Method for determining whether immune cells have acquired immunity against tumor cells

Abstract

Provided is a method for determining whether immune cells extracted from a patient have acquired immunity against tumor cells, which can reliably determine whether immune cells extracted from a patient have acquired immunity against tumor cells. [Solution] A method for determining whether immune cells have acquired immunity against tumor cells includes the steps of identifying tumor tissue from collected tissue containing tumor tissue and non-tumor tissue, allowing the immune cells to act on the tumor tissue, identifying the distribution of immune cells in the tumor tissue, and detecting the degree of accumulation of immune cells in the tumor tissue, and determining that the immune cells have acquired immunity if the degree of accumulation is equal to or greater than a reference value.

Description

[Technical Field]

[0001] The present invention relates to a method for determining whether immune cells have acquired immunity against tumor cells. [Background technology]

[0002] The main cancer treatment methods include surgery, radiation therapy, drug therapy, and immunotherapy. While surgery is effective for early-stage cancer, it is not an option when the cancer has metastasized or there is a possibility of metastasis. It also places a heavy burden on the patient and requires a long recovery time. Radiation therapy is effective for early-stage cancer, but it has significant side effects and aftereffects. Drug therapy can deliver drugs throughout the body via oral medication or intravenous drip, and is effective against metastatic cancer, but it also kills normal cells, resulting in significant side effects and the risk of infection. Immunotherapy is effective for certain cancers, but is expensive because it involves gene manipulation, and is not considered effective for cancer recurrence or solid tumors.

[0003] In contrast to conventional cancer treatment methods, a new treatment method has been proposed in which immune cells are extracted from a patient's blood using an apheresis system, an immunostimulant is mixed with the extracted immune cells, and the immune cells are allowed to act on tumor tissue to acquire immunity against the tumor cells, and then the immune cells are returned to the patient. This treatment method places a small burden on the patient and does not require genetic manipulation, allowing for multiple treatments at low cost and is expected to be effective against cancers from early to late stages, or even metastatic cancers. Such a treatment method is disclosed, for example, in Patent Document 1. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2020-530486 Summary of the Invention [Problem to be solved by the invention]

[0005] In the aforementioned treatment method, it is necessary to determine whether immune cells have acquired immunity against tumor cells. In Patent Document 1, this determination is made by visual observation, which may result in variations in the determination depending on the observer's level of skill, and may result in insufficient therapeutic effects.

[0006] The present invention has been made to solve the above-mentioned problems, and aims to provide a method for determining whether immune cells extracted from a patient have acquired immunity against tumor cells, which can reliably determine whether immune cells extracted from a patient have acquired immunity against tumor cells. [Means for solving the problem]

[0007] (1) A method according to the present invention for achieving the above-mentioned object, for determining whether immune cells have acquired immunity against tumor cells, includes the steps of identifying tumor tissue from collected tissue containing tumor tissue and non-tumor tissue, allowing immune cells to act on the tumor tissue, identifying the distribution of the immune cells in the tumor tissue, and detecting the degree of accumulation of the immune cells in the tumor tissue, and determining that the immune cells have acquired immunity if the degree of accumulation is equal to or greater than a reference value. [Effects of the Invention]

[0008] The method (1) for determining whether immune cells have acquired immunity against tumor cells, configured as described above, detects the degree of accumulation of immune cells in tumor tissue and determines whether immunity has been acquired based on the degree of accumulation, thereby making it possible to reliably and consistently determine whether immune cells extracted from a patient have acquired immunity against tumor cells.

[0009] (2) The method for determining whether immune cells have acquired immunity against tumor cells according to (1) above may further include a step of identifying the distribution of the immune cells in the non-tumor tissue. This allows the method for determining whether immune cells have acquired immunity against tumor cells to detect the absence of accumulation of immune cells in the non-tumor tissue, thereby enabling more accurate determination of whether immune cells have acquired immunity against tumor cells.

[0010] (3) The method for determining whether immune cells have acquired immunity against tumor cells according to (1) or (2) above may further include a step of setting the reference value for the accumulation degree, thereby enabling the method for determining whether immune cells have acquired immunity against tumor cells to be appropriately determined in accordance with circumstances such as the patient's condition.

[0011] (4) In any of the methods for determining whether immune cells have acquired immunity against tumor cells described in (1) to (3), the method further includes the step of preparing a medical device including a mixing chamber for placing tumor tissue and non-tumor tissue, an imaging unit for imaging the inside of the mixing chamber, and a determination unit for determining whether the immune cells have acquired immunity, wherein the determination unit identifies the tumor tissue from the image captured by the imaging unit, the mixing chamber allows the immune cells to act on the placed tumor tissue, and the determination unit identifies the distribution of the immune cells in the tumor tissue from the image captured by the imaging unit, and detects the accumulation level of the immune cells in the tumor tissue from the identified distribution of the immune cells. This allows the determination unit to detect the accumulation level of the immune cells in the tumor tissue from the image captured of the tumor tissue placed in the mixing chamber, thereby enabling the medical device to reliably and consistently determine whether the immune cells have acquired immunity against tumor cells.

[0012] (5) In the method for determining whether immune cells have acquired immunity against tumor cells according to (4), the mixing chamber may be provided with a first compartment in which the tumor tissue and the non-tumor tissue are placed, and a second compartment in which only the non-tumor tissue is placed, the imaging unit may be capable of imaging the first compartment and the second compartment, and the determining unit, in the step of determining whether immune cells have acquired immunity against tumor cells, may detect the degree of accumulation of the immune cells in the tumor tissue based on the distribution of the immune cells in the first compartment and the distribution of the immune cells in the second compartment. This allows the method for determining whether immune cells have acquired immunity against tumor cells to clearly distinguish and recognize the distribution of immune cells in the non-tumor tissue and the distribution of immune cells in the tumor tissue, thereby enabling more accurate detection of the degree of accumulation of immune cells in the tumor tissue.

[0013] (6) The method for determining whether immune cells have acquired immunity against tumor cells according to (4) or (5) above may further include a step of returning the immune cells that have acted on the tumor tissue in the mixing chamber to the blood, thereby enabling the method for determining whether immune cells have acquired immunity against tumor cells to proceed with patient treatment while confirming the immune acquisition of the immune cells. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 2 is an operation flow diagram of the medical device according to the present embodiment. [Figure 2] FIG. 1 is a configuration diagram of a medical device. [Figure 3] FIG. 2 is a perspective view of the mixing chamber, with the rotating container visible. [Figure 4] FIG. [Figure 5] FIG. 10 is a front view of a filter unit according to a modified example. [Figure 6] 6 is a cross-sectional view taken along line AA in FIG. 5 and a view showing a pressing member. [Figure 7] FIG. 10 is a perspective view of a mixing chamber according to a modified example. [Figure 8]FIG. 2 is a more detailed operational flow diagram of the medical device. [Figure 9] FIG. 1 is an image showing the initial state of contact of immune cells with tumor tissue. [Figure 10] FIG. 1 is a flow chart for determining whether immunity has been acquired. [Figure 11] This is an image showing the state in which immune cells act on and accumulate in tumor tissue. [Figure 12] (a) is an illustration of the distribution of immune cells in a section containing only non-tumor tissue, and (b) is an illustration of the distribution of immune cells in a section containing both non-tumor tissue and tumor tissue. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the dimensional proportions in the drawings may be exaggerated for the sake of explanation and may differ from the actual proportions.

[0016] The medical device 10 of this embodiment is configured as an apheresis system that collects blood from a cancer patient, separates immune cells from the blood, brings the separated immune cells into contact with tissue collected from the patient containing tumor cells to acquire immunity, and returns the immune cells that have acquired immunity to the blood and reinfuse them into the patient.

[0017] The operational flow of the medical device 10 will now be described. As shown in Figure 1, first, blood is collected from a patient (S1), and the collected blood is centrifuged (S2). White blood cells, which are immune cells, are obtained by centrifugation (S3). The immune cells are mixed with an immunostimulant (S4), and are allowed to act on tumor tissue, causing the immune cells to acquire immunity against tumor cells (S5). Once the immune cells have acquired immunity, they are mixed with the blood from which they were separated by centrifugation, and the blood is returned to the patient (S6).

[0018] Next, we will explain the configuration of the medical device 10. As shown in Figure 2, the medical device 10 has an immune cell acquisition means 20 that collects blood from a patient 200 and separates and acquires immune cells from the collected blood, a chamber section 30 that contains collected tissue containing tumor cells and has a plurality of mixing chambers 32 through which the immune cells pass, and a blood return means 60 that returns the immune cells that have passed through the mixing chambers 32 to the separated blood and returns the blood to the patient.

[0019] The medical device 10 has a reservoir tank 70 that stores blood. The reservoir tank 70 is partitioned into a first region 71 that stores blood collected from a patient, and a second region 72 that stores blood from which immune cells have been separated and mixes the blood with the immune cells after passing through the mixing chamber 32.

[0020] The immune cell acquisition means 20 is composed of a first line 90, a second line 91, a third line 92, an anticoagulant supply unit 73, a first region 71 of a reservoir tank 70, and a centrifugal separation unit 75. The first line 90, the second line 91, and the third line 92 are tubular bodies through which a fluid can flow. Similarly, a fourth line 93, a fifth line 94, a sixth line 95, a seventh line 96, and an eighth line 97, which will be described later, are also tubular bodies through which a fluid can flow. Each line is provided with a fluid driving means such as a pump, which can be automatically controlled by a control means such as a computer, thereby allowing the fluid to flow through each line at any timing and flow rate.

[0021] The first line 90 is inserted into a blood vessel to allow collection of the patient's blood and communicates with the first region of the reservoir tank 70. The second line 91 communicates with the first line 90 and is connected to an anticoagulant supply unit 73. The third line 92 connects the first region 71 of the reservoir tank 70 with the centrifugal separator 75. The anticoagulant supply unit 73 contains an anticoagulant for blood and can mix the anticoagulant 73 with the blood before it is contained in the first region 71 of the reservoir tank 70. Examples of anticoagulants that can be used include, but are not limited to, citrate and heparin salt. In this embodiment, an acid citrate dextrose (ACD) solution is used as the anticoagulant.

[0022] The centrifugal separator 75 can separate immune cells from the collected blood by centrifugation. The centrifugal separator 75 and the second region 72 of the reservoir tank 70 are connected by a fourth line 93, and the remaining blood from which immune cells have been separated in the centrifugal separator 75 is stored in the second region 72 of the reservoir tank 70 via the fourth line 93.

[0023] The chamber section 30 is connected to the centrifugal separator 75 via a fifth line 94. The fifth line 94 is connected to an immunostimulant supplying section 76 via a sixth line 95. The immunostimulant supplying section 76 stores an immunostimulant and can supply the immunostimulant to the immune cells before they flow into the mixing chamber 32 via the sixth line 95. The immunostimulant may be, for example, a cytokine preparation, specifically interleukin 2 (IL-2), but is not particularly limited thereto.

[0024] The chamber section 30 is provided with an imaging section 80 that images the interior of the mixing chamber 32, and a light source section 85 that irradiates the interior of the mixing chamber 32 with light for imaging by the imaging section 80. The imaging section 80 is connected to a determination section 82. The determination section 82 acquires the image captured by the imaging section 80 and can identify tumor tissue consisting of tumor cells from the image, as well as determine immune acquisition by immune cells. The imaging section 80 is composed of a high-resolution microscope camera that can observe minute tumor tissue and immune cells. This microscope camera may be either a transmission type or a reflection type.

[0025] The chamber unit 30 is connected to the second region 72 of the reservoir tank 70 by a seventh line 96, and can send immune cells that have acquired immunity to the second region 72 of the reservoir tank 70. Since the second region 72 of the reservoir tank 70 contains the remaining blood from which the immune cells have been separated in the centrifuge unit 75 as described above, the immune cells and the blood from which they have been separated are mixed in the second region 72 of the reservoir tank 70.

[0026] The second region 72 of the reservoir tank 70 is connected to one end of an eighth line 97. The other end of the eighth line 97 is inserted into a patient's blood vessel, and the blood stored in the second region 72 of the reservoir tank 70 can be returned to the patient. In other words, the second region 72 of the reservoir tank 70 and the eighth line 97 constitute the blood return means 60.

[0027] Next, the chamber section 30 will be described in detail. As shown in Fig. 3, the chamber section 30 has a container-shaped storage base 31 that stores a plurality of mixing chambers 32, and four mixing chambers 32 that are arranged in the storage base 31 so that their relative positions do not change. The number of mixing chambers 32 is not particularly limited. The mixing chamber 32 stores a mounting surface 50 on which the collected tissue is placed, and a pressing surface 52 that is provided to cover the mounting surface 50.

[0028] A fifth line 94 extending from the centrifugal separation section 75 is connected to a branch port 33 that branches the internal fluid into four inlet pipes 34. The inlet pipes 34 are connected to each mixing chamber 32. An outlet pipe 35 extends from each mixing chamber 32 and is connected to a collection port 36. The collection port 36 is connected to a seventh line 96 that leads to the second region 72 of the reservoir tank 70, and the fluid from each outlet pipe 35 is collected and sent to the seventh line 96.

[0029] The accommodating base 31 is surrounded by an imaging unit 80 that is positioned to capture an image of the mixing chamber 32 from the outlet pipe 35 side, and a light source unit 85 that is positioned opposite the imaging unit 80 with the accommodating base 31 in between. At least the upper and lower surfaces of the accommodating base 31 are formed of a translucent material that allows light to pass through. The imaging unit 80 and the light source unit 85 are fixed in position, and the accommodating base 31 that accommodates the mixing chamber 32 is movable relative to the imaging unit 80 and the light source unit 85. To this end, the accommodating base 31 has a drive linkage 31a that extends circumferentially on its outer periphery. The chamber drive unit 38 mechanically engages and links with the drive linkage 31a, allowing the accommodating base 31 to rotate circumferentially. The chamber drive unit 38 can be configured as a motor. This allows each of the multiple mixing chambers 32 to move circumferentially to a position where the imaging unit 80, which is in a fixed position, can capture an image of the collected tissue contained within each of the mixing chambers 32, so that the imaging unit 80 can capture an image of the collected tissue contained within each of the mixing chambers 32. The positional relationship between the imaging unit 80 and the light source unit 85 may be reversed. The light source unit 85 may also be disposed on the same side of the imaging unit 80 and the mixing chamber 32. The chamber unit 30 may also be configured so that the storage base 31 is in a fixed position and the imaging unit 80 and the light source unit 85 are movable relative to the storage base 31.

[0030] The multiple mixing chambers 32 housed in the housing base 31 are each connected to an inlet pipe 34 branched by a branch port 33, and are arranged in parallel with the flow direction of the fluid containing immune cells. Collected tissue containing tumor tissue is placed in each mixing chamber 32. The collected tissue is a piece of meat obtained in advance from a patient and has a size of approximately 1 mm to 2 mm. The arrangement conditions of the collected tissue, such as the size and arrangement density of the collected tissue, can be varied in each mixing chamber 32. The medical device 10 may also include a control unit that controls the flow of fluid in each mixing chamber 32 to vary the time that immune cells are brought into contact with the collected tissue in each mixing chamber 32. For immune cells to acquire immunity, it is desirable to maximize the effect on tumor tissue contained in the collected tissue. Arranging the mixing chambers 32 in parallel allows immune cells to act on tumor tissue under multiple conditions, thereby increasing the likelihood that immune cells will acquire immunity. Collected tissue may be placed under the same conditions in each of the parallel-arranged mixing chambers 32.

[0031] As shown in FIG. 4, the mixing chamber 32 includes a container 40 that contains collected tissue and through which a fluid containing immune cells can pass. The container 40 has a lid 41 that can be opened and closed to seal or open the interior. A threaded portion 40b is formed on the top of the container 40 so that the lid 41 can be screwed. The container 40 has an inlet portion 41a on the upper surface of the lid 41 that is connected to the inlet pipe 34, and an outlet portion 40a on the lower surface that is connected to the outlet pipe 35. Because the container 40 can be opened and closed using the lid 41, the collected tissue can be easily placed in it under sterile conditions.

[0032] The container 40 is provided with a mounting surface 50 on which the collected tissue is placed, and a pressing surface 52 arranged to sandwich the collected tissue between the mounting surface 50. The mounting surface 50 has a mesh structure 50a over its entire surface. The mesh structure 50a is a porous thin film with meshes large enough to block the collected tissue but allow immune cells to pass through. The mesh size of the mesh structure 50a is in the range of 30 μm or more and the lower limit of the size of the collected tissue, for example, 30 μm. This allows the collected tissue to be placed on the mounting surface 50. The pressing surface 52 is also a mesh-like thin film with a mesh structure over its entire surface. The pressing surface 52 allows the collected tissue to be fixed so that its position does not change before and after contact with the immune cells.

[0033] By introducing a fluid containing immune cells from the inlet 41a of the container 40, the fluid passes through the pressing surface 52 and the mounting surface 50, allowing the immune cells in the fluid to come into contact with and act on the tumor tissue. Even as the fluid passes through the mounting surface 50, the collected tissue maintains its position, and the immune cells pass through the mounting surface 50 together with the fluid and flow out from the outlet 40a.

[0034] The imaging unit 80 is disposed on the lower surface side of the container unit 40, and the light source unit 85 is disposed on the upper surface side of the container unit 40. In order to enable imaging of the collected tissue disposed inside the container unit 40, the lower surface of the container unit 40 is a light-transmitting external light-transmitting unit 40c. Furthermore, the upper surface of the lid unit 41, which forms the upper surface of the container unit 40, is a light-transmitting second external light-transmitting unit 41b so that light from the light source unit 85 is irradiated into the inside of the container unit 40. Note that the entire container unit 40, including the lid unit 41, may be light-transmitting. Furthermore, the external light-transmitting unit 40c may be provided in the form of a window in a part of the lower surface of the container unit 40 as long as it allows the imaging unit 80 to image the collected tissue disposed inside the container unit 40. Furthermore, the second external light-transmitting unit 41b may also be provided in the form of a window in a part of the upper surface of the container unit 40.

[0035] The entire surface of the mounting surface 50 has a mesh structure 50a, allowing the placed collected tissue to be viewed from the imaging unit 80. That is, the entire surface of the mounting surface 50 is an internal light-transmitting portion 50b that is translucent. In addition, the pressing surface 52 is also mesh-shaped, and therefore has an internal light-transmitting portion 52a that can transmit light from the light source unit 85.

[0036] In this way, the mixing chamber 32 has an external light-transmitting section 40c, a second external light-transmitting section 41b, and internal light-transmitting sections 50b and 52a, so that light from the light source section 85 can be irradiated onto the collected tissue placed on the placement surface section 50, and the collected tissue can be imaged by the imaging section 80.

[0037] Another embodiment of the mounting surface portion will be described. As shown in FIG. 5, the mounting surface portion 55 has a disk-shaped main body portion 55a that is impermeable to fluid and a mesh structure portion 55b that is provided around the entire periphery of the main body portion 55a. The main body portion 55a has a cross-shaped groove portion 55e and a recess portion 55c that is provided at the longitudinal intermediate position of the groove portion 55e. The groove portion 55e connects a central position facing the inlet portion 41a, which serves as an inlet for the fluid containing immune cells in the container portion 40, to a position that communicates with the mesh structure portion 50a. Therefore, the fluid introduced into the container portion 40 from the inlet portion 41a reaches the mesh structure portion 55b along the groove portion 55e and passes through the mesh structure portion 55b in the thickness direction. Note that the mesh structure portion 55b does not have to extend around the entire periphery of the mounting surface portion 55; it may be provided at least partially around the periphery as long as the fluid containing immune cells can flow through it.

[0038] As shown in FIG. 6 , the recess 55c is formed as a pocket recessed from the surface of the main body 55a, and can accommodate and place the collected tissue 100. The pressure surface 52 is placed on the placement surface 50 on which the collected tissue 100 is placed, sandwiching the collected tissue 100. The collected tissue 100 is fixed to the placement surface 55 by the recess 55c and the pressure surface 52 so that it does not move. The pressure surface 52 is mesh-shaped, but it may also be made of a transparent plate material. If the pressure surface 52 is made of a plate material, an opening must be provided in the center of the pressure surface 52 to allow fluid flowing in from the inlet 41a of the container 40 to contact the collected tissue. The fluid containing immune cells can enter between the placement surface 55 and the pressure surface 52 from the inlet 41a through the opening in the pressure surface 52 and come into contact with the collected tissue 100 placed in the recess 55c of the placement surface 55.

[0039] At least the recess 55c of the mounting surface 55 is a light-transmitting internal portion 55d. The entire main body 55a of the mounting surface 55 may be light-transmitting. This allows the imaging unit 80 to capture an image of the collected tissue 100 mounted on the recess 55c.

[0040] A modified example of the chamber section will be described. As shown in Fig. 7, the chamber section 110 is configured by connecting three mixing chambers 112 in series in the direction of passage of the fluid containing immune cells. A fifth line 94 is connected to the most upstream mixing chamber 112. A seventh line 96 is connected to the most downstream mixing chamber 112. A light source section 85 is disposed on the upper surface side of the most upstream mixing chamber 112. An imaging section 80 is disposed on the lower surface side of the most downstream mixing chamber 112 so as to face the light source section 85. At least the upper and lower surfaces of the mixing chamber 112 are translucent and serve as external translucent sections for the imaging section 80.

[0041] Each mixing chamber 112 is provided with a mounting surface 115 on which the collected tissue is placed and a pressing surface 116. The mounting surface 115 has a groove 55e and a recess 55c as shown in FIG. 5, allowing the collected tissue to be set at a predetermined position. The mounting surface 115 of each mixing chamber 112 is accommodated so that the collected tissue is positioned at a different circumferential position. Furthermore, the mounting surface 115 accommodated in the upstream mixing chamber 112 is positioned biased toward the lower surface of the mixing chamber 112, while the mounting surface 115 accommodated in the downstream mixing chamber 112 is positioned biased toward the upper surface. This minimizes the difference in distance between the mounting surface 115 accommodated in each mixing chamber 112 and the imaging unit 80, making it easier to focus the imaging unit 80 on each mounting surface 115. At least the recess 55c of the placement surface portion 115 is an internal light-transmitting portion that is light-transmitting, and the pressing surface portion 116 is mesh-like, so that it is also an internal light-transmitting portion that is light-transmitting, and therefore the collected tissue in each mixing chamber 112 can be imaged by the imaging portion 80.

[0042] One of the mixing chambers 112 has a drive linkage 113 provided circumferentially on its outer peripheral surface, and the three mixing chambers 112 connected in series can be rotated circumferentially by the chamber drive unit 38. As described above, the circumferential position at which the collected tissue is located differs in each mixing chamber 112, so by moving the circumferential position of the mixing chamber 112 with the chamber drive unit 38, the collected tissue can be imaged for all of the mixing chambers 112 by the imaging unit 80, which is in a fixed position.

[0043] In the chamber unit 110, the mixing chambers 112 are arranged in series in the direction of passage of the fluid containing immune cells, so that the immune cells can come into contact with and act on the tumor tissue multiple times. This allows the immune cells to reliably acquire immunity. Note that the chamber unit may also be arranged in parallel in the direction of passage of the fluid, with multiple mixing chambers connected in series in the direction of passage of the fluid.

[0044] In the mixing chamber 112, the fluid containing immune cells flows from the inlet to the outlet, but the outlet of each mixing chamber 112 may be temporarily blocked to allow the fluid to accumulate in the mixing chamber 112 for a certain period of time, and then the outlet may be opened to allow the fluid to flow to the next mixing chamber 112 connected in series. This increases the time that immune cells are in contact with tumor tissue, making it easier to acquire immunity.

[0045] Next, the operation of the medical device 10 will be described in detail. As shown in FIG. 8, prior to blood collection, collected tissue obtained from a patient is placed in the mixing chamber 32 and set therein (SA-1). The mixing chamber 32 is opened by opening the lid 41 under sterile conditions, and the collected tissue is placed on the mounting surface 50, with the pressing surface 52 positioned to cover the upper side of the mounting surface 50. If the mounting surface 50 has the mesh structure 50a covering the entire surface as shown in FIG. 4, multiple pieces of collected tissue are placed at any position on the mounting surface 50. If the mounting surface 55 has the recesses 55c and grooves 55e as shown in FIG. 5, the collected tissues are placed in each of the multiple recesses 55c. Once the collected tissues have been placed in the mixing chamber 32, the lid 41 is closed to seal the mixing chamber 32.

[0046] Next, the initial state of the tumor tissue contained in the mixing chamber 32 is imaged (SA-2). When the mixing chambers 32 are arranged in parallel with the direction of passage of the fluid containing immune cells as shown in Fig. 3, the collected tissue in each mixing chamber 32 is sequentially imaged by rotating the storage base 31 in the circumferential direction. When the mixing chambers 112 are arranged in series with the direction of passage of the fluid containing immune cells as shown in Fig. 7, the connected mixing chambers 112 are rotated in the circumferential direction, and the collected tissue in each mixing chamber 112 is sequentially imaged.

[0047] Determination unit 82 analyzes the image captured by imaging unit 80 to recognize tumor tissue from the collected tissue and determine whether it is in an observable state (SA-3). Tumor tissue 101 is photographed, for example, as shown in FIG. 9. In FIG. 9, tumor tissue 101 is surrounded by normal non-tumor tissue 102. Tumor cells that form tumor tissue 101 have irregular nuclei that are not regular compared to non-tumor cells 102, and therefore determination unit 82 can recognize tumor tissue from the image captured by imaging unit 80. If tumor tissue determination unit 82 determines that the tumor tissue is not in an observable state, it performs steps SA-1 and thereafter again. If determination unit 82 determines that the tumor tissue is in an observable state, it proceeds to the next step.

[0048] Next, a reference value for immune acquisition is set in the medical device 10 (SA-4). The reference value is set as a threshold value for determining whether immune cells have acquired immunity when they are applied to tumor tissue. In this embodiment, whether immune cells have acquired immunity against tumor cells is determined by the accumulation of immune cells, and the reference value is expressed as an index that quantifies the accumulation. The accumulation is expressed, for example, as a ratio of the number of tumor cells in which immune cells have accumulated to the total number of tumor cells. The reference value can preferably be an accumulation of 60% or more, 70% or more, 80% or more, or 90% or more. The reference value is set depending on the patient's condition, the type of cancer, etc. For example, if the cancer is in the early stage, the reference value is set low, and if the cancer is in the late stage, the reference value is set high.

[0049] Next, blood collection from the patient begins (SA-5). The collected blood is stored in the first region 71 of the reservoir tank 70, and then centrifuged in the centrifugal separator 75 (SA-6), and immune cells are extracted (SA-7). The separated immune cells are sent to the chamber 30, and the blood other than the immune cells is stored in the second region 72 of the reservoir tank 70.

[0050] The immune cells collected in the centrifuge section 75 are mixed with an immune stimulant from an immune stimulant supply section 76 (SA-8). The immune cells mixed with the immune stimulant come into contact with and act on tumor tissue in the mixing chamber 32 (SA-9). The immune cells that have passed through the mixing chamber 32 are collected in the second region 72 of the reservoir tank 70 (SA-10) and returned to the blood (SA-11).

[0051] Next, the imaging unit 80 images the tumor tissue in the mixing chamber 32 (SA-12). This allows imaging of the state of the tumor tissue after the immune cells have acted on it. The determination unit 82 determines from the captured image whether the immune cells have acquired immunity (SA-13). As shown in FIG. 10 , the determination unit 82 identifies the distribution of the immune cells 103 in the tumor tissue 101 from the image captured by the imaging unit 80 after the immune cells 103 have acted on the tumor tissue 101 (SB-1). Furthermore, the determination unit 82 identifies the distribution of the immune cells 103 in the non-tumor tissue 102 from the image captured by the imaging unit 80 after the immune cells 103 have acted on the tumor tissue 101 (SB-2).

[0052] As shown in FIG. 11 , when immune cells 103 are allowed to act on tumor tissue 101, the immune cells 103 gather around the tumor tissue 101, resulting in a high density of immune cells 103 in the tumor tissue 101. Since immune cells 103 do not accumulate in non-tumor tissue 102, the density of immune cells 103 in non-tumor tissue 102 is low. Based on this result and the previously set reference value, determination unit 82 determines whether immune cells 103 have accumulated in tumor tissue 101 (SB-3). Based on the results of SB-1 and SB-2, determination unit 82 detects the degree of accumulation of immune cells 103 in tumor tissue 101. If the degree of accumulation is higher than the previously set reference value, determination unit 82 determines that immune cells 103 have acquired immunity (SB-4). If the degree of accumulation is lower than the previously set reference value, determination unit 82 determines that immune cells 103 have not acquired immunity (SB-5). The degree of accumulation may be, for example, the ratio of the density of immune cells 103 in the region of the tumor tissue 101 to the density of immune cells 103 in the entire image.

[0053] The action of immune cells 103 destroys the cell membranes of tumor cells constituting tumor tissue 101, causing the cells to lose their shape. Since determination unit 82 acquires tumor tissue in an initial state before immune cells 103 are allowed to act on tumor tissue 101 using SA-2, it is possible to compare the states of tumor tissue 101 before and after immune cells 103 are allowed to act. From this comparison, determination unit 82 can calculate the percentage of tumor cells that have been phagocytosed or destroyed by immune cells 103. Determination unit 82 may determine whether immune cells 103 have acquired immunity, taking into account the degree of phagocytosis or destruction caused by allowing immune cells 103 to act on tumor tissue 101.

[0054] If the determination unit 82 determines in SA-13 that immunity has not been acquired, the steps from SA-5 are repeated again. If the determination unit 82 determines in SA-13 that immunity has been acquired, immune cells 103 that have acquired immunity have been reinfused into the patient in SA-11, and the steps are terminated.

[0055] Steps SA-12 and SA-13 may be performed before the step of returning blood in SA-11. When steps SA-12 and SA-13 are performed after immune cells 103 are collected in reservoir tank 70, if determination unit 82 determines that immunity has not been acquired, steps SA-6, starting with the centrifugation process, can be performed again for the blood contained in reservoir tank 70. If determination unit 82 determines that immunity has been acquired, the blood contained in reservoir tank 70 is returned to the patient.

[0056] If the immune cells 103 that have passed through the mixing chamber 32 can be stored in a region other than the second region 72 of the reservoir tank 70, steps SA-12 and SA-13 can also be executed at this stage. If the determination unit 82 determines that the immune cells 103 have not acquired immunity, the immune cells 103 can be re-executed from step SA-9, which involves contacting the immune cells 103 with the tumor tissue 101. If the determination unit 82 determines that the immune cells 103 have acquired immunity, the immune cells 103 are sent to the second region 72 of the reservoir tank 70 and returned to the patient.

[0057] In the example of FIG. 11 , the distribution of immune cells 103 is compared between tumor tissue 101 and surrounding non-tumor tissue 102. However, the mixing chamber 32 may be provided with a first compartment in which tumor tissue 101 and non-tumor tissue 102 are arranged, and a second compartment in which only non-tumor tissue 102 is arranged, and an image captured in the first compartment may be compared with an image captured in the second compartment. As shown in FIG. 12( a), in the image captured of non-tumor tissue 102, immune cells 103 are uniformly distributed. In contrast, as shown in FIG. 12( b), in the image including tumor tissue 101, immune cells 103 accumulate around tumor tissue 101 but do not accumulate in non-tumor tissue 102. Therefore, by comparing the distribution of immune cells 103 in these images, it is possible to more accurately determine whether immune cells 103 have accumulated in tumor tissue 101.

[0058] As described above, the method (1) for determining whether immune cells have acquired immunity against tumor cells according to this embodiment includes the steps of identifying tumor tissue from collected tissue containing tumor tissue and non-tumor tissue, applying immune cells to the tumor tissue, determining the distribution of immune cells in the tumor tissue, and detecting the accumulation of immune cells in the tumor tissue and determining that the immune cells have acquired immunity if the accumulation is equal to or greater than a reference value. The method for determining whether immune cells have acquired immunity against tumor cells thus configured detects the accumulation of immune cells in the tumor tissue and determines the acquisition of immunity based on the accumulation, thereby enabling consistent and reliable determination of whether immune cells extracted from a patient have acquired immunity against tumor cells.

[0059] (2) The method for determining whether immune cells have acquired immunity against tumor cells according to (1) above may further include a step of identifying the distribution of immune cells in non-tumor tissues. This allows the method for determining whether immune cells have acquired immunity against tumor cells to detect the absence of accumulation of immune cells in non-tumor tissues, thereby enabling more accurate determination of whether immune cells have acquired immunity against tumor cells.

[0060] (3) The method for determining whether immune cells have acquired immunity against tumor cells according to (1) or (2) above may further include a step of setting a reference value for the degree of accumulation, thereby enabling the method for determining whether immune cells have acquired immunity against tumor cells to be appropriately determined in accordance with the patient's condition and other circumstances.

[0061] (4) The method for determining whether immune cells have acquired immunity against tumor cells according to any one of (1) to (3) above may further include the step of preparing a medical device 10 including a mixing chamber 32 in which tumor tissue and non-tumor tissue are placed, an imaging unit 80 that images the inside of the mixing chamber 32, and a determination unit 82 that determines whether immune cells have acquired immunity, wherein the determination unit 82 identifies the tumor tissue from the image captured by the imaging unit 80, the mixing chamber 32 allows the immune cells to act on the placed tumor tissue, and the determination unit 82 identifies the distribution of immune cells in the tumor tissue from the image captured by the imaging unit 80, and detects the accumulation level of immune cells in the tumor tissue from the identified distribution of immune cells. This allows the determination unit 82 to detect the accumulation level of immune cells in the tumor tissue from the image captured of the tumor tissue placed in the mixing chamber 32, thereby making it possible to consistently and reliably determine whether immune cells have acquired immunity against tumor cells using the medical device 10.

[0062] (5) In the method for determining whether immune cells have acquired immunity against tumor cells described in (4) above, the mixing chamber 32 may be provided with a first compartment in which tumor tissue and non-tumor tissue are placed and a second compartment in which only non-tumor tissue is placed, the imaging unit 80 may be capable of capturing images of the first compartment and the second compartment, and the determining unit 82 may detect the degree of accumulation of immune cells in the tumor tissue based on the distribution of immune cells in the first compartment and the distribution of immune cells in the second compartment in the step of determining whether immune cells have acquired immunity against tumor cells. This allows the method for determining whether immune cells have acquired immunity against tumor cells to clearly distinguish and recognize the distribution of immune cells in non-tumor tissue and the distribution of immune cells in tumor tissue, thereby more accurately detecting the degree of accumulation of immune cells in tumor tissue.

[0063] (6) The method for determining whether immune cells have acquired immunity against tumor cells according to (4) or (5) above may further include a step of returning the immune cells that have acted on the tumor tissue in the mixing chamber 32. This allows the method for determining whether immune cells have acquired immunity against tumor cells to proceed with the treatment of the patient while confirming that the immune cells have acquired immunity.

[0064] The present invention is not limited to the above-described embodiment, and various modifications may be made by those skilled in the art within the technical spirit of the present invention. In the above-described embodiment, the reservoir tank 70 is divided into the first region 71 and the second region 72, and the first region 71 is connected to the first line 90, and the second region 72 is connected to the eighth line 97. Therefore, the first line 90 and the eighth line 97 are inserted into different parts of the patient's blood vessel. However, blood collection and return may be performed using a common flow path. In this case, the reservoir tank 70 is not divided, and the first line 90 is inserted into the patient's blood vessel. Blood collected from the first line 90 is stored in the reservoir tank 70, and the blood from the reservoir tank 70 is returned to the patient through the first line 90. [Explanation of symbols]

[0065] 10 Medical Devices 20 Immune cell acquisition means 30 Chamber section 31 Storage base 32 Mixing chamber 33 Branch Port 34 Inlet pipe 35 Outlet pipe 36 aggregate ports 38 Chamber drive unit 40 Container section 40a Exit section 40b Threaded part 40c External transparent part 41 Lid 41a Entrance 41b 2nd external transparent part 50 Placement surface section 50a Mesh structure 50b Internal transparent part 52 Pressing surface 52a Internal transparent part 55 Placement surface section 55a Main body 55b Mesh structure 55c recess 55d Internal transparent part 55e Groove 60 Blood return means 70 Reservoir Tank 73 Anticoagulant Supply Department 75 Centrifugal separation section 76 Immunostimulant Supply Department 80 Imaging unit 82 Judgment section 85 Light source section

Claims

1. Identifying tumor tissue from collected tissues including tumor tissue and non-tumor tissue; A step of allowing immune cells to act on the tumor tissue; Identifying the distribution of the immune cells in the tumor tissue; A method for determining whether immune cells have acquired immunity against tumor cells, comprising the steps of detecting the degree of accumulation of the immune cells in the tumor tissue, and determining that the immune cells have acquired immunity if the degree of accumulation is equal to or greater than a reference value.

2. The method for determining whether immune cells have acquired immunity against tumor cells according to claim 1, further comprising a step of identifying the distribution of the immune cells in the non-tumor tissue.

3. The method for determining whether immune cells have acquired immunity against tumor cells according to claim 1 , further comprising the step of setting the reference value of the accumulation degree.

4. The method further includes a step of preparing a medical device including a mixing chamber in which tumor tissue and non-tumor tissue are placed, an imaging unit that images the inside of the mixing chamber, and a determination unit that determines whether the immune cells have acquired immunity; the determination unit identifies the tumor tissue from the image captured by the imaging unit; The mixing chamber allows the immune cells to act on the placed tumor tissue, 2. The method for determining whether or not immune cells have acquired immunity against tumor cells according to claim 1, wherein the determination unit identifies a distribution of the immune cells in the tumor tissue from the image captured by the imaging unit, and detects the degree of accumulation of the immune cells in the tumor tissue from the identified distribution of the immune cells.

5. The mixing chamber is provided with a first compartment in which the tumor tissue and the non-tumor tissue are placed, and a second compartment in which only the non-tumor tissue is placed; the imaging unit is capable of imaging the first section and the second section, 5. The method for determining whether or not immune cells have acquired immunity against tumor cells according to claim 4, wherein the determination unit, in the step of determining whether or not the immune cells have acquired immunity, detects the degree of accumulation of the immune cells in the tumor tissue based on the distribution of the immune cells in the first compartment and the distribution of the immune cells in the second compartment.

6. The method for determining whether immune cells have acquired immunity against tumor cells according to claim 4 , further comprising a step of returning the immune cells that acted on the tumor tissue in the mixing chamber to the blood.

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