JP6074916B2 - Cell activation method and cell culture method - Google Patents

Description

  The present invention relates to a technique for culturing cells, and particularly to a cell activation method for adjusting cell activation in order to improve culture efficiency, and a cell culture method using the same.

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.
In such a situation, it is performed that a culture vessel (culture bag) is filled with cells and a culture solution, and a large number of cells are automatically cultured in a closed system.

  When culturing immune cells in large quantities, cell stimulation through adhesion is important for tissue distribution and functional expression of cells. That is, immune cells usually exist in a floating state in a tissue, but in order to promote their division and promote functional expression, it is necessary to activate the cells by contacting them with antibodies. In order to activate immune cells, as shown in FIG. 1, three elements of “cell”, “antibody”, and “substrate” are necessary. In the conventional immune cell culture method, generally, an antibody 2 is immobilized on a container surface 3 and activated by bringing the immune cell 1 into contact with the antibody 2 to give a stimulus. In FIG. 1, in order to clearly show these three elements, the antibody is schematically shown extremely large against cells. The same applies to FIGS.

  On the other hand, if the immune cells are excessively stimulated by contacting the immune cells with the antibody for a long time or the like, there is an adverse effect, and the proliferation efficiency is lowered. Therefore, in order to efficiently proliferate immune cells, conventionally, as shown in FIG. 8, prior to mass culture, immune cells are activated in an activation container 4 in which antibodies are immobilized on the inner surface of the container in advance. In other words, a method is employed in which activated immune cells are transferred to a culture vessel 5 and mass culture is performed.

For example, in the peripheral blood lymphocyte cell proliferation stimulating method described in Patent Document 1, after culturing with a culture substrate coated with an antibody, culturing is continued with a culture substrate coated with no antibody. Is described.
In such a conventional method, immune cells are first activated, the time when activation is sufficient (activation completion time) is judged by the human eye, and the activated cells are new to which no antibody is applied. It was necessary to transfer to a container.

Japanese Patent No. 3056230

However, since the period required for activation varies depending on the donor and its physical condition, it is difficult to determine whether or not immune cell activation has been completed, and the timing of cell transfer based on that determination is important for growth efficiency. Because of this, there is a problem that it is difficult to obtain an optimal proliferation efficiency.
That is, if the determination of the completion of activation is delayed, the immune cells are overstimulated, resulting in a state called overactivity, and the proliferation efficiency is lowered. If the activation completion is judged too early, immune cell activation becomes insufficient, and the maximum proliferation efficiency cannot be obtained.

Moreover, in the conventional cell culture method, after culturing the cells in the activation container, it is necessary to transfer the activated cells to another culture container, which increases the number of culture containers and complicates the culture procedure.
For this reason, the conventional method has a problem that the culture system becomes complicated and large.

Thus, as a result of intensive studies, the present inventors have first immobilized an antibody on the surface of an immune cell without using an activation container coated with the antibody, and then the immune cell is placed in a culture container via the antibody. By adhering to the surface, it was found that immune cells were sufficiently activated without overactivation, and excellent culture efficiency was obtained, and the present invention was completed.
That is, the present invention provides a cell activation method that allows cells to be cultured efficiently by immobilizing a physiologically active substance on the surface of cells floating in the liquid and then seeding the cells in a culture vessel, and An object is to provide a cell culture method.

  In order to achieve the above object, the cell activation method of the present invention is a method in which a cell is seeded in a culture vessel after a physiologically active substance is immobilized on the surface of the cell suspended in a liquid. At this time, it is preferable that after the physiologically active substance is immobilized, the physiologically active substance that has not been immobilized on the cells in the liquid is washed and removed, and then the cells are seeded in a culture vessel. It is also preferable to use the immune cells as the cells, use an anti-CD3 antibody as the physiologically active substance, add the anti-CD3 antibody to a solution containing the immune cells and react to carry out the above-described immobilization.

  The cell culture method of the present invention is a method of seeding cells in a culture vessel after immobilizing a physiologically active substance on the surface of the cells suspended in the liquid. At this time, after immobilizing the physiologically active substance, the cells are attached to the surface of the culture vessel via the physiologically active substance to complete the activation of the cell in the culture vessel, and the cell is cultured using the culture vessel as it is. It is preferable to do. It is also preferred that after the physiologically active substance is immobilized, the physiologically active substance that has not been immobilized on the cells in the solution is washed and removed, and then the cells are seeded in a culture vessel.

  According to the present invention, it is possible to activate and culture immune cells without the need to determine whether or not the activation of immune cells has been completed, and to transfer cells to another culture vessel. Without performing, it is possible to provide a cell activation method and a cell culture method that can prevent adverse effects on cells such as antibody residues and overactivity.

It is a figure which shows three elements required in order to activate an immune cell. It is a figure which shows the cell activation method of this invention, the cell culture method, and the conventional method. It is a figure which shows the solid-phase surface of the cell activation method and cell culture method of this invention, and a conventional method. It is a figure which shows the case where a small amount of antibody is used in the cell activation method of this invention, the cell culture method, and the conventional method. It is a figure which shows the process in the cell activation method and cell culture method of this invention. It is a figure which shows the culture magnification of the Example by the culture experiment of Experiment 1, and a comparative example. It is a figure which shows the culture magnification of the Example by the culture experiment of Experiment 2, and a comparative example. It is a figure which shows the activation container and culture container in a conventional method.

  Hereinafter, an embodiment of a cell activation method and a cell culture method of the present invention will be described in detail with reference to the drawings. First, the outline of the cell activation method and the cell culture method of the present embodiment will be described with reference to FIGS.

(Overview)
In the cell activation method and cell culture method of this embodiment, an antibody solution containing an anti-CD3 antibody is first added to a cell suspension in which cells such as lymphocytes are suspended in phosphate buffered saline. Leave it for a predetermined time. Thereby, the antibody can be bound to the CD3 antigen on the cell surface by an antigen-antibody reaction. For example, the predetermined time may be about 5 to 15 minutes.
Next, the cells having the antibody bound to the surface are sealed in a culture vessel. As a result, the cells adhere to the surface in the culture vessel via the antibody, and as shown in FIG. 2 (new method), a configuration is formed in which the three elements “cell”, “antibody”, and “substrate” are combined. The cells are activated.
And it is possible to culture | cultivate a cell efficiently by performing cell culture, without transferring from the culture vessel used for activation to another new culture vessel.

On the other hand, in the conventional cell activation method and cell culture method, an activation container in which an anti-CD3 antibody is coated and immobilized in advance is prepared on the surface of the container, and a cell suspension is prepared in this. By encapsulating, the CD3 antigen on the cell surface binds to the antibody on the surface of the container by an antigen-antibody reaction, and as shown in FIG. 2 (conventional method), the three elements of “cell”, “antibody”, and “substrate” Is formed, and the cells are activated.
And after activation of a cell is completed, these cells are transferred from an activation container to a culture container, and cell culture is performed.

As described above, in the cell activation method and cell culture method of the present embodiment, since the antibody immobilized on the cells adheres to the surface of the culture container to activate the cells, the (new method) shown in FIG. As described above, it is possible to minimize the amount of antibody adhering to the container surface.
On the other hand, in the conventional method, the antibody is coated on the surface in the activation container, and the cells are attached to the surface for activation. Therefore, as shown in (conventional method) in FIG. Many surfaces (surfaces with antibodies attached) remain. For this reason, if this container is used for cell culture as it is, the cells are overactive and have an adverse effect on proliferation. Therefore, when activation is completed, it is necessary to transfer the cells to another culture container.
In addition, it is difficult to determine whether or not the activation is completed, and the cell transfer timing based on this determination affects the proliferation efficiency, which causes a problem that the proliferation efficiency varies.

In addition, in the cell activation method and cell culture method of the present embodiment, the amount of antibody used can be greatly reduced as compared with the conventional method. That is, in this method, as shown in FIG. 4 (new method), the antibody is bound to the cell surface and is attached to the surface of the culture vessel. Therefore, the antibody is used in the minimum amount necessary for activation. That's it. In addition, since the cell membrane is flowing, it is considered that the antibody immobilized above the cell in the figure also adheres to the surface in the culture vessel when it moves downward.
On the other hand, in the conventional method, as shown in FIG. 4 (conventional method), when the same amount of antibody is applied to the surface in the activation container, cells attached to the surface on which the antibody is not immobilized are not activated.
Thus, while it is difficult to reduce the number of antibodies used in the conventional method, the cell activation method of this embodiment can achieve the same effect even if the amount of antibody used is reduced to, for example, about 1/10. It is possible.

(Immune cells)
In the cell activation method and cell culture method of the present embodiment, immune cells to be activated include lymphocytes such as T cells, B cells, and NK cells (natural killer cells), macrophages, dendritic cells, and the like. There may be mentioned leukocytes such as granulocytes such as monocytes, neutrophils, eosinophils and basophils.
Among these, in culturing lymphocytes, particularly T cells and NK cells, these can be preferably activated. In addition, even if it is a cell other than an immune cell, if there exists a cell which needs the activation by a bioactive substance in culture | cultivation, this may be cultured with the cell activation method and cell culture method of this embodiment.
In the present embodiment, the cells to be activated are not limited to humans, and various cells in vertebrates including other mammals can be targeted. In addition, if activation is required, cells of other animals, plants, microorganisms, etc. can be targeted.

(Bioactive substance)
The physiologically active substance immobilized on the surface of the immune cell in the present embodiment is not limited as long as it can be activated by giving growth stimulation to the immune cell, for example, humans, other mammals, birds, reptiles, amphibians, Various antibodies (immunoglobulin) in fish, cell adhesion glycoproteins such as fibronectin, laminin, collagen, glycolipids such as α-galactosylceramide (α-GalCer), lipopolysaccharide (LPS), and epidermal growth factor (EGF), Examples include growth factors (growth factors) such as fibroblast growth factor (FGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and the like. Even if cells other than immune cells are required to be activated by these physiologically active substances for culturing, they can be cultured by the cell activation method and cell culture method of the present embodiment using these. good.

(Culture container)
As a culture container used in the present embodiment, a closed container formed in a bag shape (back shape) can be used with a soft packaging material as a material. However, the present invention is not limited to this, such as a dish. Immune cells can be activated using an open container. In addition, when using a soft packaging material, what is described in Unexamined-Japanese-Patent No. 2009-247225 (culture container) and Unexamined-Japanese-Patent No. 2006-262876 (a culture bag, a culture medium preservation method, and a cell culture method) is mentioned, for example. Etc. can be used suitably.
Moreover, when using a closed culture container, it is preferable to use a part or all of which is transparent so that it has gas permeability necessary for cell culture and the contents can be confirmed. Examples of the culture vessel material that satisfies such conditions include polyolefins, ethylene-vinyl acetate copolymers, styrene elastomers, polyester thermoplastic elastomers, silicone thermoplastic elastomers, and silicone rubber.

(Cell activation method and cell culture method)
The cell activation method and cell culture method of the present embodiment include an immobilization step in which a physiologically active substance is added to a cell suspension to immobilize an antibody on the cell surface, and the cells are enclosed in a culture vessel. What is necessary is just to have the seeding process which attaches a cell to the surface in a culture container via a physiologically active substance, and may include another process. For example, as shown in FIG. 5, (1) cell acquisition step, (2) cell separation step, (3) first washing step, (4) immobilization step, (5) second washing step, (6) seeding step It can have. In the following description, the cell to be activated is described as a lymphocyte, and the physiologically active substance is described as an anti-CD3 antibody.

(1) Cell acquisition step First, immune cells to be activated are acquired. For example, blood containing about 20 ml of blood is collected from a donor (patient or the like) to obtain blood containing lymphocytes to be activated.
(2) Cell separation step Next, cells are separated from the obtained blood or the like. For example, density gradient centrifugation is performed on the obtained blood to separate the blood into lymphocytes, red blood cells, plasma and the like.

(3) First washing step The lymphocytes obtained by separation are washed. For example, the separated lymphocytes are washed with phosphate buffered saline to obtain a cell suspension. In addition, it is preferable to perform a washing | cleaning process fully by repeating centrifugation and resuspension repeatedly. For example, it is preferable to repeat 3 to 4 times or more together with a second cleaning step described later.
(4) Immobilization step A solution containing a physiologically active substance is added to the obtained cell suspension and left for a predetermined time. As a result, the physiologically active substance is immobilized on the cell surface. For example, by using an anti-CD3 antibody as a physiologically active substance, the anti-CD3 antibody can be reacted with the CD3 antigen on the lymphocyte surface and immobilized. In addition, various antibodies, cell adhesion glycoproteins such as fibronectin, laminin and collagen, glycolipids such as α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS) and various growth factors (growth factors) It can also be used to immobilize any of these on the cell surface. A plurality of these can also be used.

(5) Second Washing Step Next, the cell suspension is washed with phosphate buffered saline to remove the physiologically active substance released without being immobilized on the cell surface.
(6) Seeding step Finally, the washed cell suspension is sealed in a culture vessel whose inner surface is not coated with a physiologically active substance such as an antibody, so that the cells are seeded in the culture vessel and cell culture is performed. .

  On the other hand, in the conventional method, the steps (1) to (3) are performed in the same manner as in the cell activation method and cell culture method of the present embodiment. Immobilization treatment (solid phase) is performed. Then, after suspending the cell suspension in the activation container and stimulating the cells for a certain period of time to activate them, the activated cells are seeded in the culture container.

As described above, the conventional method includes a cell acquisition step, a cell separation step, a washing step, an immobilization step, and a seeding step, but the immobilization step is different from the cell activation method and the cell culture method of the present embodiment. The antibody is applied to the inner surface of the activation container, and the cells are placed in the activation container. After activation, the antibody is transferred to a culture container and cell culture is performed.
For this reason, since an activation container and a culture container are needed when performing cell culture, there exists a problem that a culture system becomes complicated and enlarged. In addition, since the period required for activation differs depending on the donor, it is necessary to observe and judge whether or not activation is completed, but this observation is not only complicated, but whether or not activation is completed. It was difficult to judge the above, and there was a problem that the culture efficiency might vary due to overactivity or insufficient activity.

  In contrast, in the cell activation method and cell culture method of the present embodiment, a physiologically active substance is immobilized on the surface of a cell to be activated, and then the cells are seeded in a culture vessel. For this reason, even if it reduces the quantity of the physiologically active substance to be used, a cell can be activated reliably. In addition, since an excessive amount of physiologically active substance is not immobilized on the inner surface of the culture vessel, cell overactivity can be prevented, and activation can be performed in the culture vessel without determining whether activation has been completed. High culture efficiency can be obtained by culturing.

  In addition, the cell activation method and the cell culture method of the present embodiment have the second washing step, but if there are many physiologically active substances added to the cell suspension in the immobilization step, It is important to sufficiently remove the physiologically active substance that has not been immobilized. If the physiologically active substance that has not been immobilized on the cell surface is not sufficiently removed, the growth efficiency decreases. In the seeding process, when a physiologically active substance that is released without being immobilized on the cell surface is present in the culture container, the antibody further acts against the cells attached to the inner surface of the culture container via the physiologically active substance. This is thought to be due to immobilization and overactivity.

  Here, the anti-CD3 antibody is known as one of the most effective immunosuppressive agents that suppresses the activation and proliferation of T cells, which are lymphocytes. It is also used clinically to suppress acute rejection symptoms that are resistant to steroids and polyclonal antibodies, and may be used prophylactically in transplantation. Further, even when immune cells were cultured with the anti-CD3 antibody in the culture solution, sufficient proliferation was not obtained. For this reason, it has been considered that such an immunosuppressant anti-CD3 antibody cannot sufficiently activate cells in a free (dissolved) state. Furthermore, when immobilizing antibodies on the substrate surface of the culture vessel, it is considered common practice to immobilize the substrate in advance before seeding the cells. In the past, it was considered that it was impossible for the antibody to react with the cell surface and to act on the culture vessel after the antibody adhered to the cell to give a stimulus to the cell. In addition, it was not known whether the antibody attached to the immune cells adhered well to the surface in the culture vessel, and it was not known whether the cells were activated as in the conventional method.

  In contrast, the present inventors focused on the fact that the opposite side of the antibody-binding site (hereinafter referred to as the C-terminus) is hydrophobic, and this is also a hydrophobic dish surface. Hypothesized that it could adhere to. Also, if the C-terminal is attached to the surface of the culture container in this way, even if the amount of antibody to be used is reduced, the cells are attached with the antibody in the culture container so that the seal is imprinted. The inventors have come up with the idea that by attaching to the surface of the cell, the activation can be reliably performed and the culture efficiency can be improved. In the conventional method, cells adhered to the surface to which no antibody is attached could not be activated. For this reason, it was speculated that if the C-terminal was attached to the surface in the culture vessel in this way, the cells could be sufficiently activated with fewer antibodies compared to the conventional method. Then, as shown in the following examples, it is proved that this hypothesis is correct, and according to the cell activation method and the cell culture method of this embodiment, the cells are activated by the above-described steps, and efficiently It became clear that it could be cultured.

  As described above, according to the cell activation method and the cell culture method of the present embodiment, it is possible to control the immune cells in a single culture container without the need to determine whether or not the activation of immune cells is complete. Activation and culture can be performed. In addition, adverse effects on cells such as antibody residues and overactivity can be prevented. For this reason, it is possible to perform highly efficient and stable cell culture without complicating the culture system.

Hereinafter, examples and comparative examples performed for evaluating the cell activation method, cell culture method, and conventional method of the present embodiment will be described with reference to the drawings.
<Experiment 1: Culture experiment using Dish>
Example 1
The blood of healthy adult males was separated into lymphocytes, erythrocytes and plasma using a blood cell separation solution (product name: Lymphoprep, manufactured by AXIS-SHIELD), and 10 ⁷ lymphocytes were obtained. This was suspended in phosphate buffered saline and divided into 4 test tubes to obtain 4 samples each having 2.5 × 10 ⁶ cells. In this example, one of these was used. To this sample, 10 μg of an anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) was added, stirred, and allowed to stand for 10 minutes to immobilize the anti-CD3 antibody on the CD3 antigen on the lymphocyte surface. The solution was then centrifuged and washed with phosphate buffered saline three times to remove the free anti-CD3 antibody without being immobilized on lymphocytes. Then, the obtained cell suspension was placed in a polystyrene 60 mm dish together with 4 ml of a culture solution (product name: AlyS505N-7, manufactured by Cell Science Laboratories) and subcultured for 215 hours. The dish is a culture container to which no anti-CD3 antibody is applied. The result is shown in FIG.

(Example 2)
Two sheets of 60 mm dish made of polystyrene were prepared, and both were used as an activation container and the other as a culture container without applying anti-CD3 antibody.
Next, 10 μg of anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) was added to one of the samples prepared in Example 1, stirred and allowed to stand for 10 minutes to give anti-CD3 antibody. It was bound to the CD3 antigen on the lymphocyte surface. Subsequently, centrifugation and washing with phosphate buffered saline were repeated 3 times for this solution to remove the free anti-CD3 antibody not immobilized on lymphocytes. Then, the obtained cell suspension was placed in an activation container together with 4 ml of a culture solution (product name: AlyS505N-7, manufactured by Cell Science Laboratories) and allowed to stand for 65 hours to activate the cells.
Next, the obtained culture solution containing activated cells was transferred to a culture vessel and subcultured for 150 hours. The result is shown in FIG.

(Comparative Example 1)
One 60 mm dish made of polystyrene was prepared, and 10 μg of anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) was applied and immobilized on the surface of the container to obtain an activated container. Moreover, in this comparative example, this was used as it is as a culture container.
For one of the samples prepared in Example 1, centrifugation and washing with phosphate buffered saline are repeated three times, and the resulting cell suspension is used as a culture solution (product name: AlyS505N-7, cell (Subsequent to Scientific Research Laboratories) 4 ml and subcultured for 215 hours in the above dish. The result is shown in FIG.

(Comparative Example 2)
Two polystyrene 60 mm dishes were prepared, one of which was coated with 10 μg of anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) and immobilized on the surface of the container to obtain an activated container. The other was used as a culture vessel without applying an anti-CD3 antibody.
For one of the samples prepared in Example 1, centrifugation and washing with phosphate buffered saline are repeated three times, and the resulting cell suspension is used as a culture solution (product name: AlyS505N-7, cell (Made by Scientific Research Laboratories) 4 ml was placed in an activation container and allowed to stand for 65 hours to activate cells.
Next, the obtained culture solution containing activated cells was transferred to a culture vessel and subcultured for 150 hours. The result is shown in FIG.

  The multiplication factor of Example 1 is almost the same as that of Example 2 in which cells were transferred to a new culture container after activation. From this result, according to the cell activation method and cell culture method of the present embodiment, even if the culture is continued using the vessel used for cell activation as it is, the solid-phased surface in the vessel can be minimized. Therefore, it can be seen that lymphocytes are not excessively activated and the culture efficiency is not lowered.

  On the other hand, from the result of Comparative Example 1, in the conventional method, when the culture is continued using the vessel used for cell activation as it is, the solid phase surface remains in the vessel, so that the lymphocytes are excessively active. It turns out that culture | cultivation efficiency will fall. For this reason, as shown in Comparative Example 2, when activation was completed, it was necessary to transfer the activated cells immediately to a culture container not coated with anti-CD3 antibody before culturing.

  Thus, in the conventional method, it was not possible to activate and culture cells in one container. In addition, since the anti-CD3 antibody is coated on the surface of the container, there is a problem that the anti-CD3 antibody cannot be saved. Furthermore, it was necessary to observe the activation state of the cells and immediately transfer the cells to a new container as soon as the activation was completed, but this was a complicated and difficult task. Then, if the determination of completion of activation is delayed, there is a problem that the culture efficiency is lowered as shown in Experiment 2 below.

<Experiment 2: Cultivation experiment during period of overactivity>
(Example 3)
From healthy adult male blood, 4 × 10 ⁶ lymphocytes were obtained in the same manner as in Example 1. This was washed with phosphate buffered saline and divided into three test tubes to obtain three samples each having the same components. In this example, one of these was used. To this sample, 5 μg of an anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) was added, stirred, and allowed to stand for 10 minutes to bind the anti-CD3 antibody to the CD3 antigen on the lymphocyte surface. Subsequently, centrifugation and washing with phosphate buffered saline were repeated 3 times for this solution to remove the free anti-CD3 antibody not immobilized on lymphocytes. Then, the obtained cell suspension was placed in an LLDPE bag together with 7 ml of a culture solution (product name: AlyS505N-7, manufactured by Cell Science Laboratories) and allowed to stand for 144 hours to activate the cells. This LLDPE bag is a culture container to which no anti-CD3 antibody is applied.
Next, the obtained culture solution containing activated cells was transferred to a polystyrene 60 mm dish not coated with anti-CD3 antibody, and subcultured for 93 hours using this dish as a culture container. The result is shown in FIG.

Example 4
For one of the samples prepared in Example 3, centrifugation and washing with phosphate buffered saline are repeated three times, and the resulting cell suspension is used as a culture solution (product name: AlyS505N-7, cell 7 ml of Scientific Research Laboratories) and 10 μg of anti-CD3 antibody (product name OKT-3, Janssen Pharma) were placed in an LLDPE bag and allowed to stand for 144 hours to activate the cells. This LLDPE bag is a culture container to which no anti-CD3 antibody is applied. In this example, unlike Example 3, washing for removing anti-CD3 antibody released without being immobilized on lymphocytes was not performed.
Next, the obtained culture solution containing activated cells was transferred to a polystyrene 60 mm dish not coated with anti-CD3 antibody, and subcultured for 93 hours using this dish as a culture container. The result is shown in FIG.

(Comparative Example 3)
An anti-CD3 antibody (product name OKT-3, manufactured by Janssen Pharma) 5 μg was applied to the LLDPE bag and immobilized on the surface of the container to obtain an activated container. Moreover, this was made into the culture container, without apply | coating an anti-CD3 antibody to polystyrene-made 60mmDish.
For one of the samples prepared in Example 3, centrifugation and washing with phosphate buffered saline are repeated three times, and the resulting cell suspension is used as a culture solution (product name: AlyS505N-7, cell The product was placed in an activation container together with 7 ml of Scientific Research Laboratories, and allowed to stand for 144 hours exceeding the time required for completion of activation to overactivate the cells.
Next, the obtained culture solution containing activated cells was transferred to a culture vessel and subcultured for 93 hours. The result is shown in FIG.

In FIG. 7, the multiplication factors of Example 3, Example 4, and Comparative Example 3 at the time of 237 hours from the start of culture are 299.7 times, 164.1 times, and 70.8 times, respectively.
From the results of Comparative Example 3, it can be seen that in the conventional method, if the determination of the completion of cell activation is delayed, the culture efficiency decreases.
In contrast, as shown in Examples 3 and 4, according to the cell activation method and cell culture method of the present invention, cells are activated without coating the container with an anti-CD3 antibody. Therefore, it is not necessary to be aware of whether or not the activation is completed, and it can be seen that high culture efficiency can be obtained by continuing the culture using the vessel used for the activation as it is.

  In addition, the results of Example 4 indicate that the proliferation efficiency is lower than that of Example 3 when the antibody is added and then cultured without washing. This is presumably because a part of the antibody added to the culture solution continues to be released without being immobilized on the cell surface, thus adversely affecting the growth. However, according to the cell activation method and the cell culture method of the present invention exemplified in Example 4, it is possible to suppress overactivity more than the conventional method shown in Comparative Example 3, and proliferate more than Comparative Example 3. It can be seen that the efficiency is high.

  Furthermore, according to the cell activation method and the cell culture method of the present invention exemplified in Example 3, after the antibody is attached to the cell surface, the free antibody is removed by performing a washing step. The activity can be prevented and there is no adverse effect on cell proliferation.

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, in the embodiments and examples, the case of activating immune cells with an antibody has been described. However, the present invention can be appropriately changed, for example, when other cells are activated with a physiologically active substance other than an antibody.

  The present invention can be suitably used when culturing immune cells and the like in large quantities using a culture vessel.

DESCRIPTION OF SYMBOLS 1 Immune cell 2 Antibody 3 Container surface 4 Activation container 5 Culture container

Claims (9)

  After immobilizing the physiologically active substance on the surface of the cell suspended in the liquid and before stimulating the cell to activate it, the cell is seeded in a culture vessel, and the cell is then physiologicly A method for activating cells, characterized in that the cells are stimulated to be activated in the culture container by attaching them to the surface of the culture container via an active substance.   2. The cell according to claim 1, wherein after the physiologically active substance is immobilized, the physiologically active substance that has not been immobilized on the cells in the liquid is washed and removed, and then the cells are seeded in a culture vessel. Cell activation method. The physiologically active substance is an antibody and / or cell-adhesive glycoprotein and / or glycolipid and / or growth factor, and the antibody and / or cell-adhesive glycoprotein and / or glycolipid and / or Or the growth factor is added and made to react, and the said fixation is performed. The cell activation method of Claim 1 or 2 characterized by the above-mentioned. The cell is an immune cell, the physiologically active substance is an anti-CD3 antibody, the anti-CD3 antibody is added to a solution containing immune cells and reacted to perform the immobilization. 3. The cell activation method according to 3.   5. The cell activation method according to claim 3, wherein the washing is performed by adding a buffer solution to the solution subjected to the immobilization reaction, and repeating centrifugation and resuspension a predetermined number of times. . The cell activation method according to claim 4, wherein the immune cell is a lymphocyte . The cell activation method according to claim 6 , wherein the cells obtained by culturing the activated immune cells are at least one of T cells and NK cells.   After immobilizing the physiologically active substance on the surface of the cell suspended in the liquid and before stimulating the cell to activate it, the cell is seeded in a culture vessel, and the cell is then physiologicly Attaching to the surface of the culture vessel through an active substance to stimulate the cells in the culture vessel to complete activation, and culturing the cells using the culture vessel as it is A cell culture method.   9. The cell according to claim 8, wherein after the physiologically active substance is immobilized, the physiologically active substance that has not been immobilized on the cells in the liquid is washed and removed, and then the cells are seeded in a culture vessel. Cell culture method.

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