JP3936714B2 - Cell processing method - Google Patents

Description

The present invention relates to a process how the cells by applying a magnetic field to the cell group different cells are mixed treatment of cells.

Conventionally, as a method for treating this type of cell, for example, the one described in Patent Document 1 (Japanese Patent Laid-Open No. 2004-49105) is known.
As shown in FIG. 7, magnetic beads Ba having an antibody capable of specifically binding to an antigen of the target cell are applied to a target cell Sa such as a cancer cell in a cell group in which different types of cells are mixed. The cells are combined, and a pulse magnetic field of at least one of a single pulse and a multi-pulse is applied to the cell group to selectively kill the target cell Sa.
The principle of this death is that when the magnetic beads Ba are stimulated by a pulsed magnetic field, the magnetic beads Ba move instantaneously, and the target cells Sa are pulled by the magnetic beads more strongly than Ba, and are physically crushed and killed. It has been.

JP 2004-49105 A

  By the way, in this conventional cell processing technique, the target cell Sa is killed using the magnetic beads Ba. However, in such cell processing using magnetism, the target cell is not necessarily intended to be killed. In some cases, it is desired to make the object survive, and in this case, there is a problem that the method cannot cope with it.

The present invention has been made in view of the above-described problems, and enables magnetic beads to be used not for killing purposes but for survival purposes, and by using magnetic beads to improve the survival rate of specific cells, the cell group is subjected to magnetic field treatment. and to provide a process how the cells to be.

  In order to solve such a problem, the cell processing method of the present invention is a cell processing method in which a magnetic field is applied to a cell group in which different types of cells are mixed. A magnetic bead having an antibody capable of specifically binding to an antigen of a specific cell is bound, a sinusoidal fluctuation magnetic field is applied to the cell group, and the survival rate of the specific cell is determined as a non-specific cell other than the specific cell. It is configured to process by increasing the survival ratio.

The inventors of the present application, in a variable magnetic field condition, in particular, in a sinusoidal variable magnetic field, by binding the magnetic beads to specific cells, contrary to the conventional pulse magnetic field, rather, the cells that do not bind the magnetic beads. It was found that the inactivation of specific cells was suppressed as compared with.
As the mechanism, in cells placed in a sinusoidal fluctuation magnetic field, the nuclear material mainly composed of ions is subjected to a magnetic force repeatedly when moving in the fluctuation magnetic field, thereby deviating from the normal division process. Cell division ends abnormally, leading to cell death, induction of cell membrane structural abnormality, or other effects such as DNA synthesis failure, but in specific cells with magnetic beads bound Is a characteristic of a sinusoidal magnetic field, and the direction of the magnetic field lines suddenly reverses repeatedly, making it difficult for the magnetic beads to follow the movement. Therefore, the magnetic field is directly applied to intracellular organelles or cytoskeletal proteins. In order to absorb (or buffer) the effect of the magnetic field and reduce the magnetic field damage effect on the cells, it is considered that these damages are suppressed. That.

  With this configuration, for example, magnetic beads are bound to specific cells other than malignant cells such as cancer cells having a relatively high division speed, and magnetic beads are not bound to non-specific cells of malignant cells. To increase the survival rate of specific cells, inactivate the ability to divide non-specific cells, promote death or reduction, and is expected to be used in the medical field .

  If necessary, the applied magnetic field strength is set to 0.01 ≦ T (Tesla) ≦ 1. Desirably, 0.05 ≦ T (Tesla) ≦ 0.7. Within this range, the survival rate of specific cells can be effectively increased.

  In addition, the frequency of the sinusoidal fluctuation magnetic field is set to 1 to 300 Hz as necessary. Desirably, it is 5 to 100 Hz. Within this range, the survival rate of specific cells can be effectively increased.

  Further, the temperature t of the cells is set to 4 ° C. ≦ t ≦ 37 ° C. as necessary. Desirably, the cell temperature t was set to 25 ° C. ≦ t ≦ 37 ° C. Thereby, when a cell is processed with the blood of a human body etc., the influence which it has on a good quality cell is reduced. That is, when the temperature is lower than 4 ° C. or higher than 37 ° C., the influence on specific cells increases.

  Furthermore, the magnetic field is applied for 5 min to 240 min. In this case, if the time is shorter than 5 minutes, a sufficient effect cannot be obtained. On the other hand, when the time is longer than 240 min, the influence on specific cells increases.

  And it is set as the structure which adds the division rate promoter which accelerates | stimulates the division rate of the said non-specific cell as needed. Thereby, non-specific cells are activated (cell division is stimulated), and the viability of non-specific cells can be reduced more effectively and effectively.

Examples of the mitotic rate promoter include phytohemagglutinin (PHA), concanavalin A (ConA), lymphocyte-specific peptide, lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglyceol, tetradecanoyl phorbol acetate (TPA) It is effective to use at least one of the following substances. These substances are mainly substances that stimulate the cells of the immune system and usually do not always have a bad action. For example, lymphocyte-specific monoclonal antibodies are also used as therapeutic agents for leukemia.
In general, each immune cell (T cell or NK cell) is activated by a specific molecule (peptide, protein, antibody or sugar, lipid) but is hardly activated by other molecules. Therefore, if you activate specific immune cells (cells) that have caused abnormalities and load a variable magnetic field (providing stimulation for division and proliferation), only abnormal immune cells can be selected without damaging normal immune cells. In addition, it can be removed without side effects.

In addition, in order to realize the cell processing method of the present invention, the cell processing apparatus has an antibody capable of specifically binding to the antigen of the specific cell in the specific cell in the cell group in which different types of cells are mixed. A magnetic field strength of 0.01 to a container in which a processed product containing the cell group formed by binding the magnetic beads is placed, a container holding unit for holding the container, and a processed product in the container held by the container holding unit. The thing of the structure provided with the fluctuation | variation magnetic field generation | occurrence | production part which applies the sinusoidal fluctuation | variation magnetic field of <= T (Tesla) <= 1,1-300Hz is mentioned. Thereby, a sinusoidal fluctuation magnetic field can be reliably applied to the cells.

  And, if necessary, the variable magnetic field generating unit is arranged with a pair of rotating disks arranged and rotated with the container held by the container holding unit interposed therebetween, and a sinusoidal variable magnetic field provided on each rotating disk by rotation. And a motor for rotationally driving the rotating disk. The apparatus can be made relatively simple and inexpensive, the process can be simplified and simplified, and versatility can be improved.

According to the process how the cells of the present invention, can be processed is increased from survival ratio of the non-specific cell survival ratio of specific cells. Therefore, for example, a magnetic bead is bound to a specific cell other than a malignant cell and a magnetic bead is not bound to a non-specific cell of the malignant cell, and a sinusoidal magnetic field is applied to increase the survival rate of the specific cell. Thus, the ability to divide non-specific cells can be inactivated and death or reduction can be promoted, and application to medical fields such as purification of blood for transfusion can be expected.

Hereinafter, a cell processing method and a cell processing apparatus according to embodiments of the present invention will be described with reference to the accompanying drawings.
Since the cell processing method according to the embodiment is realized by the cell processing apparatus according to the embodiment, its operation will be described. The processing apparatus according to this embodiment can process, for example, cells in blood collected from a human body as a processed product. Examples of the cells include various cancer cells such as leukemia, cells containing malignant cells such as virus-infected cells, and the like.

  As shown in FIGS. 1 to 3, the processing apparatus K includes a container 1 that stores a processed product including a cell group in which different types of cells to be processed are mixed. As the container 1, for example, as shown in FIG. 2, a test tubular resin body 2 in which a lid 3 is integrally provided by a resin hinge 4 is used. As shown in FIG. 4, a magnetic bead B having an antibody capable of specifically binding is bound to an antigen of a specific cell S in the cell group. As the magnetic beads B, appropriate ones are used depending on the specific cells.

  The container 1 is held by a resin container holding unit 10. As shown in FIG. 2, the container holding unit 10 includes a base 11 that is detachably fixed to the machine base 5 with screw means 14 (FIG. 1), and the like, and the container 1 is fitted and held on the base 11. A plurality of concave portions 12 are provided on a predetermined circumference (four in the embodiment), and a retaining plate 13 is detachably attached to both surfaces of the retaining plate 13 with clips 15 or the like and fitted into the concave portion 12. A pair of presser plates 16 that prevent the container 1 from dropping off are provided. In the center of the holding plate 13 and the presser plate 16, a recess 17 is formed in which one end through which a rotating shaft 22 of a variable magnetic field generating unit 20 described later is inserted is opened.

The machine base 5 is provided with a variable magnetic field generator 20. Fluctuating magnetic field generating unit 20 applies magnetic field intensity 0.01 ≦ T (Tesla) ≦ 1, 0.5T, 1 to 300 Hz in the embodiment, to the processed material in container 1 held in container holding unit 10. Then, a sinusoidal fluctuating magnetic field of 50 Hz is applied.
Specifically, the variable magnetic field generation unit 20 includes a pair of rotating disks 21A and 21B that are arranged and rotated with the container 1 held by the container holding unit 10 interposed therebetween. The turntables 21 </ b> A and 21 </ b> B are supported by one rotary shaft 22. Both ends of the rotating shaft 22 are rotatably supported by bearing stands 23 and 24.

  As shown in FIG. 3, each of the turntables 21 </ b> A and 21 </ b> B is provided with a permanent magnet 25 that generates a sinusoidal fluctuation magnetic field by rotation. Specifically, on the side facing the container holding portion 10 of one of the turntables 21A, a permanent magnet having alternating magnetic poles in an equiangular relationship on the circumference corresponding to the circumference where the concave portion 12 is provided. Sixty-five 25 are provided. On the side facing the container holding portion 10 of the other rotating plate 21B, the magnetic poles are alternately changed in an equiangular relationship on the circumference corresponding to the circumference where the concave portion 12 is provided, and the one rotating plate 21A The six permanent magnets 25 are provided in a positional relationship with one permanent magnet 25 shifted in phase. That is, six pairs of permanent magnets 25 having different magnetic poles are alternately arranged across the holding plate 13.

  One bearing stand 24 is provided with a motor 26 that rotationally drives the rotary shaft 22 of the rotary disks 21A and 21B. The number of rotations of the motor 26 is 1000 RPM, whereby a 50 Hz sinusoidal varying magnetic field can be applied.

  Furthermore, the processing apparatus K is placed in a thermostat (not shown) that keeps the temperature in the container 1 within a certain range. The temperature in the thermostatic bath is maintained, for example, in a range of 4 ° C. ≦ t ≦ 37 ° C., preferably 25 ° C. ≦ t ≦ 37 ° C. In the embodiment, for example, t = 25 ° C. is set.

  And the container 1 is hold | maintained by the processing apparatus K in the container holding | maintenance part 10, the holding plate 13 of the container holding | maintenance part 10 is located between the rotating discs 21A and 21B of the fluctuation magnetic field generation | occurrence | production part 20, and it is a sine wave form for 5 to 240 minutes. Apply a fluctuating magnetic field. In the embodiment, for example, it is set to 120 min.

  Therefore, when a cell group is processed by the processing apparatus K according to this embodiment, for example, the magnetic beads B are bound to specific cells S other than malignant cells such as cancer cells having a relatively high division rate, and malignant. The magnetic beads B are not bound to non-specific cells. That is, as shown in FIG. 4, the magnetic beads B having an antibody that can specifically bind to the antigen of the specific cell S are bound in advance. Then, the cell group is put in the container 1. Then, the container 1 is held by the container holding unit 10, the holding plate 13 of the container holding unit 10 is positioned between the rotating disks 21 </ b> A and 21 </ b> B of the variable magnetic field generating unit 20, and the motor 26 is operated to keep the magnetic field in the variable magnetic field for a predetermined time. Hold on.

As a result, when a fluctuating magnetic field acts on a malignant cell having a high division rate, a sinusoidal fluctuating magnetic field is applied to the cell group, and the death or reduction of the malignant cell is promoted. In other words, when a malignant cell is stimulated to divide, the cell is differentiated with the division of the nucleus, but when nuclear substances mainly composed of ions move in the magnetic field, they receive magnetic force from the magnetic field and deviate from the normal division process. As a result, cell damage occurs, and the division ability is deactivated. And since malignant cells have a high division rate and the opportunity of magnetic field stimulation at the time of division increases, the malignant cells die or decline because disordered increase in division is prevented.
On the other hand, in the specific cells S other than the malignant cells, the survival rate of the specific cells S is increased by the action of the magnetic beads B. That is, effects such as changes in immune function and cytotoxicity are prevented.

  In the above-described embodiment, it is effective to add a mitotic rate promoter that accelerates the mitotic rate of non-specific cells as necessary. For example, when there are specific immune cells (cells) that have abnormalities in the cells of the immune system and it is desired to remove them, phytohemagglutinin (PHA), concanavalin A (ConA), lymphocyte specific It is effective to use at least one substance such as a specific peptide, a lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglycol, tetradecanoylphorbol acetate (TPA).

  In general, each immune cell (T cell or NK cell) is activated by a specific molecule (peptide, protein, antibody or sugar, lipid) but is hardly activated by other molecules. Therefore, if you activate specific immune cells (cells) that have caused abnormalities and load a variable magnetic field (providing stimulation for division and proliferation), only abnormal immune cells can be selected without damaging normal immune cells. In addition, it can be removed without side effects. Therefore, by adding this mitotic rate accelerator, cells are activated (stimulation of cell division), and abnormal cells can be removed more powerfully and effectively.

  Moreover, in the above-described embodiment, it is effective to add a deactivation auxiliary agent that deactivates non-specific cells. For example, in order to remove cancer cells, vitamin A, thyroid hormone, glucocorticoid, cyclophosphamide, azathioprine, adriamycin, bleomycin, antimetabolite, alkylating agent, plant alkaloid (bin alkaloid) It is effective to use at least one substance of cytoskeletal disorder drug. These are known as drugs used for the treatment of cancer, and the addition of these substances makes it possible to mainly inactivate cancer cells with high efficiency. That is, in addition to cell deactivation by a magnetic field, deactivation by a deactivation auxiliary agent is also performed, so the effect is synergistic and the cell removal efficiency is further improved.

Experimental example

Next, an experimental example is shown.
As shown in FIG. 5, CD8 · T cells and CD4 · T cells were used as cells. As the magnetic beads B, those to which a CD3 antibody was bound were used. In these cells, magnetic beads B bonded and non-bonded are prepared, a mitotic rate accelerator is added (PHA administration), and the above processing apparatus K is used to perform the measurement under a sinusoidal magnetic field. The survival ratio of each cell was compared.

Test conditions are
Frequency: 50Hz
Magnetic field strength: 0.5T
Holding temperature: t = 25 ° C.
Magnetic field application time: 120 min.

The results are shown in FIG. In CD4 · T cells, the survival rate of those not bound with magnetic beads B was 65%, whereas the binding rate of magnetic beads B was 86%, and the increase in survival rate was noticeable.
In addition, in CD8 · T cells, those without magnetic beads B have a survival rate of 83%, whereas those with magnetic beads B have a binding rate of 91%, and this also increases the survival rate. It was seen.

  Thus, it can be seen that binding of the magnetic beads B may reduce cell damage. That is, conventionally, it has been difficult to modulate only the function of a specific cell, but a substance that specifically recognizes a target cell using a substance (drug or monoclonal antibody) that recognizes a specific cell ( In the experiment, a complex in which magnetic beads are bound to an anti-CD3 monoclonal antibody) is prepared, and the CD3 antibody-magnetic bead complex is bound to leukocytes prepared from human peripheral blood. In this case, since the CD3 antibody binds only to T lymphocytes (CD4 lymphocytes and CD8 lymphocytes), only T lymphocytes are affected by the magnetic field and magnetic beads. Therefore, the cell can be protected against the target cell by adjusting the magnetic field strength (variable magnetic field). Under a strong magnetic field, the survival rate of T lymphocytes decreases, but the survival rate improves with T lymphocytes bound with magnetic beads.

  The cells may be any cells other than blood, and may be changed as appropriate.

It is a perspective view which shows the processing apparatus of the cell which concerns on embodiment of this invention. It is a perspective view which shows the structure of a container and a container holding part in the processing apparatus of the cell which concerns on embodiment of this invention. In the cell processing apparatus which concerns on embodiment of this invention, the structure of the rotating disk of a fluctuation | variation magnetic field generation | occurrence | production part is shown, (a) is A view in FIG. 1, (b) is B view in FIG. It is a figure which shows the relationship between a specific cell and a magnetic bead. It is a figure which shows the relationship between the specific cell and magnetic bead in an experiment example. It is a graph which shows the result of having experimented the influence of the magnetic bead which gives to T cell in sinusoidal magnetic field application environment, and shows the increase / decrease in the number of cells by making the number of cells of a control group 100%. It is a figure which shows the relationship between the target cell and the magnetic bead for the purpose of the conventional death.

Explanation of symbols

K processing apparatus S specific cell B magnetic bead 1 container 10 container holding part 20 fluctuating magnetic field generating part 21A, 21B rotating disk 22 rotating shafts 23, 24 bearing stand 25 permanent magnet 26 motor

Claims (7)

In the cell processing method of applying a magnetic field to a cell group in which different types of cells are mixed,
A magnetic bead having an antibody capable of specifically binding to an antigen of the specific cell is bound to a specific cell of the cell group, and a sinusoidal fluctuation magnetic field is applied to the cell group, so that the survival rate of the specific cell A method for treating cells, characterized by increasing the survival rate of non-specific cells other than the specific cells.   2. The method for treating cells according to claim 1, wherein the applied magnetic field intensity is 0.01 ≦ T (Tesla) ≦ 1.   The cell processing method according to claim 1 or 2, wherein the frequency of the sinusoidal fluctuating magnetic field is 1 to 300 Hz.   4. The method for treating cells according to claim 1, wherein the temperature t of the specific cells is 4 ° C. ≦ t ≦ 37 ° C.   5. The method for treating cells according to claim 1, wherein a magnetic field is applied for 5 min to 240 min.   The method for treating cells according to claim 1, 2, 3, 4 or 5, wherein a mitogenic rate promoter for accelerating the division rate of the non-specific cells is added.   Examples of the mitogenic rate promoter include phytohemagglutinin (PHA), concanavalin A (ConA), lymphocyte-specific peptide, lymphocyte-specific monoclonal antibody, galactosylceramide, ceramide, ganglioside, diacylglyceol, tetradecanoyl phorbol acetate (TPA) 7. The method for treating cells according to claim 6, wherein at least one substance is used.

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