JP2019122261A - Cell preparation method and cell culture vessel - Google Patents

Abstract

To achieve practical use of antigen-specific immune cell treatment, and preferably prepare an antigen-specific immune cell by mass production, efficiently at low cost.SOLUTION: A culture vessel 1 in which a plurality of recesses 4 being cell culture parts are provided in a dent state on one surface of an inner wall face of the vessel, is used, then cells are precipitated to recesses 4 for preparing target cells.SELECTED DRAWING: Figure 1

Description

  The present invention particularly relates to a cell preparation method capable of efficiently preparing immune cells bearing specific immune responses against viruses, bacteria, or cancer and other immune diseases, and a cell culture vessel used therefor About.

  In recent years, with the progress of elucidation of human immune system, an interest in immunotherapy for treating and preventing diseases such as cancer and viral infection using immune reaction is increasing. In particular, since the discovery of a virus-specific antigen and a tumor-specific antigen, research on antigen-specific immunotherapy as a new antiviral and anticancer therapeutic method has been advanced, and clinical trials have been conducted worldwide. Among them, immune cell therapy using autologous or allogeneic antigen specific immune cells and antigen specific gene modified immune cells is an opportunistic infection after hematopoietic cell transplantation, lymphoma, melanoma, nasopharyngeal cancer, blood cancer Effective clinical data for diseases such as autoimmunity are being accumulated.

  Antigen-specific immune cell therapy has been the most rapid medical technology, TIL therapy using tumor infiltrating cells, CTL therapy using cytotoxic T cells, Perper T cells for the past twenty years HTL therapy, CAR-T using genetically modified immune cells, CAR-NK therapy, TCR-T therapy and the like have been developed and are in clinical trials.

  Unlike so-called non-specific immune cell therapy including conventional LAK therapy and CIK therapy, the core technology of antigen-specific immune cell therapy is to efficiently carry out only immune cells that are responsible for specific immune responses outside the body It is to be induced or transformed and grown to a sufficient amount.

For example, in CTL therapy or HTL therapy, antigen-specific cytotoxic T cells (CTLs) or helper T cells (HTLs) are induced and proliferated from patient's own peripheral blood mononuclear cells collected from patients. Transfuse the patient to In many clinical trials using it, the number of cells transfused into a patient is ideally 1 × 10 ⁷ or more at one time. Therefore, for example, when the abundance ratio of antigen-specific immune cells is 10%, the total number of cells at the end of culture needs to be at least 1 × 10 ⁸ (several hundreds of millions) or more. And, in order to reduce the burden on the subject of blood collection, it is required to induce and proliferate more antigen-specific immune cells by one operation.

  However, in peripheral blood, antigen-specific cytotoxic T cells (CTLs) and helper T cells (HTLs) are present only at a very low frequency, which is less than 1/100 of the peripheral blood mononuclear cell fraction. Without it, it is very difficult to prepare it in large quantities outside the body.

  Furthermore, the frequency of presence of these antigen-specific immune cells is biased according to individual, antigen type and allele. For example, the viral antigen HCMV which is one of the therapeutic targets in opportunistic infections after hematopoietic stem cell transplantation is exemplified. The number of HLA-A24-restricted HCMV-specific CTL frequently found in Asians in peripheral blood is about one-hundredth of that in HLA-A2-restricted HCMV-specific CTLs, which are found frequently in Westerners. This is considered to be one of the reasons why the in vitro induction and proliferation of HLA-A24-restricted HCMV-specific CTL is difficult. Therefore, there have been few reports on methods for preparation outside the body for clinical application for specific CTLs other than HLA-A2-restricted.

  In Asia, the extension of average life expectancy is expected to further increase the number of virus-infected and cancer patients, and there is a demand for immune cell therapy to increase the number of HLA types to be treated more than ever. The inability to efficiently and universally prepare antigen-specific immune cells has become a rate-limiting factor in transition to clinical trials and commercialization.

Although many studies have been conducted so far, the methods for preparing antigen-specific immune cells that have been reported have not been applied as business because of the problems listed below.
(1) In order to induce antigen-specific immune cells, for example, in the case of antigen-specific CTL, preparation of antigen-presenting cells is required. Dendritic cells are often used as antigen-presenting cells, but preparation of dendritic cells is complicated and expensive.
(2) In the case of further proliferating the induced antigen-specific CTL, antigen-presenting cells are used (see Non-Patent Document 1). Therefore, the same problem as the above (1) occurs. In addition, in order to prepare antigen-presenting cells to be used for proliferation, it is necessary to collect blood from the donor again. Furthermore, during proliferation, the burden on the donor is large because a large number of antigen-presenting cells are required compared to the time of induction.
(3) Nonspecific stimulation by OKT3 or lectin may be used as another means of further proliferating the induced antigen-specific CTL. However, this method is relatively inefficient because cells other than antigen-specific CTL tend to proliferate. Also, the REM method is known as a relatively efficient proliferation method. The REM method is not a practical method for commercialization because it requires a large amount of peripheral blood mononuclear cells and EBV-LCL, and there is a major problem such as virus contamination.

  Under such circumstances, the present inventors have developed peripheral blood mononuclear cells without preparing antigen-presenting cells, in order to realize practical application of cell therapy with antigen-specific immune cells, in particular, antigen-specific CTL. We propose a preparation method that can induce antigen-specific immune cells from peripheral blood mononuclear cells rapidly and easily by co-culturing a sphere with an antigen-specific peptide and a stimulatory substance.

  Moreover, since the culture method of the antigen specific immune cell currently performed until now passes a complicated step, 24 well plane plate (refer nonpatent literature 2), flat plate flask (refer nonpatent literature 3), stirring type culture We have to rely on open culture vessels such as tanks (see Non-Patent Document 4).

There are many unsolved problems when using an open culture vessel. For example, a culture method using EBV-LCL is given as an example of a relatively efficient preparation method. Specifically, in this culture method, EBV-LCL cells are pulsed with an antigen peptide and then inactivated by X-ray (40 Gy) irradiation. Thereafter, it is mixed with 1 × 10 ⁶ peripheral blood mononuclear cells collected at a ratio of 40: 1 and seeded in a 24-well flat plate. At that time, a maximum of 2 mL of culture medium is added per well. During the period of cell culture, when restimulation with EBV-LCL cells becomes necessary, or whenever the medium is depleted, the cells need to be repeatedly collected by centrifugation, resuspended in fresh medium and seeded.

  Since the proportion of antigen-specific immune cells in total cells changes dynamically in the growth phase of antigen-specific immune cells, particularly in the late growth phase, as compared to the first half of induction, it is necessary to use essential nutrients and cytokines in appropriate amounts. Lee must control.

  Therefore, culture in the open system requires frequent media exchange work, and it is necessary to carry out culture in a cell processing facility in order to prevent contamination of bacteria, viruses, etc. from the outside. I need it.

Usually, the number of cells finally obtained in a 24-well flat plate is 1 × 10 ⁷ or less. If you do not scale up with multiple plates, you will not get enough cell numbers for treatment. Furthermore, culture vessels such as flat plates and flat flasks are limited in volume and are very unsuitable for mass preparation of cells.

Furthermore, there are fundamentally insurmountable problems in non-air-permeable plastic culture vessels such as flat plates and flat flasks. That is that efficient gas exchange and maintenance of stable buffer capacity can not be simultaneously adjusted. Previous research reports have shown that it is important to strictly control the gas exchange rate (O ₂ / CO ₂ ) and pH conditions of the culture medium for the culture of antigen-specific immune cells ( Non-Patent Document 5). In order to ensure sufficient gas exchange, empirically 1 mL volumes of culture medium must be kept to a surface area of 1 cm ² or more. That is, the shallower the medium covering the cells, the better the gas exchange rate. On the other hand, due to the accumulation of lactic acid, carbon dioxide, etc., which are metabolic wastes of cells, the shallower the medium covering the cells, the lower the ability to deliver nutrients and the pH buffering ability tend to be. This dilemma can only be overcome with non-breathable plastic culture vessels such as flat plates and flat flasks.

  On the other hand, in order to improve the gas exchange rate, a stirred culture vessel has been devised. However, the shear stress generated by the stirrer tends to damage the cells, resulting in an increase in the dead cell rate and not a general-purpose culture vessel.

  In addition, in order to solve some problems of the above-mentioned open system culture vessel, in recent years, in the culture of nonspecific immune cells such as LAK therapy and CIK therapy, a closed system culture system using a flat culture bag Is beginning to be used. However, when such flat culture bags are used for antigen-specific immune cells, low induction efficiency and high dead cell rate become problems.

The film material of the flat culture bag is very soft, and it is contained by the distortion of the film generated when the culture container is placed at an angle, or when it is left still, and also by slight vibration from the surrounding environment or the incubator. The cells in the center are concentrated around a part or the periphery of the container, causing damage due to physical collisions between cells and uneven cell density in the liquid phase, which causes cell proliferation to be reduced. ing.
In addition, although it is possible to prevent the concentration in the vicinity of the periphery by providing a partition, etc., it is impossible to disperse, concentrate or control the cells in each area, and as a result, the antigen It has been difficult to efficiently induce specific immune cells.

J. 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 adaptive immunotherapy., J Hematother Stem Cell. 2003 Feb; 12 (1): 93-105. Leen AM, Christin A, Myers GD, Liu H, Cruz CR, Hanley PJ, Kennedy-Nasser AA, Leung KS, Gee AP, Kranje RA, Brenner MK, Heslop HE, Rooney CM, Bollard CM. Cytotoxic T therapy for donor T cells preventins and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation., Blood. 2009 Nov 5; 114 (19): 4283-92. 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. 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. 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 (72): 72 -86.

  The present invention has been made in view of the above circumstances, and in order to realize antigen-specific immune cell therapy, the present invention has both simplicity and safety, yet efficiently at low cost and in large quantities. It is an object of the present invention to provide a cell preparation method which can be suitably prepared, and a cell culture vessel used therefor.

  The cell preparation method according to the present invention is a cell preparation method for preparing an antigen-specific immune cell by co-culture of peripheral blood mononuclear cells and an antigen-specific peptide or transformation virus, which comprises: As a method of preparing target cells, cells are sedimented in the recess by using a culture container in which a plurality of recesses are formed on one surface of the inner wall surface of the container.

  The cell culture vessel according to the present invention is a cell culture vessel used in the above-described cell preparation method, and comprises a vessel body made of a plastic film having gas permeability and a port 3 for pouring and discharging, and the vessel body Has a bulging shape in which the peripheral portion is sealed, and a plurality of bowl-shaped concave portions having an opening diameter of 1.5 mm or more serving as a cell culture portion are provided on the bottom surface of the container body.

  According to the cell preparation method of the present invention, a target cell can be prepared by a simple operation without requiring complicated operations. In addition, the time required for preparation is also very short.

  In addition, according to the cell culture vessel of the present invention, all culture steps can be performed in a closed system. This leads to improved safety. By ensuring high safety, the level of equipment required can be reduced and the risk of contamination can be reduced.

It is explanatory drawing which shows the outline of the cell culture container which concerns on embodiment of this invention, (a) is a top view, (b) is a side view, (c) is a bottom view. It is a microscope image which shows the sedimentation state of the cell in the evaluation example of an Example. It is a graph which shows the change of a total viable cell number in the induction | guidance | derivation process of the evaluation example of an Example. It is a graph which shows the stained image and the CTL cell number which show the positive rate of HCMV specific CTL in the induction | guidance | derivation process of the evaluation example of an Example. It is a graph which shows the change of the total viable cell number in the induction | guidance | derivation process of the evaluation example of an Example, and a proliferation process. It is a graph which shows the stained image which shows the positive rate of HCMV specific CTL in the proliferation process of the evaluation example of an Example, and the number of CTL cells.

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

[Culture vessel]
The cell culture container 1 shown in FIG. 1 is provided with a container main body 2 made of a plastic film having gas permeability, and a pouring port 3 made of a tubular member through which a medium, cells and the like can flow.

  The container main body 2 has a bulging shape in which the peripheral portion is sealed and expanded like a pillow, and the bottom surface 2 b of the container main body 2 has an opening diameter r of 1.5 mm or more as a cell culture portion. A plurality of bowl-shaped recesses 4 are provided. On the other hand, the top surface 2a of the container body 2 is a flat surface.

  Although it is preferable to form in a U-shaped cross-section so that cells may be easily collected at the bottom of the bottom 2b of the container body 2, the shape is not limited to this. In order to facilitate cell concentration at the bottom, any diameter reducing structure may be used as long as the area of the bottom is smaller than the opening area. Specifically, in addition to the U-shaped cross section, the semicircular cross section, V cross section , May be inverted conical.

  Furthermore, in order to suppress migration of cells in the container body 2 so that cells in culture remain in one cage-like recess 4, the depth d of the cage-like recess 4 is The ratio d / r of the depth d to the diameter r is preferably 0.05 to 1.

  Further, the arrangement of the ridge-like recesses 4 is preferably staggered as shown so that the area occupied by the ridge-like recesses 4 occupied on the bottom surface 2b is as large as possible, but they may be arrayed in a grid if necessary. May be

  Further, the size of the container body 2 is not particularly limited, but preferably 50 to 500 mm in length and 50 to 500 mm in width.

Such a cell culture vessel 1 can be manufactured, for example, as follows.
First, a top plastic film to be the top surface 2 a of the container body 2 and a bottom plastic film to be the bottom 2 b of the container body 2 are prepared. The bottom-side plastic film is molded so as to leave the peripheral portion, and the ridge-like concave portion 4 is formed in the bulging portion. These can be formed by general vacuum forming, pressure forming, etc., and by appropriately adjusting the mold, the formed shape and the shape of the ridge-like recess 4 become the desired shape. can do.

  Next, the bottom side plastic film molded as described above and the top side plastic film are overlapped, and the peripheral portion is melted by sandwiching the tubular member forming the injection / discharge port 3 at a predetermined position. Seal by wearing and trim the periphery if necessary. Thereby, the cell culture container 1 as shown in FIG. 1 can be manufactured.

The gas permeability of the plastic film forming the container body 2 is 3000 ml / (m ² · day · atm) of oxygen permeability measured at a test temperature of 37 ° C. according to the gas permeability test method of JIS K 7126. Or more, and more preferably 6000 ml / (m ² · day · atm) or more.
Moreover, it is preferable that the said plastic film has transparency partially or entirely. By doing this, the progress of cell culture, the state of cells, etc. can be confirmed by visual observation or microscopic observation from the outside, as needed, without removing cells from the container.

  The material used for the plastic film forming the container body 2 is not particularly limited as long as it has desired gas permeability. For example, thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, tetrafluoroethylene-hexafluoropropylene copolymer (FEP) and the like can be mentioned. It is also possible to use silicone. These may be used as a single layer, or may be used by laminating the same or different materials, but in view of the heat-sealing property when sealing the periphery, it has a layer that functions as a sealant layer Is preferred.

  Moreover, the thickness of the plastic film used to form the container main body 2 is 30 to 200 μm so as to have an appropriate shape-retaining property that does not cause the depressions 4 to be crushed while having flexibility. Is preferred.

  Further, as described above, the injection / delivery port 3 is formed of a tubular member through which the culture medium or cells can flow. The tubular member forming the injection port 3 is formed into a predetermined shape by injection molding, extrusion molding or the like using a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, FEP, etc. However, the specific form of the injection port 3 is not particularly limited. For example, a port having an injection port made of an elastic material such as rubber may be used, or a luer lock type port may be used so that injection by a needle can be performed.

  The infusion port 3 is also used for the infusion of cells such as peripheral blood mononuclear cells, and also for the delivery of desired cells such as prepared antigen-specific immune cells. In the example shown in the figure, the injection port 3 is one, but a plurality of ports may be provided to cope with an operation error. In this case, by providing a cap for each port, contamination can be prevented, and identification can be performed by making the color, shape, and the like of the cap different, so that erroneous operation can be prevented more reliably. .

  To prepare target cells by using such a cell culture vessel 1 to induce or transform antigen-specific immune cells from, for example, peripheral blood mononuclear cells, the cells are connected to the port 3 for injection and delivery. Peripheral blood mononuclear cells or other cells isolated from the patient's peripheral blood while maintaining a closed system through a fluid transfer tube, an antigen-specific peptide prepared according to the patient's HLA type, a stimulator, and A predetermined culture medium or the like is injected into the container body 2 at a predetermined blending ratio.

  The peripheral blood mononuclear cells and the antigen-specific peptide injected into the container body 2 are precipitated in the culture medium and divided into each caged recess 4 and collected at the bottom of each, thereby suppressing the movement in the container . Thus, peripheral blood mononuclear cells and an antigen-specific peptide can be co-cultured in a state in which the cells are in close contact with each other, and effective antigen presentation to very low frequency of antigen-specific immune cells is achieved. It becomes possible to realize and efficiently induce antigen-specific immune cells.

  In addition, by making each ridge-shaped recess 4 into substantially the same shape, it is possible to obtain a substantially uniform cell density for each ridge-shaped recess 4. Then, the cells collected in the bowl-shaped recess 4 due to distortion of the film generated when the cell culture vessel 1 is placed at an angle, or when the cell culture vessel 1 is left standing, and also due to slight vibration from the surrounding environment or incubator. Thus, it becomes possible to form a fixed cohesion within each bowl-shaped recess 4 without moving to another bowl-shaped recess 4. As a result, the cell density can be kept in a state where it can be efficiently induced, and cells can be induced and proliferated without causing a decrease in proliferation rate due to crowding or a decrease in induction efficiency due to dispersion. As a result, homogeneous cell growth can be realized, which is effective in reducing dead cells and improving induction efficiency.

  In addition, the top surface 2a of the container body 2 is flat, and is sedimented in each of the bowl-shaped concave portions 4 by pushing down the top surface 2a to make contact with the bottom of the bowl-shaped concave portion 4 using flexibility. Cells can be easily resuspended. As a result, even if the cell density of each bowl-shaped recess 4 is biased, cells can be dispersed substantially equally in each bowl-shaped recess 4.

  Also, at the time of recovery of proliferated cells, for example, the contents liquid can be easily discharged from the injection / delivery port 3 by inverting and tilting the container, as in the case of using a plastic container with a 24-well flat plate. Using a pipette, the time-consuming operation of injecting and recovering cells per well can be omitted. Furthermore, since the cells can be filled and the proliferated cells can be recovered while maintaining the closed system, contamination of microbes such as bacteria and mycoplasma and viruses can be reduced. Therefore, culture of cells transfused or buried in the body or attached to the injured part can be performed more suitably.

  And since the container main body 2 consists of a plastic film which has gas permeability, even if it comprises a closed system, it can supply sufficient quantity of oxygen to the cell in culture | cultivation.

  As described above, according to the cell culture vessel 1 of the present embodiment, all culture steps can be performed in a closed system. This leads to improved safety. By ensuring high safety, the level of equipment required can be reduced and the risk of contamination can be reduced. Furthermore, since the container body 2 is formed using a plastic film, it is lightweight even if its volume is increased, and is not bulky, so it is suitable for mass cell culture, and the container body 2 is A sufficient amount of culture medium can be injected.

[Cell preparation method]
Next, an embodiment of the cell preparation method of the present invention using the above-described cell culture vessel 1 will be described.

  Cells targeted by the present invention can be cells capable of inducing antigen-specific immune cells by co-culture with antigen-specific peptides and stimulators, cells capable of transforming into antigen-specific genetically modified immune cells by co-culture with viral solution It is. In particular, it is suitable for peripheral blood mononuclear cells.

  First, the medium and cells, and antigen-specific peptide or transformation virus solution are injected into the cell culture vessel 1 from the injection / delivery port 3, and cells etc. which precipitate in the medium gather at the bottom of each scaly recess 4 Let's do it. At this time, the culture medium is poured so as to cover not only the bowl-shaped recess 4 but also the bottom surface 2 b of the container body 2.

  As a culture medium, a liquid culture medium is used, and it selects suitably according to the kind of cell. Media suitable for culturing antigen-specific immune cells (eg, RPMI 1640, AIM-V, Dulbecco's modified eagle medium), X-VIVO (manufactured by Bio Whittaker), ALyS505N (Cell Science Laboratories) A cytokine-containing medium can be prepared by adding IL-2 or the like to KBM series, KBM series lymphocyte culture medium (Cordin Bio) or the like. Serum, plasma, serum albumin, antibiotics, L-glutamine and the like may also be added. As IL-2, it is preferable to use recombinant human IL-2 (for example, PROLEUKIN provided by Chiron, or theseleukin provided by Shionogi & Co., Ltd.). The content of IL-2 is, for example, 10 to 1,000 IU / mL.

Antigen-specific peptides include viruses (cytomegalovirus, Epstein-Barr virus, adenovirus, etc.), tumor antigens (WT1, hTERT, MN / CA9, gp100, MART1, TRP1, TRP2, tyrosinase, MAGE1, MAGE2, MAGE3, MAGE6) , NY-ESO-1, MUM1, BAGE, GAGE1, GAGE2, CEA, PSA, etc.). Usually, only one type of antigen-specific peptide is used, but it does not prevent the use of two or more types of antigen-specific peptides.
Viruses for transformation include retrovirus vectors (including onchoretrovirus vectors, lentivirus vectors, and pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai. Viral vectors such as viral vectors, Epstein-Barr virus (EBV) vectors, HSV vectors and the like can be used. As the above-mentioned viral vector, one which is deficient in replication ability so as to be unable to replicate in infected cells is preferable.

The number of cells to be seeded in each cage-like recess 4 is preferably about 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL, although it depends on the size of the cage-like recess 4. If the cell density is less than 1 × 10 ⁴ cells / mL, the induction efficiency is low because the probability that the cells meet will be low. On the other hand, when the cell density exceeds 1 × 10 ⁶ cells / mL, the cell death rate increases, and thus the cell growth efficiency decreases.

(1) Step of inducing antigen-specific immune cells After the above operation, the cell culture is carried out in an incubator set to conditions (typically, 37 ° C., 5% CO ₂ ) suitable for culturing the antigen-specific immune cells. Transfer container 1. After a predetermined time (for example, 1 to 4 days) has elapsed, the cell culture vessel 1 is removed from the incubator, and a cytokine-containing medium is injected from the injection / delivery port 3. After culture in the incubator for a predetermined time (for example, 2 to 7 days), the cytokine-containing medium is again injected into the cell culture vessel 1 from the injection / discharge port 3. Thereafter, the culture in the incubator is continued. The cell culture vessel 1 may be rotated in a state of containing a new medium to culture while adjusting the cell density by resuspension. In this way, media can be added without changing the container.

(2) Step of proliferating antigen-specific immune cells A small amount of cells are collected from cell culture vessel 1 in cell culture, and the frequency and amount of antigen-specific immune cells are determined using the method for quantifying antigen-specific immune cells described later. Measure Thereafter, it proceeds to the proliferation step.
In this step, induced antigen-specific immune cells are proliferated to prepare a large amount of antigen-specific immune cells. When the positive rate of antigen-specific immune cells is sufficiently obtained in the above-mentioned induction step, the conditions suitable for culture of antigen-specific immune cells are continued (typically, 37 ° C., 5% CO ₂ ) Incubate in the incubator set to.
After a predetermined time (for example, 1 to 4 days) has elapsed, the cell culture vessel 1 is taken out of the incubator, and the growth medium is injected from the injection / delivery port 3 into the cell culture vessel 1. After culture in the incubator for a predetermined time (for example, 2 to 7 days), the growth medium is again injected from the injection / discharge port 3. Thereafter, the culture in the incubator is continued. After this, it proceeds to the cell recovery operation. The interval from the injection operation of the growth medium to the next injection operation of the growth medium is, for example, 1 to 6 days. The interval between the first injection operation and the second injection operation may be 3 days, and the interval between the second injection operation and the third injection operation may be 2 days. It is not necessary to unify the intervals here.

  The growth medium contains cytokines such as IL-2 or 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.

  In the above-mentioned induction step, when the positive rate of antigen-specific immune cells is relatively low, any one of the following operations (a) to (e) in order to promote the growth of antigen-specific immune cells: Alternatively, two or more may be used in combination, and then the growth step may be performed.

(A) Add an antigen specific peptide.
Further addition of an antigen specific peptide promotes the expression of cell surface stimulatory factor. As a result, in the subsequent proliferation step, the proliferation rate of the induced antigen-specific immune cells becomes fast, and can be proliferated with high efficiency. The amount of antigen specific peptide in the medium by addition is, for example, 0.01 to 100 μg / mL.

(B) Add anti-CD3 antibody.
The addition of anti-CD3 antibody promotes the expression of costimulatory factors on the cell surface. As a result, in the subsequent proliferation step, the proliferation rate of the induced antigen-specific immune cells becomes fast, and can be proliferated with high efficiency.
The anti-CD3 antibody to be used can be prepared by an immunological procedure using a CD3 molecule or a part thereof. Commercially available anti-CD3 antibodies can also be used. An example of a commercially available anti-CD3 antibody is OKT-3 antibody (manufactured by Janssen Pharma). From the viewpoint of safety, it is preferable to use an OKT-3 antibody that has received regulatory approval.
The anti-CD3 antibody may be a monoclonal antibody or a polyclonal antibody. However, in view of specificity and efficiency, it is preferable to use an anti-CD3 antibody which is a monoclonal antibody. It is also possible to use two or more anti-CD3 antibodies in combination. The amount of anti-CD3 antibody in the medium is, for example, 0.01 to 10 μg / mL.

(C) Add anti-PD-1 antibody.
By adding an anti-PD-1 antibody, it is possible to block an immune checkpoint PD-1 molecule that inhibits the proliferation of antigen-specific immune cells, and to proliferate induced antigen-specific immune cells with high efficiency. it can.
The anti-PD-1 antibody to be used can be prepared by an immunological procedure using PD-1 molecule or a part thereof. Commercially available anti-PD-1 antibodies can also be used.
An example of a commercially available anti-PD-1 antibody is nivolumab (manufactured by Ono Pharmaceutical Co., Ltd.). From the viewpoint of safety, it is preferable to use an anti-PD-1 antibody that has received regulatory approval.
The anti-PD-1 antibody may be a monoclonal antibody or a polyclonal antibody. However, in view of specificity and efficiency, it is preferable to use an anti-PD-1 antibody which is a monoclonal antibody. It is also possible to use two or more anti-PD-1 antibodies in combination. The amount of anti-PD-1 antibody in the medium is, for example, 0.01 to 10 μg / mL.
In addition to PD-1, immune checkpoint molecules that inhibit the proliferation of antigen-specific immune cells include PD-L1, CTLA-4, TIGHT, TIM-3, LAG-3, B7-H3, B7-H4, There are many BTLA, VISTA, etc. Antibodies to those molecules can be used as well to media. It is also possible to use two or more anti-immune checkpoint molecule antibodies in combination.

(D) Add an immune checkpoint molecule or a small molecule inhibitor that inhibits the expression / metabolic pathway of regulatory T cells (Treg) or improves the expression of HLA molecules.
Examples of the substance that inhibits the expression of the immune checkpoint molecule include low molecular weight compounds such as Nafamostat Mesylate, which is a serine protease inhibitor that suppresses the expression of PD-L1. Examples of substances that suppress Treg expression include Lenalidomide low molecular weight compounds that inhibit only effector Tregs (CD4 + FOXP3 high CD45 RA low) cells, or INCB024360 low molecular weight compounds that are indoleamine 2,3-dioxygenase inhibitors that inhibit Treg-like cells. Etc. Examples of substances that improve the expression of HLA molecules include Lapatinib Ditosylate, which is a MAPK inhibitor. It is preferable to add any one or more of the aforementioned low molecular weight compounds to the growth medium. The amount of low molecular weight compound in the medium is, for example, 1 pM to 1 μM.

(E) Add a substance that activates immunity via an immune receptor such as Toll-like receptor (TLR) or RIG-I-like receptor.
Examples of the immunostimulatory component include mushroom components (extract and raw powder etc.) having immunostimulatory activity, seaweed (such as Mekabu and mozuku) components (extract and raw powder etc.) and the like. These may use only 1 type and may use 2 or more types together.
In addition, components of pathogens and substances that mimic them, as so-called Immune Potentiators (immunostimulators), lipoproteins (ligands for TLR1), peptidoglycans (ligands for TLR2), Poly (I: C) (Polyinosinic-polycytidylic acid sodium) Examples include dsRNA (a ligand for TLR3), lipopolysaccharide (LPS) (a ligand for TLR4), ssRNA (a ligand for TLR7), Cp G DNA (a ligand for TLR9), and the like, as represented by salt.
Immunostimulatory components are important not only in the regulation of the innate immune system but also in the initiation and control of the acquired immune system. TLRs are expressed on T cells themselves and possess the ability to directly control T cell functions. For example, CD8 positive T cells express TLR2 and costimulation with antigen and TLR2 ligand directly enhances cell proliferation and cell viability and reduces the need for indirect dendritic cell-derived costimulation Help to make In addition, stimulation with a TLR ligand (especially TLR3, TLR7 or TLR9) increases the ability to "cross-present" foreign antigens by MHC class I, and specifically antigen-specific CD8-positive T cells are cytotoxic T cells (T Can be effectively differentiated.

(3) Recovery step of antigen-specific immune cells A small amount of cells are collected from the cell culture vessel 1 in cell culture, and the frequency and amount of antigen-specific immune cells are determined using the method for quantifying antigen-specific immune cells described later. Measure Thereafter, the process proceeds to the recovery step.

  The port 3 for injection and delivery of the cell culture vessel 1 is used for the recovery operation. That is, the proliferated antigen-specific immune cells are exported from the infusion port 3. After the cells are removed, a washing step of the cells by centrifugation may be incorporated. Such washing steps may be repeated (for example, 2 to 4 times).

(4) Quantification of Antigen-Specific Immune Cells In each step of preparing an antigen-specific immune cell by applying the cell preparation method of the present invention, it is necessary to know the variation of the amount of antigen-specific immune cell by adjusting the process period. It is very important in deciding whether to finish or not, the interval of medium addition, etc.
Quantitatively measuring an antigen-specific immune cell intended for clinical treatment has the same meaning as measuring its effect component. Furthermore, it is also important in the quality assay which measures the active ingredient amount of the final preparation provided using the antigen specific immune cell prepared by applying the cell preparation method of this invention. The quantification of antigen-specific immune cells can be performed by the following three methods.

・ Quantification method 1 (MHC-multimer method etc.)
Using MHC-monomer and / or MHC-multimer (hereinafter collectively referred to as MHC-multimer etc.) produced using the same peptide as the antigen-specific peptide used for inducing antigen-specific immune cells, Antigen-specific immune cells prepared or prepared can be quantified. The quantification can be performed, for example, as follows.
The sample collected from the cell culture solution is reacted with an MHC-multimer or the like at an appropriate concentration. The antigen-specific immune cells bound to the MHC-multimer and the like are stained with a labeling dye, so they are counted using a flow cytometer, a microscope or the like. When reacting with an MHC-multimer etc., the cell subset of antigen-specific immune cells is also reacted by reacting an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody etc. labeled with a dye different from the MHC-multimer etc. It can judge simultaneously.

・ Quantification method 2
By stimulating collected cells with an antigen-specific peptide, it is possible to quantify IFN-gamma (interferon gamma) produced by antigen-specific immune cells, TNFα (tumor necrosis factor alpha), cytokines such as interleukins, and / or chemokines and / or chemokines. is there. The following specifically shows IFNγ as an example.

2-1) Method 1 (cytokine IFNγ-producing cell assay) by cytokine assay
The harvested cells are suspended in an appropriate medium at a cell concentration of about 2 × 10 ⁶ / mL, and an antigen-specific peptide is added. Furthermore, an intracellular protein transport inhibitor (eg, Brefeldin A, Monensin etc.) is added, and the cells are cultured in a 5% CO ₂ thermostat at 37 ° C. for 5 to 16 hours. After culture, the cells are reacted with T cell marker antibody (anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody), or MHC-multimer, etc., fixed to cells, permeabilized, and reacted with dye-labeled anti-IFNγ antibody Let It analyzes using a flow cytometer etc., and quantifies the percentage of IFNγ positive cells in all cells, in T cells or in MHC-multimer etc positive cells.

2-2) Method 2 by cytokine quantification (Erispot assay)
The collected cells are seeded on a 96-well MultiScreen-HA plate (Millipore) on which an anti-IFNγ antibody is immobilized. Thereafter, an antigen-specific peptide is placed in each well and cultured in a 5% CO ₂ thermostat at 37 ° C. for 20 hours. The next day, the plate is washed and reacted in the order of anti-IFNγ antibody and peroxidase-labeled anti-IgG antibody. Furthermore, a substrate for peroxidase is added, and the IFNγ spot is visualized by color development, and quantified by counting using a stereomicroscope or an ELISPOT analyzer (manufactured by C.T.L.).

2-3) Method 3 by cytokine quantification (method for quantifying IFNγ secreted in culture supernatant)
The prepared cell sample is suspended in an appropriate medium at a cell concentration of about 2 × 10 ⁶ / mL, and an antigen-specific peptide is added. Incubate for 24 to 48 hours at 37 ° C. in a 5% CO ² thermostat. After culture, the IFNγ concentration contained in the culture supernatant is quantified using a commercially available ELISA kit (eg, Quantikine ELISA Human IFNγ Immunoassay from R & D Systems).

・ Quantification method 3
The quantification is performed using a cell surface protein specific antibody. Antigen-specific immune cells that specifically recognize an antigen-specific peptide can be stimulated by binding to the peptide to enhance expression of cell surface proteins (eg, CD137, CD107a, CD107b, CD63, CD69, etc.) It has been reported. Therefore, by mixing cells stimulated with an antigen-specific peptide and the like with a labeled antibody that specifically recognizes cell surface proteins, antigen-specific immune cells bind to the labeled antibody and are stained with the labeling dye. The stained antigen-specific immune cells can be counted and quantified using a flow cytometer, a microscope or the like. Furthermore, by adding an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody or the like labeled with a dye different from the labeled antibody, a cell subset of antigen-specific immune cells can be determined simultaneously.

As described above, the cell culture vessel of the present invention is used to mix and culture peripheral blood mononuclear cells and an antigen-specific peptide to induce and proliferate antigen-specific immune cells from peripheral blood mononuclear cells. It can be suitably used as a cell culture vessel. In addition, although the time required for preparation of the target antigen-specific immune cells is naturally affected by the therapeutic purpose and the condition of the patient, according to the cell preparation method of the present invention, the isolated peripheral blood mononuclear cells The preparation of the antigen-specific immune cells of the necessary amount for treatment becomes possible in about 14 days to about one month.
In addition, the cell culture vessel of the present invention can also be used as a culture vessel used for coculturing peripheral blood mononuclear cells and a specific virus to produce and propagate genetically modified immune cells from peripheral blood mononuclear cells.

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

Example 1
A cell culture vessel shown in FIG. 1 using a gas-permeable plastic film (made by Toyo Seikan Group Holdings Co., Ltd .: the same material as a polyethylene-based special multilayer film used for the cell culture bag “CCB-st”) A culture vessel similar to 1 was produced.
The bowl-shaped recessed part 4 formed in the bottom part 2b of the container main body 2 is a spherical crown having a diameter r6 mm and a depth d3 mm (d / r = 0.5), and 96 pieces so that the occupied area on the bottom surface 2b is 64%. The bowl-shaped recess 4 was formed.

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 that the occupied area occupied on the bottom surface 2b is 51%. In the same manner as in Example 1, a culture vessel was produced.

Comparative Example 1
A culture vessel was manufactured in the same manner as in Example 1 except that the bottom 2b of the container body 2 was not formed in the bottom 2b of the container body 2 and the bottom 2b was a flat surface similar to the top surface 2a.

[Example of evaluation]
HLA-A24-restricted CMV pp65 antigen-specific CTL was prepared using the culture vessels of Example 1, Example 2, and Comparative Example 1 according to the following steps.

(1) Antigen-specific CTL induction step (a) Isolation of peripheral blood mononuclear cells 20 mL of peripheral blood is collected from HLA-A24 positive healthy persons using a syringe to which a small amount of heparin is added, and Ficoll specific gravity centrifugation Peripheral blood mononuclear cells were isolated (3 × 10 ⁷ ).

(B) Collection of plasma When isolating peripheral blood mononuclear cells, plasma was collected, heat-treated at 56 ° C. for 20 minutes, and centrifuged at 2500 rpm for 10 minutes.

(C) Suspension of peripheral blood mononuclear cells in RPMI 1640 medium After washing peripheral blood mononuclear cells 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 collected and suspended with peripheral blood mononuclear cells. Furthermore, 3 mL of autologous plasma was added. Subsequently, HLA-A24-restricted HCMV-specific CTL peptide QYD (sequence: QYDPVAALF, manufactured by Medical and Biological Research Institute) was added to a final concentration of 10 μg / mL, and thoroughly mixed (cell density: 2 × 10 ⁶ / mL).

(D) Induction of CTL The peripheral blood mononuclear cells suspended in the induction medium are divided into three equal parts, 5 mL each using a lure lock syringe from the injection port, and each of Example 1, Example 2, and Comparative Example 1 Into the culture vessel of Thereafter, it was transferred to a 37 ° C., 5% CO ₂ CO ₂ incubator (culture start).

  After standing for a while in the incubator, an image of the cells was obtained by a microscope system equipped with a digital camera, and the sedimentation state of the cells in the culture vessels of Example 1 and Example 2 was confirmed. The microscope used inverted fluorescence phase contrast microscope BZ-X700 (made by Keyence). Magnification used 4x and 40x lenses. The results are shown in FIG.

  In FIG. 2, it was found that the injected cells were sedimented in the center of the bottom of the U-shaped cross section of the wedge-shaped concave portion to be the cell culture portion. While relatively high density of cells are concentrated at the bottom center of the U-shaped cross section, almost no cells can be confirmed in other areas. Thus, the cells in the container body are divided into a plurality of bowl-shaped depressions, and the movement of the container wall in the extending direction is suppressed, and the cells are sedimented at the bottom center of the U-shaped cross section It was possible to culture the cells in close contact with each other, and prepared an environment capable of efficiently inducing antigen-specific immune cells against very low frequency of antigen-specific immune cells.

  The culture vessel was removed 2 days after the start of culture. Next, 1 mL of IL-2-containing induction medium (IL-2 content: 300 IU / mL) was injected into the culture vessel from the injection port. After culturing for 5 days in a 5% CO 2 incubator at 37 ° C., 1 mL of IL-2-containing induction medium (IL-2 content: 50 IU / mL) was injected into the culture vessel from the injection port. By the above operation, antigen-specific CTL was induced in a short time of 7 days from the start of culture (except for the time required for preparation of peripheral blood mononuclear cells and plasma).

  Seven days after induction, a fixed number of cells were taken out from the injection port of each culture vessel, and a part of the cells was counted using a hemocytometer to count the total number of cells. Another part reacted PE-labeled HCMV-specific MHC tetramer with PC5-labeled CD8 antibody, and used a flow cytometer to measure the induction rate of HCMV-specific CTL. Specifically, for an appropriate number of cells, 10 μL of PE-labeled MHC-tetramer reagent (manufactured by Medical Biology Research Institute) and 20 μL of FITC (fluorescein isothiocyanate) -labeled T cell surface antibody CD8 (manufactured by Beckman Coulter, Inc.) Added. Thereafter, they were mixed gently and allowed to stand at 2-8 ° C. for 60 minutes or at room temperature for 30 minutes. After adding and stirring 1.5 mL PBS, it centrifuged at 3,000 rpm for 5 minutes. After the supernatant was aspirated and discarded, the cells were resuspended in 400 μL PBS. At this time, in order to exclude nonspecific fluorescence due to dead cells, 7-AAD viability Dye (dead cell detection reagent, manufactured by Medical Biology Research Institute) was added. Analysis was made with a flow cytometer within 24 hours. The results are shown in FIG. 3 and FIG.

In FIG. 3, the cell suspension and trypan blue staining solution were mixed 1: 4 and the number of cells was immediately measured from a hemocytometer. As a result, while the number of living cells obtained in the culture container of Comparative Example 1 was 1.04 × 10 ^{7, the} number of living cells obtained in the culture containers of Example 1 and Example 2 was They were 1.182 × 10 ⁷ and 1.20 × 10 ⁷ , respectively. In the culture vessels of Example 1 and Example 2 provided with a bowl-shaped recess, more viable cells were obtained than when cultured in the culture vessel of flat-bottom type Comparative Example 1. In addition, the larger the number of bowl-shaped depressions, the larger the number of viable cells was observed.

On the other hand, the results of quantification of antigen-specific CTL are shown in FIG. In the left column of FIG. 4, the numbers in the dot plot development view indicate the area obtained by dividing the development view into four parts as UL (upper left), UR (upper right), LL (lower left), and LR (lower right) The percentage of UR for (UR + LR) minutes is shown. The dot plot developed view shows the fluorescence intensity for CD8 on the X axis and the MHC-tetramer mixed reagent on the Y axis on the log scale.
As apparent from the results shown in FIG. 4, although the HCMV-specific CTL induced in the culture container of Comparative Example 1 had a positive rate of 0.13%, the HCMV-specific CTL induced in the culture container of Example 1 was Is a positive rate of 0.54%, and a positive rate of 1.13% was detected in Example 2.

Further, in the right column of FIG. 4, the number of CTL cells was calculated. The number of CTL cells induced up to 7 days after the start of culture was 4.16 × 10 ⁵ (Comparative Example 1), 7.10 × 10 ⁵ (Example 1), 10 in three types of culture vessels, respectively. 80 × 10 ⁵ (Example 2). With respect to the culture container of Comparative Example 1, 1.7 times of CTL cells were obtained in the culture container of Example 1, and 2.6 times in the culture container of Example 2. It was found that the frequency of tetramer positive cells and the number of CTL cells of the example shown in FIG. 4 were higher than those of Comparative Example 1 despite the induction experiment from peripheral blood mononuclear cells of the same donor. From this, it is clear that a U-shaped cross section of the U-shaped cross section is effective for inducing antigen-specific CTL.

(2) Antigen-Specific CTL Proliferation Step In the above-described step of inducing antigen-specific immune cells, a positive ratio of antigen-specific immune cells is sufficiently obtained, and conditions that are suitable for culture of immune cells continuously (typical In the incubator set at 37 ° C., 5% CO ₂ ) (growth culture start). Thereafter, culture (for 2 days) and injection of growth medium from the injection / delivery port were performed in this order. Thereafter, the culture in the incubator is continued. By the above operation, antigen-specific CTL was prepared in a short time of 14 days from the start of culture in the steps (1) and (2). The resulting cells were subjected to cell counting and flow cytometry analysis. The results are shown in FIG. 5 and FIG.

As shown in FIG. 5, the total number of viable cells obtained on the 14th day from the start of culture is 2.99 × 10 ⁵ (comparative example 1) and 3.17 × 10 ⁵ (example 1). , 3.32 × 10 ⁵ (Example 2). Although there is no significant difference in the total number of viable cells, as shown in FIG. 6, the number of CTL cells is 8.97 × 10 ⁵ (comparative example 1) and 29.51 × 10 ⁵ (example 1). 52.15 × 10 ⁵ (Example 2), and it was found that they were greatly different. With respect to the culture container of Comparative Example 1, CTL cells of about 3.3 times in the culture container of Example 1 and about 5.8 times in the culture container of Example 2 were obtained. Further, as compared with the flow cytometric analysis results in the left column of FIG. 6, the positive rate of CTL was about 15.7 times that of the culture container of Comparative Example 1 in the culture container of Example 1, It was found that the culture vessel was about 17.8 fold. Higher purity CTL cells were obtained by conventional culture.

  That is, despite the short period of 14 days from the start of culture in steps (1) and (2) (except for the time required for preparation of PBMC and plasma), it succeeded in preparing antigen-specific CTL in high purity and in large amounts. did.

(3) Antigen-Specific CTL Recovery Step An example of the recovery procedure after preparing the antigen-specific CTL is shown. First, the contents (ie, antigen-specific CTL) are dispensed into an appropriate container using the injection port of the culture container. Next, after centrifuging, discard the supernatant (culture solution) and inject a cryopreservation solution (8% human serum albumin added CP-1: manufactured by Kyoto Pharmaceutical Industries Co., Ltd. etc.) to suspend the cells . The cell suspension is subsequently transferred to a suitable cryopreservation container and cryopreserved (e.g. -130 to -150 <0> C). Then, the cells are thawed at the time of use, the cells are washed, and then the cells are transfused to the patient using the transfusion bag.

  Although the present invention has been described with reference to the preferred embodiments, it is needless to say that the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the present invention.

  For example, in the embodiment described above, the container body 2 has a rectangular shape, and the injection / discharge port 3 is provided on one side of the short side thereof, but is not limited thereto. The shape of the container main body 2 may be square, oval, circular, or the like, and may be various shapes as needed. The position provided with the injection port 3 can also be changed as appropriate.

  The present invention can be used particularly in the field where it is necessary to obtain a large amount of cells in a short period by enhancing the induction efficiency of cells and the proliferation rate of cells, culture of cells for regenerative medicine and the like.

1 culture vessel 2 vessel main body 2b bottom 3 injection port 4 indented recess

(C) Suspension of peripheral blood mononuclear cells in RPMI 1640 medium After washing peripheral blood mononuclear cells 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 collected and suspended with peripheral blood mononuclear cells. Furthermore, 3 mL of autologous plasma was added. Subsequently, HLA-A24-restricted HCMV-specific CTL peptide QYD (SEQ ID NO : 1 : QYDPVAALF, manufactured by Medical and Biological Research Institute) was added to a final concentration of 10 μg / mL, and thoroughly mixed (cell density: 2 × 10 ⁶ / mL).

Claims (7)

A cell preparation method for preparing antigen-specific immune cells by co-culture of peripheral blood mononuclear cells and an antigen-specific peptide or transformation virus,
A cell preparation method comprising: preparing a target cell by precipitating cells in the recess by using a culture container in which a plurality of recesses serving as a cell culture portion are provided on one surface of the inner wall of the container. The cell preparation method according to claim 1, wherein the following procedures (a) to (e) are performed in combination with any one or two or more to promote the proliferation of antigen-specific immune cells.
(A) adding an antigen-specific peptide (b) adding an anti-CD3 antibody (c) adding an anti-PD-1 antibody (d) an immune checkpoint molecule, inhibiting expression / metabolic pathways of regulatory T cells or HLA Add small molecule inhibitors to improve the expression of molecules (e) Add substances that activate immunity through immune receptors A cell culture vessel for use in the cell preparation method according to claim 1 or 2, comprising:
A container body made of a plastic film having gas permeability and a port for injecting and discharging,
The container body has a bulging shape whose peripheral portion is sealed,
A cell culture vessel, wherein a plurality of bowl-shaped concave portions having an opening diameter of 1.5 mm or more, which is a cell culture portion, are provided on the bottom surface of the container body. The cell culture vessel according to claim 3, wherein the oxygen permeability of the plastic film is 3000 mL / (m ² · day · atm) or more.   The cell preparation method according to claim 3 or 4, wherein the area of the bottom of the bowl-shaped recess is smaller than the area of the opening.   The cell preparation method according to claim 5, wherein the bowl-shaped recess is U-shaped in cross section.   The cell culture vessel according to any one of claims 3 to 6, wherein the ratio d / r of the depth d to the diameter r of the bowl-shaped recess is 0.05 to 1.