs (CD4 + FOXP3highCD45RAlow) cells, or indoleamine 2,3-dioxidase that inhibits Treg-like cells Inhibitor INCB024360 low molecular compounds and so on. Examples of substances that enhance the expression of HLA molecules include MAPK inhibitor Lapatinib Ditosylate. It is preferable to add any one or a plurality of the aforementioned low-molecular compounds to the growth medium in advance. The amount of the low-molecular compound in the culture medium is, for example, 1 pM to 1 μM.

(e) Adding substances such as Toll-like receptor (TLR) or RIG-I-like receptor (RIG-I-like receptor) to activate immunity through immune receptors.

Examples of the immune-activating component include an immuno-active shiitake mushroom component (extract and raw material powder, etc.), a seaweed (wakame root bud, seaweed, etc.) component (extract and raw material powder, etc.) and the like. These may be used alone or in combination of two or more.

In addition, as a constituent component of the pathogen or its mimetic substance, so-called Immune Potentiator (immunostimulator), lipoprotein (TLR1 Ligand), peptidoglycan (ligand for TLR2), dsRNA (ligand for TLR3) representing Poly (I: C) (polyinosinic-polycytidylic acid sodium salt) , Lipopolysaccharide (LPS) (ligand for TLR4), ssRNA (ligand for TLR7), Cp GDNA (ligand for TLR9), etc.

The immune-activating ingredients are important not only in regulating the innate immune system, but also in activating and controlling the acquired immune system. TLR is expressed in the T cells themselves and has the ability to directly control the function of T cells. For example, CD8-positive T cells express TLR2, which directly enhances cell growth and cell viability through the co-stimulation of antigen and TLR2 ligand, which helps reduce the need for indirect co-stimulation from dendritic cells. In addition, TLR ligands (especially TLR3, TLR7, or TLR9) can be used to increase the "cross-presentation" ability of the foreign antigen MHC type I, and the antigen specifically makes CD8-positive T cells effectively move towards cytotoxic T Cell (CTL) differentiation.

(3) Recovery steps of antigen-specific immune cells

A small number of cells were collected from the cell culture container 1 in the cell culture, and the frequency and number of the antigen-specific immune cells were measured by a quantitative method of the antigen-specific immune cells described later. Thereafter, the process proceeds to a recovery step.

In the recovery operation, the injection port 3 for the cell culture container 1 is used. That is, the grown antigen-specific immune cells are removed from the injection / exit nozzle 3. After removing the cells, a cell washing step using centrifugation can also be added. Such a cleaning step may be repeated (for example, 2 to 4 times).

(4) Quantification of antigen-specific immune cells

In each step of applying the cell preparation method of the present invention to prepare antigen-specific immune cells, understanding the change in the number of antigen-specific cells is very important for determining the adjustment during the step, judging whether it can be completed, the interval of adding medium, etc. important.

Quantitative measurement of antigen-specific immune cells for clinical treatment has the same meaning as measuring its effect components. Moreover, it is also important for quality identification to measure the amount of active ingredient of the final preparation provided by using the antigen-specific immune cells prepared by applying the cell preparation method of the present invention. Quantification of antigen-specific immune cells can be performed by the following three methods.

‧Quantitative method 1 (MHC-multimer method, etc.)

MHC-monomers and / or MHC-multimers produced using the same peptides as the antigen-specific peptides used for induction of antigen-specific immune cells can be used (hereinafter, sometimes collectively referred to as MHC-poly Body, etc.) to quantify or prepare the antigen-specific immune cells. The quantification can be performed in the following manner, for example.

The sample taken from the cell culture solution is reacted with an appropriate concentration of MHC-multimer or the like. Since the antigen-specific immune cells bound to the MHC-multimer or the like are stained with a marker pigment, they can be counted using a flow cytometer, a microscope, or the like. When reacting with MHC-multimers, etc., they can be determined simultaneously by reacting them with anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, etc., which are labeled with different pigments from MHC-multimers. Antigen A subpopulation of heterogeneous immune cells.

‧Quantitative Method 2

It is a kind of cytokines and / or chemotaxis such as IFNγ (interferon gamma), TNFα (tumor necrosis factor alpha), interleukin, etc. produced by antigen-specific cells by stimulating the cells taken by antigen-specific peptide Hormone approach. The method is specifically shown below by taking IFNγ as an example.

2-1) Method 1 for quantification of cytokines (quantification method for intracellular IFNγ producing cells)

The collected cells were floated in an appropriate medium at a cell concentration of about 2 × 10 ⁶ / mL, and an antigen-specific peptide was added. Intracellular protein transport inhibitors (for example, Brefeldin A, Monensin, etc.) are further added, and cultured in a constant temperature bath at 5% CO ₂ at 37 ° C. for 5 to 16 hours. After culturing, it is reacted with T cell-labeled antibodies (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody) or MHC-multimer, etc. After the cells are fixed, membrane permeation treatment is performed, and pigment-labeled anti-IFNγ antibodies are reacted. . Flow cytometry analysis was used to quantify the rate of IFNγ positive cells in whole cells, T cells, or positive cells such as MHC-multimers.

2-2) Method 2 for quantification of cytokines (ELISA-linked spots (ELISPOT analysis)

The collected cells were seeded in a 96-well MultiScreen-HA culture plate (manufactured by Millipore) in which the IFNγ antibody was immobilized. Thereafter, the antigen-specific peptide was added to each well, and cultured at 37 ° C for 20 hours in a 5% CO ₂ thermostat. The next day, the culture plate was washed and reacted in the order of anti-IFNγ antibody and peroxidase-labeled anti-IgG antibody. A peroxidase substrate was further added, and IFNγ spots were visualized by coloration, and were quantified by counting using a stereo microscope or an ELISPOT analyzer (manufactured by CTL).

2-3) Method 3 for quantifying cytokines (Method for quantifying IFNγ secreted into culture supernatant)

The prepared cell sample was floated in an appropriate medium at a cell concentration of about 2 × 10 ⁶ / mL, and an antigen-specific peptide was added. Incubate at 37 ° C for 24 to 48 hours in a 5% CO ₂ thermostat. After incubation, a commercially available ELISA kit (for example, Quantikine ELISA Human IFNγ Immunoassay from R & D Systems, Inc.) is used to quantify the IFNγ concentration contained in the culture supernatant.

‧Quantitative Method 3

Quantification was performed using cell surface protein-specific antibodies. The report indicates that immune cells that specifically recognize an antigen-specific peptide are stimulated by binding to the peptide, and the expression of cell surface proteins (such as CD137, CD107a, CD107b, CD63, CD69, etc.) is enhanced. Therefore, by mixing cells stimulated with an antigen-specific peptide or the like with a marker antibody that specifically recognizes cell surface proteins, the antigen-specific immune cells will bind to the marker antibody and be stained with the marker pigment. Stained antigen-specific immunity The cells can be counted and quantified using a flow cytometer, a microscope, and the like. Furthermore, by adding an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, etc., which are labeled with a pigment different from the labeled antibody, the cell subgroup of the antigen-specific immune cells can also be determined at the same time.

As described above, the cell culture container of the present invention can be suitably used as a cell culture container for culturing peripheral blood mononuclear cells and antigen-specific peptides to induce and grow antigen-specific immune cells from peripheral blood mononuclear cells. . In addition, the time required to prepare the specific antigen-specific immune cells is inevitably affected by the purpose of treatment and the state of the patient. However, with the cell preparation method of the present invention, it can start from isolated peripheral blood mononuclear cells in about 14 days ~ A sufficient amount of antigen-specific immune cells are prepared for treatment in about one month.

In addition, the cell culture container of the present invention can also be used as a culture container for co-culturing peripheral blood mononuclear cells and specific viruses, and generating and growing genetically modified immune cells from peripheral blood mononuclear cells.

[Example]

Hereinafter, the present invention will be described in more detail by giving specific examples, but the present invention is not limited thereto.

[Example 1]

Gas-permeable plastic film (the same material as the polyethylene-based special multilayer film used in the cell culture bag "CCB-st" manufactured by Toyo Can Group Holdings Co., Ltd.) is used to produce the cell culture shown in Figure 1. container 1 Same culture container.

The bowl-shaped recess 4 formed on the bottom 2b of the container body 2 is formed into a spherical crown with a diameter r 6mm and a depth d 3mm (d / r = 0.5), and 96 bowls are formed so as to occupy 64% of the occupied area of the bottom surface 2b.状 槽 部 4。 Concave portion 4.

[Example 2]

Except that the diameter r of the bowl-shaped recess 4 is 3 mm, the depth d is 1 mm (d / r = 0.33), and 330 bowl-shaped recesses 4 are formed so as to occupy 51% of the occupied area of the bottom surface 2b. Example 1 produced a culture container in the same manner.

[Comparative Example 1]

A culture container was manufactured in the same manner as in Example 1 except that the bowl-shaped recess 4 was not formed at the bottom 2b of the container body 2 and the bottom 2b was made the same flat surface as the top 2a.

[Evaluation example]

Using each culture container of Example 1, Example 2, and Comparative Example 1, HLA-A24-restricted CMV pp65 antigen-specific CTL was prepared through the following steps.

(1) Antigen-specific CTL induction step

(a) Isolation of peripheral blood mononuclear cells

20 mL peripheral blood was collected from HLA-A24-positive healthy normal people using a syringe with a small amount of heparin, and peripheral blood mononuclear cells (3 × 10 ⁷ ) were separated by Ficoll density centrifugation.

(b) Taking of plasma

Plasma was collected when the peripheral blood mononuclear spheres were separated, and after heat treatment at 56 ° C. for 20 minutes, centrifugation was performed at 2500 rpm for 10 minutes.

(c) Suspension of peripheral blood mononuclear cells in RPMI 1640 medium

After peripheral blood mononuclear cells were washed with an induction medium (Hepes buffered RPMI 1640 medium containing 0.01% human serum albumin and 10 μg / mL Gentacin), 12 mL of induction medium was suspended with the peripheral blood mononuclear cells. Further 3 mL of autologous plasma was added. Next, add HLA-A24-restricted HCMV-specific CTL peptide QYD (SEQ ID NO: 1: QYDPVAALF, manufactured by Institute of Medical Biology) to a final concentration of 10 μg / mL, and mix well (cell density: 2 × 10 ⁶ / mL).

(d) CTL induction

Peripheral blood mononuclear cells suspended in the induction medium were divided into three equal portions, and 5 mL of each was injected into each of the culture containers of Example 1, Example 2, and Comparative Example 1 from the injection inlet / outlet port using a lock adapter syringe. After that, it was moved to a CO ₂ incubator at 37 ° C. and 5% CO ₂ (culture start).

After leaving it in the incubator for a while, obtain an image of the cells using a microscope system equipped with a digital camera, and confirm the results of Examples 1 and 2. The state of cell sedimentation in the culture vessel. As the microscope, an inverted fluorescence phase difference microscope BZ-X700 (manufactured by Keyence Corporation) was used. Magnification uses 4x and 40x lenses. The results are shown in Fig. 2.

As can be seen from FIG. 2, the injected cells settle to the center of the U-shaped bottom of the cross section of the bowl-shaped concave portion that is the cell culture portion. Relative to the central part of the U-shaped bottom of the section, there is a relatively high density of cell aggregation, and there are almost no cells in other regions. Therefore, the cells in the container body are divided into a plurality of bowl-shaped recesses, and the movement in the container wall extension direction is suppressed, and the cells settle to the center of the U-shaped bottom section. The cells can be cultured while closely contacting each other. For antigen-specific immune cells with a very low frequency, an environment that can effectively induce antigen-specific immune cells is prepared.

The culture container was removed 2 days after the start of the culture. Next, 1 mL of an induction medium containing IL-2 (IL-2 content: 300 IU / mL) was injected into the culture container from the injection port. After culturing in a CO ₂ incubator at 37 ° C. and 5% CO ₂ for 5 days, 1 mL of an induction medium containing IL-2 (IL-2 content: 50 IU / mL) was injected into the culture container from the injection port. Through the above operations, antigen-specific CTLs were induced within a short period of 7 days from the start of culture (except for the time required to prepare peripheral blood monocytes and plasma).

Seven days after the induction, a certain number of cells were removed from the injection / exit nozzles of each culture container, and a part of them was counted using a hemocytometer to count the total number of cells. The other part was reacted with PE-labeled HCMV-specific MHC tetramer and PC5-labeled CD8 antibody, and the induction rate of HCMV-specific CTL was measured using a flow cytometer. Specifically, adding the right amount of cells 10 μL of PE-labeled MHC-tetramer reagent (manufactured by the Institute of Medical Biology) and 20 μL of FITC (fluorescein isothiocyanate) -labeled T cell surface antibody CD8 (manufactured by Beckman Coulter, Inc.). Thereafter, it is gently mixed and left to stand at 2 to 8 ° C. for 60 minutes, or at room temperature for 30 minutes. After 1.5 mL of PBS was added and stirred, centrifugation was performed at 3,000 rpm for 5 minutes. After the supernatant was aspirated and discarded, the cells were resuspended in 400 μL of PBS. At this time, in order to remove non-specific fluorescence from dead cells, 7-AAD viability Dye (dead cell detection reagent, manufactured by Medical Biology Research Institute Co., Ltd.) was added. Analysis by flow cytometry within 24 hours. The results are shown in FIGS. 3 and 4.

In FIG. 3, the cell suspension and trypan blue staining solution were mixed at a ratio of 1: 4, and the number of cells was immediately measured using a hemocytometer. As a result, the number of viable cells obtained in the culture container of Comparative Example 1 was 1.04 × 10 ⁷ cells, and the number of viable cells obtained in the culture containers of Example 1 and Example 2 were 1.182 × 10 ⁷ cells and 1.20 each. × 10 ⁷ pcs. When cultured in the culture containers of Examples 1 and 2 provided with bowl-shaped recesses, more living cells were obtained when cultured in the culture container of the comparative example 1 of the flat bottom type. In addition, the larger the number of bowl-shaped recesses, the higher the number of living cells was observed.

On the other hand, the quantitative results of the antigen-specific CTL are shown in FIG. 4. In the left section of FIG. 4, the numbers in the dot plot expansion represent when the expanded view is divided into quarters by UL (upper left), UR (upper right), LL (lower left), and LR (lower right). UR + LR) percentage of UR. X-axis represents the fluorescence intensity of CD8 on a logarithmic scale, and Y-axis represents the fluorescence intensity of MHC-tetramer mixed reagent on a logarithmic scale. Icon.

As can be seen from the results shown in FIG. 4, the positive rate of HCMV-specific CTL induced by the culture container of Comparative Example 1 was 0.13%, but the positive rate of HCMV-specific CTL induced by the culture container of Example 1 was detected. 0.54%, and the positive rate detected in Example 2 was 1.13%.

In addition, the number of CTL cells was calculated in the right section of FIG. 4. The number of CTL cells induced from the start of culture to 7 days later was 4,160 (Comparative Example 1), 17,730 (Example 1), and 36,000 (Example 2) in each of the three culture vessels. Compared with the culture container of Comparative Example 1, the culture container of Example 1 obtained 4.26 times, and the culture container of Example 2 obtained 8.65 times CTL cells. It can be seen that even in a peripheral blood mononuclear cell induction experiment from the same donor, the frequency of tetramer positive cells and the number of CTL cells in the example shown in FIG. 4 are still higher than those in Comparative Example 1. From this, it can be seen that the U-shaped bowl-shaped recessed section is effective for induction of antigen-specific CTL.

(2) Antigen-specific CTL growth steps

In order to obtain a sufficient positive rate of antigen-specific immune cells in the aforementioned antigen-specific immune cell induction step, continue to directly set the conditions suitable for the culture of immune cells (typically 37 ° C, 5% CO ₂ ). Culture in an incubator (start of growth culture). Thereafter, the culture was performed in the order of two days, and the growth medium was injected from the injection inlet / outlet orifice. Thereafter, the culture in the incubator was continued. Through the above operations, the antigen-specific CTL was prepared within a short period of 14 days from the start of the culture in steps (1) and (2). The obtained cells were subjected to cell counting and flow cytometry analysis. The results are shown in Figs. 5 and 6.

As shown in FIG. 5, the total number of living cells on the 14th day from the start of the culture was 2.99 × 10 ⁷ (Comparative Example 1), 3.17 × 10 ⁷ (Example 1), and 3.32 × 10 ⁷ (Example 2). Although there was no significant difference in the total number of living cells, as shown in FIG. 6, the number of CTL cells was 0.27 × 10 ⁵ (Comparative Example 1), 3.90 × 10 ⁵ (Example 1), and 5.22 × 10 ⁵ (Example 2) The difference is very large. Compared with the culture container of Comparative Example 1, the culture container of Example 1 obtained about 14.4 times, and the culture container of Example 2 obtained about 19.3 times CTL cells. In addition, from the comparison of the results of flow cytometry analysis in the left section of FIG. 6, it is known that the positive rate of CTL of the culture container of Example 1 is about 15.7 times, and the CTL of the culture container of Example 2 is about 15.7 times compared to the culture container of Comparative Example 1. The positive rate was about 17.8 times. CTL cells having a higher purity than the conventional culture container (Comparative Example 1) were obtained.

That is, in a short period of 14 days from the start of the culture in steps (1) and (2) (however, the time required to prepare PBMC and plasma was excluded), the antigen-specific CTLs were prepared in high purity and in large quantities. Success.

(3) Recovery steps for antigen-specific CTL

An example of recovery operation after preparation of antigen-specific CTL is shown. First, the contents (i.e., antigen-specific CTL) are dispensed into an appropriate container using the injection / exit nozzle of the culture container. Next, the supernatant (culture medium) was discarded after centrifugation, and a cryopreservation solution (CP-1 supplemented with 8% human serum albumin: manufactured by Jidong Pharmaceutical Industry Co., Ltd.) was injected to suspend the cells. Next remove the cell suspension to an appropriate cryopreservation volume Store in a freezer (for example, -130 ~ -150 ° C). Then, the cells are thawed during use, and the cells are washed with the infusion bag and then infused into the patient.

As mentioned above, although the preferred embodiment was demonstrated and demonstrated about this invention, this invention is not limited to the said embodiment, Of course, various changes can be implemented within the scope of this invention.

For example, in the foregoing embodiment, the container body 2 is formed in a rectangular shape, and the short side is provided with the injection inlet / outlet nozzle 3, but the invention is not limited to this. The shape of the container body 2 may be made into a square, an ellipse, a circle, or the like, and may be made into various shapes as required. The position where the injection / exit nozzle 3 is provided can also be changed as appropriate.

[Industrial availability]

In particular, the present invention can be used in the fields where cell induction efficiency and cell growth rate need to be improved, and a large number of cells can be obtained in a short period of time, the culture of regenerative medical cells, and the like.

<110> Toyo Can Manufacturing Group Co., Ltd. Medical Biology Research Institute Co., Ltd.

<120> Cell preparation method and cell culture container

<130> TSKMBL-1649-PCT

<150> JP2016-100798

<151> 2016-05-19

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HLA-A24 restricted epitope

<400> 1

1‧‧‧ culture container

2‧‧‧ container body

2a‧‧‧Top

2b‧‧‧ underside

3‧‧‧ nozzle for injection

4‧‧‧ bowl-shaped recess

Claims (8)

A cell preparation method, which is a method for preparing an antigen-specific immune cell by co-culturing with peripheral blood mononuclear cells and an antigen-specific peptide or a transformation virus, and is characterized in that it is used on one side of the inner wall surface of a container A plurality of culture vessels serving as recessed portions of the cell culture portion are provided in a recessed manner, and the cells are allowed to settle to the recessed portions to prepare target cells. If the cell preparation method of claim 1 is to perform any one of the following steps (a) to (e), or to combine two or more of them to promote the growth of antigen-specific immune cells: (a) additional antigen Specific peptide (b) Add anti-CD3 antibody (c) Add anti-PD-1 antibody (d) Add low molecules that suppress immune checkpoint molecules, suppress the performance of regulatory T cells, metabolic pathways, or improve the performance of HLA molecules The inhibitor (e) adds a substance that activates immunity through an immune receptor. A cell culture container, which is the cell culture container used in the cell preparation method of claim 1 or 2, and is characterized in that it is provided with a container body composed of a gas-permeable plastic film and an injection inlet / outlet nozzle. The aforementioned container body has a convex shape in which a peripheral portion is sealed, The bottom surface of the container body is provided with a plurality of bowl-shaped recesses having an opening diameter of 1.5 mm or more as a cell culture portion. For example, the cell culture container of claim 3, wherein the oxygen permeability of the aforementioned plastic film is 3000 mL / (m ² ‧day‧ atm) or more. The cell preparation method according to claim 3 or 4, wherein the bottom area of the bowl-shaped recess is smaller than the opening area. The method for preparing a cell according to claim 5, wherein the bowl-shaped recess is U-shaped in cross section. For example, the cell culture container of claim 3, 4 or 6, wherein the ratio d / r of the depth d to the diameter r of the bowl-shaped recess is 0.05 to 1. The cell culture container according to claim 5, wherein the ratio d / r of the depth d of the bowl-shaped recess to the diameter r is 0.05 to 1.