JP2015116152A - Culture vessel, and culturing method for lymphocyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable lymphocytes to be activated and proliferated efficiently with only single culture vessel.SOLUTION: The invention provides a culture vessel for culturing lymphocytes formed of a gas permeable film, in which antibodies are solid phased on only one of inner surfaces of the vessel facing each other, thereby a solid phase surface and a non-solid phase surface are provided. In the solid phase surface, anti-CD3 antibodies are solid phased at a concentration of 10-300 ng/cm. Lymphocytes and a culture medium are sealed in this culture vessel, the culture vessel is arranged so that the solid phase surface in the culture vessel becomes the bottom and the lymphocytes are activated, then the culture vessel is reversed thereby the culture vessel is arranged so that the non-solid phase surface in the culture vessel becomes the bottom and the lymphocytes are amplified.

Description

  The present invention relates to a cell culture technique, and more particularly to a culture vessel for culturing lymphocytes and a lymphocyte culture method.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
Under such circumstances, cells and a culture solution are sealed in a culture bag formed from a gas permeable film, and a large number of cells are automatically cultured in a closed system.

By the way, in order to proliferate lymphocytes, it is necessary to first activate the lymphocytes. For this reason, lymphocytes are activated on a base material on which an anti-CD3 antibody is immobilized, and then the activated lymphocytes are enclosed in a culture bag for proliferation.
At this time, as shown in FIG. 8, generally, a flask with an anti-CD3 antibody immobilized on the bottom surface is used for lymphocyte activation, and the lymphocyte is activated and then transferred to a culture bag. Thus, lymphocytes were cultured. The reason is that lymphocytes need to be stimulated with an anti-CD3 antibody for proliferation, but have the property of being difficult to proliferate if they continue to be stimulated with an anti-CD3 antibody. For this reason, after lymphocyte activation, it was necessary to perform culture in a container in which the anti-CD3 antibody was not immobilized.

  Examples of the culture container for activating lymphocytes include the closed cell culture container described in Patent Document 1. In this closed cell culture container, an anti-CD3 antibody is immobilized on the entire surface of the container (paragraphs 0048 and 0057), and lymphocytes can be efficiently activated.

JP 2007-175028 A

However, when lymphocytes are cultured using only such a closed cell culture vessel, since the lymphocytes continue to be stimulated by the anti-CD3 antibody even after the lymphocytes are activated, the lymphocytes are excessively stimulated to proliferate. It will be suppressed. For this reason, in order to efficiently cultivate lymphocytes in large quantities, it is desirable to use this closed cell culture vessel as a dedicated activation vessel, and to proliferate cells in a separate culture vessel.
Therefore, in this conventional technique, when lymphocytes are efficiently cultured in large quantities, there are problems that the complexity of transferring activated cells and the risk of contamination occur.

Therefore, the present inventors have intensively studied, and are culture vessels for culturing lymphocytes, which are made of a gas permeable film, and an anti-CD3 antibody is immobilized on only one side of the opposing inner surface of the vessel to fix the lymphocytes. The one with phase and non-solid phase was developed. Then, the culture vessel is arranged so that the solid-phased surface becomes the bottom surface and the lymphocytes are activated, and then the culture container is arranged so that the non-solid-phased surface becomes the bottom surface, By proliferating, it was possible to efficiently activate and proliferate lymphocytes in a single container.
That is, an object of the present invention is to provide a culture container and a lymphocyte culture method that can efficiently activate and proliferate lymphocytes with a single culture container.

In order to achieve the above object, the culture container of the present invention is a culture container for culturing lymphocytes, which is composed of a gas permeable film, and an antibody is immobilized on only one side of the opposing container inner surface. The solid-phased surface and the non-solid-phased surface are provided, and the anti-CD3 antibody is solid-phased at a concentration of 10 to 300 ng / cm ^{2 on} the solid-phased surface.

  The lymphocyte culture method of the present invention is a lymphocyte culture method using the culture vessel, wherein the lymphocyte and the culture solution are sealed in the culture vessel, and the solid-phased surface in the culture vessel is An activating step of activating lymphocytes by disposing the culture container so as to be a bottom surface, and inverting the culture container so that the non-solid phase surface in the culture container is a bottom surface And a proliferation step of amplifying lymphocytes.

  According to the present invention, it is possible to efficiently activate and proliferate lymphocytes using only a single culture vessel and without using a dedicated culture apparatus or the like. For this reason, it is possible to eliminate the complexity of transfer for preventing excessive stimulation of lymphocytes by antibodies and the risk of contamination.

It is a figure which shows the culture container which concerns on embodiment of this invention. It is a figure which shows the graph of the movement result of the antibody at the time of storing the culture container which concerns on embodiment of this invention in the state which bonded the container inner surface. It is a figure which shows the storage form of the culture container which concerns on embodiment of this invention. It is a figure which shows the culture | cultivation method of the lymphocyte which concerns on 1st embodiment of this invention. It is a figure which shows the culture | cultivation method of the lymphocyte which concerns on 2nd embodiment of this invention. It is a schematic diagram showing the Example and comparative example of the culture container which concern on embodiment of this invention, and the culture | cultivation method of a lymphocyte. It is a figure which shows the graph of the experimental result of the Example and comparative example of the culture container which concerns on embodiment of this invention, and the culture | cultivation method of a lymphocyte. It is a figure which shows the conventional culture | cultivation method of a lymphocyte.

  Hereinafter, embodiments of the culture container and the lymphocyte culture method of the present invention will be described in detail. First, an embodiment of the culture container of the present invention will be described with reference to FIG. In the figure, a schematic view of the culture vessel placed on a loading table (not shown) and viewed from above and a schematic view viewed from the side are shown.

[Culture container]
As shown in FIG. 1, the culture vessel 10 according to the embodiment of the present invention has a vessel wall facing up and down. One of the inner surfaces of the container is a solid-phased surface 11 (the bottom surface in the container of FIG. 1 (2)) on which the antibody 20 is solid-phased. Further, the other inner surface of the container is a non-solid phase surface 12 (the upper surface in the container of FIG. 1B) where the antibody 20 is not solid phased. The culture vessel 10 is provided with a tube 13, whereby the lymphocytes and the culture solution are enclosed in the culture vessel 10, and the cultured lymphocytes and the culture solution are collected. In the example of the figure, one tube 13 is provided in the culture vessel 10, but two or more tubes may be provided.

The culture vessel 10 is formed into a bag shape (back shape) using a film having gas permeability necessary for cell culture. As a material for such a culture vessel 10, a polyolefin resin such as polyethylene or polypropylene can be suitably used.
As the antibody 20 to be immobilized on the immobilization surface 11, an anti-CD3 antibody is preferably used. Lymphocytes can be activated and expanded by anti-CD3 antibodies.

The concentration of immobilized antibody 20 in the immobilized surface 11 is preferably in a 10~300ng / cm ^2, and more preferably a 10~40ng / cm ^2.
This is because if the concentration of the immobilized antibody 20 is 10 ng / cm ² or more, lymphocytes can be activated effectively, and lymphocytes can be efficiently proliferated thereafter. Further, even if the concentration of the immobilized antibody 20 is higher than 40 ng / cm ² , there is no significant difference in the proliferation efficiency of lymphocytes, and the use of the antibody excessively increases the cost of the container.

The closest packing concentration of the anti-CD3 antibody on the solid-phased surface 11 is 300 ng / cm ² , and up to this concentration range, the lymphocytes are sufficiently activated, and then the lymphocytes are efficiently proliferated. Can be made. On the other hand, when the concentration of the immobilized anti-CD3 antibody exceeds 300 ng / cm ² , the anti-CD3 antibody may float in the culture medium in the culture vessel 10 and give excessive stimulation to lymphocytes. It is not preferable.

The culture container 10 of this embodiment can be manufactured as follows, for example.
First, a low density polyethylene is extruded using a plastic extrusion molding apparatus to form a film. And the bag-shaped culture container 10 is manufactured from this film using an impulse sealer. As shown in FIG. 1, the culture vessel 10 is manufactured by attaching a tube 13.

  Next, the culture vessel 10 is placed on a loading table, and a predetermined amount of gas is sealed, and a buffer solution in which the antibody 20 is dissolved is continuously sealed. Then, by shaking the culture vessel 10 or the like, the droplet of the buffer solution is moved on the bottom surface in the culture vessel 10, and the antibody 20 contained in the buffer solution is attached on the bottom surface in the culture vessel 10. Thereby, the antibody 20 is attached to only the bottom surface in the culture vessel 10 to form the solid-phased surface 11. At this time, the upper surface in the culture vessel 10 is formed as a non-solid phased surface 12 to which the antibody 20 is not attached.

Next, the storage form of the culture container 10 of this embodiment is demonstrated.
It is preferable that the culture container 10 of this embodiment is held and stored in a form in which the solid-phased surface 11 and the non-solid-phased surface 12 do not contact each other.
That is, when the culture vessel 10 is held at 37 ° C. for 2 hours under a load of 1.6 kg with the solid-phased surface 11 and the non-solid-phased surface 12 in contact with each other, as shown in FIG. About 20% of the antibodies 20 were transferred from the solid-phased surface 11 to the non-solid-phased surface 12. Thus, the antibody 20 that has moved to the non-solid-phased surface 12 can cause excessive stimulation of the lymphocytes and decrease the proliferation rate when the lymphocytes are activated and then proliferated. Therefore, it is preferable to suppress the movement of the antibody 20 from the solid-phased surface 11 to the non-solid-phased surface 12 as much as possible.

Therefore, when storing the culture container 10 of the present embodiment, a predetermined amount of gas is sealed in the culture container 10 to maintain a form in which the solid-phased surface 11 and the non-solid-phased surface 12 do not contact each other. preferable.
Specifically, the amount of gas to be sealed is preferably 0.01 to 4 ml per 1 cm ² of the bottom area in the culture vessel 10. If the amount of gas to be sealed is within such a range, it is possible to prevent the solid-phased surface 11 and the non-solid-phased surface 12 from contacting each other.
The gas to be sealed is not particularly limited, but is preferably an inert gas, and air, nitrogen gas, or the like can be used. Such gas can be inject | poured into the culture container 10 through the tube 13, for example with a gas supply apparatus.

As a specific storage form of the culture container 10 of the present embodiment, it is preferable to hold the shape of the culture container 10 by a rigid outer container 60 as shown in FIG. When the culture container 10 is stored using such an outer container 60, the culture container 10 can be stored so that the antibody 20 is not turned over only by sealing a small amount of gas in the culture container 10.
Moreover, as shown in (2) of FIG. 3, it is also preferable that the culture container 10 is filled with gas and stored in the packing container 70. In this way, a plurality of culture containers 10 can be simply packed and stored so that the antibody 20 is not turned over.

As described above, the culture vessel 10 of the present embodiment is made of a gas permeable film, and the antibody 20 is immobilized on only one surface of the opposing container inner surface, so that the immobilized surface 11 and the non-solid phase are immobilized. The surface 12 is provided. Moreover, the lymphocytes enclosed in the culture container 10 gather at the bottom of the container.
Therefore, when lymphocytes are activated, the culture vessel 10 is used so that the solid-phased surface 11 becomes the bottom surface, and when lymphocytes are grown, the culture vessel 10 is set so that the non-solid-phased surface 12 becomes the bottom surface. By using it, it is possible to prevent excessive stimulation of lymphocytes by the antibody 20, and to efficiently activate and proliferate lymphocytes in a single container. As a result, complicated transfer and contamination risks can be eliminated. Moreover, since the transition from the activation to the growth process is a simple operation of inverting the container, it can be easily performed without using a dedicated device or the like.
In addition, by enclosing a predetermined amount of gas in the culture vessel 10 of the present embodiment and maintaining a form in which the solid-phased surface 11 and the non-solid-phased surface 12 do not contact each other, an effect of preventing excessive stimulation to lymphocytes is achieved. The culture vessel 10 can be stored without reducing the above.

  The conventional flask used for the activation of lymphocytes has an anti-CD3 antibody immobilized on the bottom surface only, thereby enabling activation of lymphocytes. However, the flask cannot be used in the growth process after activation of lymphocytes in a single container. The reason is that the flask has a problem that the number of cells per area cannot be maintained at an appropriate density. That is, since lymphocytes proliferate after activation, it is necessary to expand the culture area. In this embodiment, for example, it is possible to enlarge a culture area by partitioning a part of a culture container with a clip or a roller and moving or removing the clip or the roller according to cell growth. The flask is used for lymphocyte activation, but is not suitable for growth.

[Method for culturing lymphocytes (first embodiment)]
Next, a first embodiment of the lymphocyte culture method of the present invention will be described with reference to FIG. As shown in the figure, the lymphocyte culture method of this embodiment has (1) an activation step and (2) a proliferation step.

(1) Activation process The activation process in the method for culturing lymphocytes of the present embodiment includes culturing the container 10 so that the lymphocyte 30 and the culture solution 40 are enclosed in the culture container 10 and the solid-phased surface 11 is the bottom surface. To activate the lymphocyte 30.
As described above, the antibody 20 is immobilized on the immobilized surface 11, and an anti-CD3 antibody is preferably used as the antibody 20.
The type of lymphocyte 30 is not particularly limited, and NK cells, B cells, T cells, mononuclear cells, and the like can be targeted for culture.
As the culture solution 40, those generally used for culturing lymphocytes 30 can be used. For example, a culture solution to which interleukin-2 is added can be suitably used.
In this activation step, the lymphocyte 30 in the culture vessel 10 is activated by stimulating the receptor molecule CD3 by the anti-CD3 antibody immobilized on the immobilization surface 11.

(2) Proliferation step The proliferation step in the method for culturing lymphocytes of this embodiment comprises inverting the culture vessel 10 and placing the culture vessel 10 so that the non-solid phased surface 12 becomes the bottom surface. This is the step of amplification.
Thus, by arranging the culture vessel 10 so that the non-solid phased surface 12 becomes the bottom surface, the lymphocyte 30 can be cultured on the non-solid phased surface 12 side in the culture vessel 10.
Thereby, the lymphocyte 30 can be proliferated without being stimulated by the anti-CD3 antibody immobilized on the immobilization surface 11. For this reason, it is possible to prevent a decrease in proliferation efficiency that occurs when the lymphocyte 30 is overstimulated from the anti-CD3 antibody.

[Method for culturing lymphocytes (second embodiment)]
Next, a second embodiment of the lymphocyte culture method of the present invention will be described with reference to FIG. The lymphocyte culture method of the present embodiment includes (1) an activation step, (2) a first proliferation step, (3) a volume expansion step, and (4) a second proliferation step, as shown in FIG. doing.
That is, the lymphocyte culture method of the present embodiment has a volume expansion step in the middle of the proliferation step, which can further improve the proliferation efficiency of lymphocytes compared to the first embodiment. It has become. Therefore, in the present embodiment, the proliferation process is subdivided into the above three processes (2) to (4). About another point, it can be set as the thing similar to 1st embodiment.

(1) Activation Step In the activation step in the lymphocyte culture method of the present embodiment, first, the culture vessel 10 is partitioned by using the partition member 50, whereby the culture section 10-1 and the expandable section 10-2. And divided into
The culture unit 10-1 is a chamber that encloses the lymphocyte 30 and the culture solution 40 and activates the lymphocyte 30.
The expandable portion 10-2 is a chamber in which the lymphocyte 30 and the culture solution 40 are not enclosed, and is used as a space for expanding the culture portion 10-1 as the lymphocyte 30 grows.
The lymphocyte 30 and the culture solution 40 cannot pass between the culture part 10-1 and the expandable part 10-2.

Here, in general, a cell has a property that it is difficult to proliferate if there is no cell density above a certain level in the initial stage of culture. For this reason, it is preferable to adjust the volume of the culture part 10-1 to be small at the initial stage of culture and to be large thereafter.
Further, FIG. 5 shows a process in which the culture container 10 is partitioned using a clip as the partition member 50, and then the clip is removed to make the entire culture container 10 the culture unit 10-1. The method of partitioning the container 10 is not limited to this. For example, a roller may be used as the partition member 50 so that the culture unit 10-1 can be changed continuously, or the culture unit 10-1 can be changed to an arbitrary size a plurality of times.

Next, the lymphocyte 30 and the culture solution 40 are sealed in the culture unit 10-1 in the culture vessel 10, and the culture vessel 10 is arranged so that the solid-phased surface 11 in the culture unit 10-1 becomes the bottom surface. The sphere 30 is activated.
In this activation step, the lymphocyte 30 in the culture unit 10-1 is activated by the anti-CD3 antibody immobilized on the immobilized surface 11.

(2) First Proliferation Step Next, the culture vessel 10 is inverted, and the culture vessel 10 is arranged so that the non-solid phased surface 12 becomes the bottom surface, and the lymphocytes 30 are amplified.
That is, by disposing the culture vessel 10 so that the non-solid phased surface 12 becomes the bottom surface, the lymphocyte 30 can be cultured on the non-solid phased surface 12 side in the culture unit 10-1, The sphere 30 can be grown without being stimulated by the anti-CD3 antibody immobilized on the immobilized surface 11. For this reason, it is possible to efficiently proliferate lymphocytes in a single container.

(3) Volume Expansion Step The volume expansion step is a step of expanding the volume of the culture unit 10-1 by moving or removing the partition member 50.
Thereby, the volume of the culture unit 10-1 can be adjusted according to the number of cells of the proliferated lymphocyte 30. Therefore, the proliferation efficiency of the lymphocyte 30 can be further improved.

(4) Second proliferation step The second proliferation step is a step in which the lymphocyte 30 is continuously cultured in a state where the culture unit 10-1 in the culture vessel 10 is expanded. At this time, the culture vessel 10 is in a state where it is arranged so that the non-solid phased surface 12 becomes the bottom surface.
As a result, the lymphocyte 30 can be proliferated without being stimulated by the anti-CD3 antibody immobilized on the immobilized surface 11, and the density of the lymphocyte 30 in the culture unit 10-1 is too high. It is possible to prevent the proliferation efficiency of the lymphocyte 30 from being lowered.
In addition, it is also possible to use a plurality of clips or rollers as the partition member 50 and repeat the steps (3) and (4) a plurality of times to increase the volume of the culture unit 10-1 stepwise.

As described above, according to the lymphocyte culture method according to the embodiment of the present invention, the culture vessel 10 according to the embodiment of the present invention is used so that the solid-phased surface 11 is the bottom surface. The lymphocyte 30 can be activated, and then the lymphocyte 30 can be proliferated by arranging the non-solid phased surface 12 to be the bottom surface.
For this reason, it is possible to prevent the lymphocyte 30 from being overstimulated by the antibody 20, and it is possible to efficiently activate and proliferate the lymphocyte 30 in a single container.
It is also possible to further increase the proliferation efficiency of the lymphocyte 30 by adjusting the volume of the culture unit 10-1 in the culture vessel 10 according to the number of cells of the proliferated lymphocyte 30.

  Hereinafter, examples and comparative examples of a culture container and a lymphocyte culture method according to an embodiment of the present invention will be described with reference to FIGS. 6 and 7. FIG. 6 is a schematic diagram showing an example and a comparative example, and FIG. 7 is a graph showing experimental results of the example and the comparative example. In addition, FIG. 6 is for showing the difference between an Example and a comparative example, and expansion of the volume of a culture part is abbreviate | omitted.

[Experiment 1: Production of culture vessel]
Example 1
Using a plastic plast mill (produced by Toyo Seiki Seisakusho Co., Ltd.) as a plastic extrusion molding apparatus, low density polyethylene was extruded to form a film having a thickness of 100 μm. Next, an 11 cm × 22.5 cm (about 225 cm ² ) bag was produced from the film using an impulse sealer, and the bag was sterilized by γ-ray and used for the experiment.

Next, about 600 ml of air was sealed in this bag, and 10 ml of a phosphate buffer solution (manufactured by Life Technologies Japan) in which 50 μg of anti-CD3 antibody (made by Miltenyi Biotech) was dissolved was continuously sealed. At this time, the droplets of the phosphate buffer were in contact with only one side (solid phase surface) of the bag inner surface.
Then, the bag is swung manually at 26 ° C. for 1 minute, and the phosphate buffer droplets are moved on the bottom surface of the bag at a speed of 10 m / min to be solidified in the bag. A surface was formed to produce a culture vessel.
Two culture vessels were produced, one of which was used for measurement of the solid phase concentration, and the other was used for a lymphocyte culture test. The same applies to other examples and comparative examples.

Measurement of the solid phase concentration was performed as follows.
First, the liquid in the culture vessel was removed, and 500 μl of a phosphate buffer solution (same as above) containing 1% sodium dodecyl sulfate (manufactured by Sigma-Aldrich Japan LLC) was brought into contact with the solid-phased surface and allowed to stand for 30 minutes. The adsorbed antibody was peeled off by applying strong vibration with a present mixer (manufactured by Taitec Corporation). The amount of antibody in the stripping solution was measured using Micro BCA ™ Protein Assay Kit (manufactured by Thermo Fisher Scientific Co., Ltd.), and the adsorption concentration was calculated by dividing by the solid phase area.

The culture container of Example 1 has an anti-CD3 antibody immobilized on only one inner surface thereof.
As a result of measurement of the immobilized concentration of the culture container of Example 1, the concentration of the anti-CD3 antibody on the immobilized surface was 40 ng / cm ² .

(Example 2)
In the same manner as in Example 1, a bag of 11 cm × 22.5 cm (about 225 cm ² ) was produced.
Next, about 600 ml of air was sealed in the bag, and 10 ml of a phosphate buffer (Life Technologies Japan) in which 5 μg of anti-CD3 antibody (Milteny Biotech) was dissolved was continuously sealed. About the other, the process similar to Example 1 was performed and the culture container of Example 2 was manufactured.

In the culture container of Example 2, the anti-CD3 antibody is immobilized on only one inner surface.
As a result of the measurement of the immobilized concentration of the culture container of Example 2, the concentration of the anti-CD3 antibody on the immobilized surface was 11 ng / cm ² .

(Comparative Example 1)
In the same manner as in Example 1, a bag of 11 cm × 22.5 cm (about 225 cm ² ) was produced.
Next, 10 ml of phosphate buffer solution (manufactured by Life Technologies Japan Co., Ltd.) in which 50 μg of anti-CD3 antibody (manufactured by Miltenyi Biotech Co., Ltd.) is dissolved is enclosed in this bag, and a droplet of phosphate buffer solution is placed in the bag. It was made to contact both upper and lower surfaces, and left still at 26 degreeC for 60 minutes. And the inside of a bag was wash | cleaned 3 times by 40 ml of phosphate buffers, and the culture container of the comparative example 1 was manufactured.

The culture container of Comparative Example 1 has an anti-CD3 antibody immobilized on both inner surfaces thereof.
As a result of measuring the solid phase concentration of the culture container of Comparative Example 1, the concentration of the anti-CD3 antibody on the inner surface of the container was 25 ng / cm ² .

(Comparative Example 2)
In the same manner as in Example 1, a bag of 11 cm × 22.5 cm (about 225 cm ² ) was produced.
Next, about 600 ml of air was sealed in the bag, and 10 ml of a phosphate buffer (Life Technologies Japan) in which 5 μg of anti-CD3 antibody (Milteny Biotech) was dissolved was continuously sealed. At this time, the droplets of the phosphate buffer were in contact with only one side (solid phase surface) of the bag inner surface.
Then, the bag is swung manually at 26 ° C. for 1 minute, and the phosphate buffer droplets are moved on the bottom surface of the bag at a speed of 10 m / min to be solidified in the bag. A surface was formed. Further, the inside of the bag was washed three times with 10 ml of a phosphate buffer solution to produce a culture container of Comparative Example 2.

The culture container of Comparative Example 2 has an anti-CD3 antibody immobilized on only one inner surface.
As a result of measurement of the immobilized concentration of the culture container of Comparative Example 2, the concentration of the anti-CD3 antibody on the immobilized surface was 8 ng / cm ² .

[Experiment 2: Culture of lymphocytes]
(Activation process)
The culture containers obtained in Examples 1 and 2 and Comparative Examples 1 and 2 of Experiment 1 were partitioned using clips so that the culture part was 5 cm × 11 cm, respectively.
Subsequently, 5.8 × 10 ⁴ human peripheral blood mononuclear cells (manufactured by Cell Applications) were added to ALYS505N-7 medium (manufactured by Cell Science Research Institute, Inc.) containing 10% fetal calf serum (manufactured by Life Technologies Japan). 4 ml was sealed in a culture vessel. And it left still at 37 degreeC for 75 hours as it was, and the activation process of the lymphocyte was performed.
At this time, Examples 1 and 2 and Comparative Example 2 were performed with the solid phased surface of the culture vessel facing down. As described above, the culture container of Comparative Example 1 has the same amount of anti-CD3 antibody immobilized on both the upper and lower surfaces inside thereof, and distinguishes between upper and lower, that is, a solid-phased surface and a non-solid-phased surface. There is no distinction.

(Proliferation process)
4 ml of the above medium was added to the culture vessel in which the activation process was completed, the culture vessel was turned upside down, and allowed to stand at 37 ° C. for 26 hours to proliferate lymphocytes (the first embodiment in the second embodiment). One growth step).
Next, the clip is removed, the volume of the culture part of the culture vessel is expanded (volume expansion process in the second embodiment), 20 ml of the above medium is added, and left as it is for 62 hours at 37 ° C. Proliferation was continued (second proliferation step in the second embodiment).
At the time of each operation from the start of culture (75 hours, 75 + 26 (= 101) hours, 75 + 26 + 62 (= 163) hours), and 18 hours after removing the clip (75 + 26 + 18 (= 119)) The number of lymphocytes in the culture vessel was counted. The result is shown in FIG.

As shown in FIG. 7, in Examples 1 and ² , in which 40 ng / cm ² and 11 ng / cm ² of anti-CD3 antibody were immobilized on only one side of the culture container, lymphocytes proliferated remarkably in the proliferation process. It is possible to do. At this time, there was no significant difference in the growth efficiency between the solid phase concentration of 40 ng / cm ² (Example 1) and 11 ng / cm ² (Example 2).
On the other hand, in Comparative Example 1 in which the anti-CD3 antibody is solid-phased on both surfaces in the culture vessel, it can be seen that the proliferation efficiency of lymphocytes in the proliferation process is significantly lower than in Examples 1 and 2.
Further, in Comparative Example 2 in which the concentration of the anti-CD3 antibody to be immobilized is slightly reduced as compared with Example 2, lymphocytes can hardly be proliferated in the proliferation process. From this, it can be seen that the concentration of the anti-CD3 antibody immobilized on the immobilization surface of the culture vessel is preferably about 10 ng / cm ² or more.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, the size of the culture vessel, the type of lymphocyte, and the culture medium can be changed as appropriate, for example, different from those in the examples.

  The present invention can be suitably used for culturing lymphocytes in large quantities while eliminating the complexity of transferring activated cells and the risk of contamination by using a single culture vessel. Is possible.

DESCRIPTION OF SYMBOLS 10 Culture container 11 Solid phase surface 12 Non-solid phase surface 13 Tube 20 Antibody 30 Lymphocyte 40 Culture solution 50 Partition member 60 Exterior container 70 Packing container

Claims (5)

A culture vessel for culturing lymphocytes, comprising a gas permeable film, wherein an antibody is immobilized on only one side of the inner surface of the opposite container to provide a solid phased surface and a non-solid phased surface An anti-CD3 antibody is solid-phased at a concentration of 10 to 300 ng / cm ^{2 on} the solid-phased surface.   The culture vessel according to claim 1, wherein the solid-phased surface and the non-solid-phased surface are held in a form that does not contact each other. The culture vessel according to claim 2, wherein 0.01 to 4 ml of gas is sealed per 1 cm ^{2 of the} bottom area of the vessel. A lymphocyte culture method using the culture vessel according to any one of claims 1 to 3,
An activating step of enclosing lymphocytes and a culture solution in the culture vessel, locating the culture vessel so that the solid-phased surface in the culture vessel becomes a bottom surface, and activating lymphocytes;
Inverting the culture vessel, arranging the culture vessel so that the non-solid phased surface in the culture vessel becomes the bottom surface, and a proliferation step for amplifying lymphocytes. Culture method. In the activation step, by dividing the culture vessel by a partition member, a culture part is formed in the culture container, and lymphocytes and a culture solution are enclosed in the culture part,
As the proliferation step,
Inverting the culture vessel, placing the culture vessel so that the non-solid phase surface in the culture vessel is the bottom, a first growth step of amplifying lymphocytes,
A volume expansion step of moving or removing the partition member to expand the volume of the culture part;
The method for culturing lymphocytes according to claim 4, further comprising a second proliferation step of amplifying lymphocytes after expanding the volume of the culture part.

Images