Method for manufacturing cell pellets

Abstract

To provide a method for producing cell pellets that can improve the visibility of the cell pellet, which is a mass of RI-labeled cells to be obtained. [Solution] A method for producing a cell pellet according to one aspect of the present invention includes: a preparation step of preparing a cell suspension containing cells in a physiological buffer; a labeling step of labeling the cells contained in a mixture of the cell suspension and a solution of a radionuclide with the radionuclide; and a separation step of separating the mixture into a cell pellet, which is a mass of the labeled cells, and a residual RI solution containing the radionuclide that was not taken up by the cells. The diameter of the cells contained in the mixture is 10 μm or more, and the number of cells contained in the mixture is 1.2 × 10⁻⁶. 7 It is more than just cells.

Description

[Technical Field]

[0001] This invention relates to a method for producing cell pellets. [Background technology]

[0002] Immunotherapy, also known as cellular immunotherapy, which aims to treat cancer by extracting immune cells that attack cancer cells in the body from a patient, culturing them outside the body, and then administering the cultured immune cells (cultured cells) back to the patient, is attracting attention, and its research and development are actively underway. In order to estimate the effectiveness of cancer treatment with such cellular immunotherapy, it is necessary to evaluate the in vivo distribution of cultured cells administered to the patient. As an effective method for this evaluation, a method is being investigated in which cultured cells are labeled with radioisotopes (RI), that is, by cell labeling using RI, and the in vivo distribution of these RI-labeled cultured cells (hereinafter referred to as RI-labeled cells) is evaluated using positron emission tomography (PET), etc.

[0003] Generally, in evaluating the in vivo distribution of RI-labeled cells, the radiation emitted from RI-labeled cells in the body is imaged using a PET scanner or similar device, and the in vivo distribution of the target cells can be indirectly estimated from the obtained image. Therefore, from the viewpoint of improving evaluation accuracy, higher spatial resolution imaging is desired when imaging radiation from RI-labeled cells. Conventionally, single-photon emission computed tomography (SPECT) has been the mainstream imaging method, and as the RI, for example, indium-111( 111 Zirconium-89 (I) was frequently used. However, in recent years, there has been a trend to replace it with positron emission tomography (PET), which has better spatial resolution, and as the above RI, zirconium-89 ( with a longer lifespan) has become popular. 89 The replacement with Zr is being considered. 89 One example of a cell labeling technique that uses Zr to label various cells, such as cultured cells, is disclosed in Non-Patent Document 1. [Prior art documents]

Non-Patent Literature

[0004]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In cell labeling using a radioisotope (RI), in a mixed solution containing target cultured cells and the RI, the cultured cells are labeled with the RI by reacting these cultured cells with the RI. In order to obtain the cultured cells labeled in this way (i.e., RI-labeled cells) so that they can be used for the above evaluations and the like, the RI-labeled cells in the above mixed solution are precipitated and collected by centrifugation. As a result, a cell pellet, which is a mass of the RI-labeled cells, is formed in the container of the above mixed solution. At the same time, the above mixed solution is separated into the cell pellet and a supernatant liquid in which the RI not taken up by the cultured cells remains. Thereafter, it is necessary to remove the supernatant liquid from the container while leaving the cell pellet in the container.

[0006] However, in the working process from the above cell labeling to the removal of the supernatant, due to the special circumstances of handling samples such as solutions containing RI, operations must be carried out while taking sufficient radiation protection measures, such as covering the sample containing RI with a shielding plate. As a result, the operations necessary for obtaining RI-labeled cells may become difficult.

[0007] For example, when the RI for labeling cultured cells is 89 Zr, 89 since Zr emits gamma rays with a strong energy of 909 keV, 89 it is not preferable to perform the above working process while directly visually observing a cell pellet composed of RI-labeled cells containing Zr. Therefore, from the perspective of radiation protection, in the above working process, while visually observing the cell pellet through thick lead glass capable of shielding radiation, or while visually observing the image of the cell pellet in a container covered with a shielding plate or the like through a screen, it is necessary to perform operations such as removing the supernatant other than the cell pellet from the container. That is, compared to directly visually observing the cell pellet, it becomes more difficult to visually observe the cell pellet through lead glass or through a screen, making it difficult to obtain the target RI-labeled cells in the form of a cell pellet. Therefore, in the above working process, it is extremely important to enhance the visibility of the target cell pellet while taking sufficient radiation protection measures.

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for manufacturing a cell pellet capable of enhancing the visibility of a cell pellet, which is a mass of RI-labeled cells to be obtained.

Means for Solving the Problems

[0009] In order to solve the above problems and achieve the object, a method for producing a cell pellet according to the present invention includes a preparation step of preparing a cell suspension containing cells in a physiological buffer solution, a labeling step of labeling the cells contained in a mixture of the cell suspension and a solution of a radionuclide with the radionuclide, and a separation step of separating the mixture into a cell pellet which is a mass of the labeled cells and a RI residual solution in which the radionuclide not incorporated into the cells remains. The diameter of the cells contained in the mixture is 10 μm or more, and the number of the cells contained in the mixture is 1.2×10 7 cells or more, which is characterized in that.

[0010] Further, the method for producing a cell pellet according to the present invention is the above invention, wherein the concentration of the cells in the cell suspension is 1.0×10 7 cells / mL or more, which is characterized in that.

[0011] Further, the method for producing a cell pellet according to the present invention is the above invention, wherein the concentration of the cells in the cell suspension is 1.0×10 9 cells / mL or less, which is characterized in that.

[0012] Further, the method for producing a cell pellet according to the present invention is the above invention, wherein the number of the cells contained in the mixture is 1.2×10 9 cells or less, which is characterized in that.

[0013] Further, the method for producing a cell pellet according to the present invention is the above invention, wherein the radionuclide is zirconium 89, which is characterized in that.

Effect of the Invention

[0014] According to the present invention, there is an effect that the visibility of a cell pellet which is a mass of RI-labeled cells to be obtained can be enhanced.

Brief Description of the Drawings

[0015] [Figure 1]Figure 1 is a schematic diagram showing an example of a cell pellet according to an embodiment of the present invention. [Figure 2] Figure 2 is a flowchart showing an example of a method for producing a cell pellet according to an embodiment of the present invention. [Figure 3] Figure 3 is a schematic diagram showing the flow from the preparation step to the separation step included in the method for producing cell pellets according to the present invention. [Figure 4] Figure 4 is a schematic diagram showing an example of the acquisition process included in the method for producing cell pellets according to the present invention. [Modes for carrying out the invention]

[0016] Hereinafter, preferred embodiments of the method for producing cell pellets according to the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the embodiments described below. Furthermore, it should be noted that the drawings are schematic, and the dimensional relationships and ratios of each element may differ from those in reality. Even between drawings, there may be parts where the dimensional relationships and ratios differ from those of other drawings.

[0017] (Cell pellet) First, a cell pellet according to an embodiment of the present invention will be described. Figure 1 is a schematic diagram showing an example of a cell pellet according to an embodiment of the present invention. The cell pellet 10 according to this embodiment is a collection of multiple RI-labeled cells 1 that are suspended (dispersed) in a liquid, so that they can be visually distinguished from the liquid; in other words, as shown in Figure 1, it is a mass (aggregate) of RI-labeled cells 1.

[0018] Each of the multiple RI-labeled cells 1 that form the cell pellet 10 emits high-energy radiation (e.g., gamma rays) due to the action of the RI (radioactive nuclide) it contains. Therefore, in the manufacturing process of the cell pellet 10, from the standpoint of radiation protection, it is undesirable for workers to directly visually inspect the cell pellet 10 formed in the liquid inside the container. Accordingly, workers must work while viewing the cell pellet 10 in the container placed in a radiation-shielded space through a radiation shielding plate such as lead glass, or while viewing it through a screen displaying images from a camera.

[0019] Generally, viewing objects such as RI-labeled cells through radiation shielding plates or screens, as described above, is more difficult than direct visual inspection. For example, when viewing RI-labeled cells 1 contained in a liquid in a container through lead glass, the lead glass is generally yellowish and has a thickness (approximately 5 cm) necessary to protect against radiation from the RI-labeled cells 1. Therefore, visibility through lead glass (i.e., the visibility of lead glass) is low. Furthermore, the distance from the worker's eye to the RI-labeled cells 1 in the container through the lead glass (line of sight distance) is approximately 1 m. Consequently, viewing the RI-labeled cells 1 in the container through lead glass is extremely difficult. Moreover, manual operation is restricted because it involves working through radiation shielding plates or using a glove box inside a hot cell device.

[0020] In contrast, in the embodiment of the present invention, the RI-labeled cells 1 to be visualized are made to have a cell diameter of a predetermined value or greater to form the cell pellet 10. Therefore, even through a radiation shielding plate or screen, the cell pellet 10 can be easily visualized as RI-labeled cells 1. This protects workers from radiation from the RI-labeled cells 1 and allows them to work while visually inspecting the cell pellet 10. Details of the method for manufacturing such a cell pellet 10 will be described later.

[0021] The RI-labeled cells 1 that make up the cell pellet 10 are cells labeled with a predetermined radioisotope (RI). Examples of cells used for the RI-labeled cells 1 include live cells collected from a subject such as a patient, and cultured cells obtained by culturing such live cells outside the body. Examples of the live cells include immune cells that fight against abnormal cells such as cancer cells and foreign substances (antigens) such as viruses in the body. Furthermore, the cultured cells may be suspension cells or adherent cells.

[0022] The radioisotopes (RIs) used in RI-labeled cells 1 are substances that emit radiation that can be imaged by radiation imaging devices such as PET scanners. Such RIs include indium-111( 111 In) and Zirconium-89 89 While there are many different types of radioisotopes (RIs), such as Zr, among them, those suitable for imaging with higher spatial resolution (images of radiation) are particularly suitable. 89 Zr is preferred.

[0023] The RI-labeled cells 1 described above are useful, for example, for evaluating the in vivo distribution of cultured immune cells, which is done to estimate the effectiveness of cancer treatment by cell immunotherapy. Specifically, immune cells collected from a patient are cultured (enhanced and proliferated), and the resulting cultured cells (immune cells with enhanced immunity) are labeled with an RI to generate multiple RI-labeled cells 1. These multiple RI-labeled cells 1 are administered into the patient's body and attack the patient's cancer cells. Furthermore, the radiation emitted from each of the multiple RI-labeled cells 1 in the body is imaged by a radiation imaging device such as a PET scanner, and the in vivo distribution of RI-labeled cells 1 in the patient's body can be evaluated from the obtained images. The obtained in vivo distribution of RI-labeled cells 1 is used as useful information for estimating the effectiveness of cancer treatment by cell immunotherapy.

[0024] (Method for manufacturing cell pellets) Next, a method for producing a cell pellet according to an embodiment of the present invention will be described. Figure 2 is a flowchart showing an example of a method for producing a cell pellet according to an embodiment of the present invention. The method for producing a cell pellet according to an embodiment of the present invention (hereinafter sometimes abbreviated as "cell pellet of the present invention") includes, for example, a culture step in step S101, a preparation step in step S102, a labeling step in step S103, a separation step in step S104, and an acquisition step in step S105, as shown in Figure 2. The cell pellet of the present invention (for example, cell pellet 10 shown in Figure 1) is produced by sequentially performing each of these steps S101 to S105.

[0025] More specifically, as shown in Figure 2, the method for producing cell pellets of the present invention first involves a culture step in which the cells of the subject are cultured outside the body (step S101). In this step S101, cells previously collected from the subject are cultured in a culture medium in a dish. At this time, the cell culture is carried out so that the diameter of the growing cells (cell diameter) becomes 10 μm or more, and is continued until the coverage rate of the culture medium by the growing cells exceeds a predetermined value. As a result, multiple spherical cultured cells having a cell diameter of 10 μm or more are obtained (specifically, 1.2 × 10⁻⁶ 7 More than 10 cells can be obtained. From the perspective of efficiently culturing the required number of cells, the quantity of cultured cells (cell count) obtained from the culture medium in one dish is 1.2 × 10⁶. 7 Over 1.2 × 10⁻¹ cells 9 It is preferable that the cell size is less than or equal to the number of cells. Furthermore, it is preferable that the upper limit of the cell diameter of the cultured cells is 30 μm or less.

[0026] Furthermore, known measurement methods can be used to measure cell diameter, such as calculating the cell diameter of each cell based on images of cells captured using an optical microscope or electron microscope. For example, the cell diameter of a single cell may be the maximum value (maximum diameter), minimum value (minimum diameter), or average value (average diameter) of multiple measurements obtained by measuring the length of line segments connecting the center of the target cell and two points on its outer circumference at multiple locations.

[0027] The cells to be cultured in step S101 above are living cells that can be collected from the body of the subject (e.g., blood). These living cells are preferably immune cells that fight against abnormal cells such as cancer cells and foreign substances (antigens) such as viruses in the patient's body. Examples of such immune cells include white blood cells. White blood cells, when injected into the bloodstream, have the property of gathering around cancer cells in the body. Therefore, white blood cells are preferred as immune cells that fight cancer cells. When the cells to be cultured are immune cells, it is preferable that these immune cells proliferate and become activated (immune function is strengthened) during the culture process in step S101. Furthermore, the cell culture performed in step S101 may be suspension culture, where suspension cells are cultured, or adherent culture, where adherent cells (also called attached cells) are cultured. In other words, the cells cultured in step S101 may be suspension cells or adherent cells.

[0028] After step S101 is performed, a preparation step is carried out to prepare a cell suspension containing the target cells, as shown in Figure 2 (step S102). In step S102, the cells cultured in step S101 (cultured cells) are transferred to a container along with their culture medium, and the liquid in the container (liquid containing cultured cells and culture medium) is subjected to centrifugation. This separates the liquid in the container into cultured cells and supernatant (liquid containing culture medium, etc.). Next, the supernatant is removed from the container by aspiration or the like, and then a physiological buffer such as phosphate-buffered saline (PBS) is added to the container. This replaces the culture medium in the container with the physiological buffer, while suspending the cultured cells in the physiological buffer. Subsequently, the same process from centrifugation to the addition of physiological buffer is repeated as appropriate for this cultured cell suspension. In this way, a cell suspension containing the target cells (cultured cells in this embodiment) in a physiological buffer is prepared.

[0029] Figure 3 is a schematic diagram showing the flow from the preparation step to the separation step included in the method for producing cell pellets of the present invention. In step S102, for example, as shown in Figure 3, a cell suspension 2 containing cultured cells in physiological buffer is prepared in a centrifuge tube 20. The concentration of cultured cells in the prepared cell suspension 2 is 1.0 × 10⁻⁶. 7 It is preferable that the concentration is 1.0 × 10⁻⁶ cells / mL or higher. The lower limit of the cultured cell concentration is 1.0 × 10⁻⁶. 7 If the concentration is 1.0 × 10⁻¹⁰ or higher, it becomes easier to secure the required number of cells (cell count) in the container such as the centrifuge tube 20 for the labeling step S103 and separation step S104 described later. Furthermore, if there is an excessive amount of cultured cells in the cell suspension 2 (concentration), it may cause cell death of the cultured cells in the cell suspension 2. Therefore, the upper limit of the cultured cell concentration should be 1.0 × 10⁻¹⁰. 9 It is preferable that the cell density is less than or equal to cells / mL. As shown in Figure 3, the cell suspension 2 in the centrifuge tube 20 is processed in the next step, the labeling step.

[0030] After step S102 is performed, a labeling step is carried out in which the target cells are labeled with a predetermined radioisotope (RI), as shown in Figure 2 (step S103). In step S103, 1.2 × 10⁶ cells of the target cells are extracted from the cell suspension prepared in step S102 (hereinafter referred to as the first cell suspension). 7 A cell suspension containing more than one cell (hereinafter referred to as the second cell suspension) is weighed out. For example, the cells targeted in this embodiment are cultured cells contained in the second cell suspension weighed out from the first cell suspension. The volume M1 [mL] of this weighed-out second cell suspension is a value that satisfies the following formula using the cell concentration C [cells / mL] in the first cell suspension. M1≧1.2×10 7 / C

[0031] The above concentration C corresponds to the concentration of cultured cells in the cell suspension prepared by step S102, and is 1.0 × 10⁻⁶. 7 cells / mL or more, 1.0×109 It is preferable that the concentration is less than or equal to cells / mL. This also applies to the concentration of cultured cells in the second cell suspension measured from the first cell suspension. Furthermore, since the cultured cells contained in the second cell suspension are the cells cultured in step S101 above, the cell diameter of these cultured cells is 10 μm or larger (preferably 30 μm or smaller). The liquid volume M1 of the second cell suspension contains 1.2 × 10⁶ cultured cells with a cell diameter of 10 μm or larger in the second cell suspension. 7 The concentration C is adjusted according to the above-mentioned concentration so that it contains more than 1.2 × 10 cells. That is, the lower limit of the number of cultured cells contained in the second cell suspension is 1.2 × 10⁶ 7 The number of cells is greater than or equal to 10. Furthermore, the upper limit for the number of cultured cells contained in the second cell suspension is 1.2 × 10⁶. 9 It is preferable that the size be smaller than or equal to the number of cells.

[0032] Next, the second cell suspension in volume M1 and the RI solution in volume M2 are injected into a container such as a centrifuge tube to prepare a mixture of the second cell suspension and the RI solution. In step S103, the cell diameter and number of cultured cells contained in the mixture are the same as those in the second cell suspension in volume M1. That is, the cell diameter of the cultured cells is 10 μm or more (preferably 30 μm or less), and the number of cultured cells contained is 1.2 × 10⁻⁶. 7 cells or more (preferably 1.2 × 10) 9 (The cells are below.)

[0033] Furthermore, the RI solution is a solution containing a predetermined RI in a ligand solution. For example, the RI may be: 89Examples include Zr. The ligand solution is a solution containing ligands that bind around the RI and is used to allow the RI to pass through the cell membrane. Examples of such ligands include highly hydrophobic ligands (hydrophobic ligands) such as 8-quinolinol. Specifically, an example of such a ligand solution is an oxinate solution containing 8-quinolinol as a ligand. In the above RI solution, the ligands bind around the RI, thereby complexing the RI. The complexed RI, together with the bound ligands, can pass through the cell membrane of the target cell (in this embodiment, cultured cells in the second cell suspension) and be taken into the interior of the cell.

[0034] Furthermore, in step S103, the volume M2 [mL] of the RI solution to be mixed with the second cell suspension should be an amount sufficient to confirm that the cultured cells in the mixture of the second cell suspension and the RI solution have been successfully labeled by the RI. For example, the volume M2 of the RI solution is preferably 1 / 30 of the volume M1 of the second cell suspension. In other words, the volume of the RI solution to be mixed with the second cell suspension is preferably 1 / 30 of the volume of the second cell suspension.

[0035] Subsequently, the container containing the above mixture is placed in a heating device such as a tube heater, and the mixture in the container is heated while being vibrated for a predetermined time to react the cultured cells and RI in the mixture. This causes the cultured cells contained in the mixture to take up the RI, thereby labeling the cultured cells with the RI. In this way, RI-labeled cells, which are cultured cells labeled with the RI, are produced in the mixture. Next, a physiological buffer such as PBS is added to the mixture containing the RI-labeled cells to suspend the RI-labeled cells in the mixture. As a result, a mixture 3 containing the suspended RI-labeled cells 1 is produced in the centrifuge tube 20, as shown in Figure 3, for example. This mixture 3 contains the RI-labeled cells 1 generated in the labeling step and RI (not shown) that was not taken up by the cultured cells, in a dispersed state. This mixture 3 containing the RI-labeled cells 1 in the centrifuge tube 20 is processed in the next step, the separation step.

[0036] Furthermore, considering that the RI-labeled cells prepared by step S103 above may be used in experiments, the radioactivity of the RI solution (specifically, the radioactivity of the RI contained in the RI solution) is preferably 0.2 MBq or more and 5 GBq or less. For example, when introducing RI-labeled cells into a living organism such as a mouse and performing experiments on the RI-labeled cells (experiments to confirm cell labeling of target cells by RI), the minimum radioactivity required for RI-labeled cells introduced into one living organism is 0.1 MBq. Since experimental results have shown that the radioactivity yield in cell labeling by RI is about 50%, the lower limit of the radioactivity of the RI solution is preferably 0.2 MBq or more, which corresponds to more than twice the above minimum value. Also, if the radioactivity of the RI solution exceeds 5 GBq, there is a risk that the cultured cells contained in the mixture of the RI solution and the second cell suspension will die. Therefore, the upper limit of the radioactivity of the RI solution is preferably 5 GBq or less. In particular, the RI in the RI solution is 89 If it is Zr, 89 When introducing RI-labeled cells into the body of a mouse, it is preferable that the radioactivity of Zr be within the range of 0.1 MBq to 100 MBq per mouse.

[0037] After step S103 is performed, as shown in Figure 2, a separation step is performed to separate the mixture containing the RI-labeled cells into the target cell pellet and the RI residue to be excluded (step S104). In step S104, the mixture containing the RI-labeled cells suspended in step S103 is sealed (stopped) in a container such as a centrifuge tube, and the mixture in the container is subjected to centrifugation using a centrifuge. This causes the RI-labeled cells in the mixture to settle (settle) to form a cell pellet, and separates the mixture in the container into the cell pellet and the RI residue.

[0038] In step S104, the cell pellet is a mass (aggregate) of RI-labeled cells that have settled in the mixture as described above. The residual RI solution is the liquid in which the RI that was not taken up by the target cells (specifically, cultured cells) in the labeling step of step S103 remains, i.e., the supernatant liquid obtained by the centrifugation process described above. In step S104, as shown in Figure 3 for example, the mixture 3 from step S103 is separated into the cell pellet 10 and the residual RI solution 11 by centrifugation. At this time, the cell pellet 10 is formed to settle on the lower inner wall surface of the centrifuge tube 20.

[0039] After the execution of step S104, as shown in Figure 2, an acquisition step is performed to obtain the cell pellet formed in step S104 (step S105), and the production of the cell pellet of the present invention is completed.

[0040] In step S105, the RI residue is removed from the container while leaving the cell pellet formed in step S104 inside the container. Figure 4 is a schematic diagram showing an example of the acquisition process included in the method for producing cell pellets of the present invention. For example, as shown in Figure 4, the suction port of a syringe or the like is inserted into the centrifuge tube 20 after the separation step in step S104. Then, while leaving the cell pellet 10 inside the centrifuge tube 20, the RI residue 11 is aspirated and removed from the centrifuge tube 20 by the suction action of the suction device 30. At this time, the operator performs the removal of the RI residue 11 while visually observing the cell pellet 10 inside the centrifuge tube 20, as shown in Figure 4.

[0041] Here, as shown in Figure 1, the cell pellet 10 is a mass of RI-labeled cells 1, and therefore, in order to protect the human body from the powerful energy radiation from the contained RI, it is placed in a radiation shielding space and in a state where direct viewing by the worker can be avoided. For this reason, in step S105, the worker needs to work while viewing the cell pellet 10 in the centrifuge tube 20 through a radiation shielding plate such as lead glass, or through a screen displaying images from a camera, as shown in Figure 4. For example, if lead glass is interposed between the worker and the cell pellet 10 in the centrifuge tube 20, the line of sight distance to view the cell pellet 10 will be about 1 meter.

[0042] In contrast, in the present invention, the cell diameter of the RI-labeled cells 1 constituting the cell pellet 10 is 10 μm or larger, similar to the cultured cells that are the source of the RI-labeled cells 1 (i.e., cells before labeling with RI). Furthermore, the cell pellet 10 is 1.2 × 10 7 The cell pellet 10 is formed by centrifugation of a mixture containing more than 100 RI-labeled cells 1, causing these RI-labeled cells 1 to precipitate and form clumps. A cell pellet 10 with this structure is sufficiently visible, even through a radiation shielding plate or screen, to the extent that it does not interfere with the operation in step S105.

[0043] In step S105, as described above, the residual RI solution is removed from the container to obtain the cell pellet remaining in the container. Then, a physiological buffer such as PBS is added to the container, and the cell pellet is suspended in this physiological buffer. This separates the cell pellet into multiple RI-labeled cells, and these multiple RI-labeled cells become dispersed (suspended) in the physiological buffer in the container. In this way, the target RI-labeled cells can be collected as a solution containing the RI-labeled cells. The collected solution of RI-labeled cells can be introduced into the body, for example by injection, and used in the treatment of patients with cell immunotherapy or in experiments using animals such as mice.

[0044] As described above, the method for producing a cell pellet according to an embodiment of the present invention includes a preparation step of preparing a cell suspension containing cells in a physiological buffer, a labeling step of labeling the cells contained in a mixture of the cell suspension and a solution of a radionuclide with the radionuclide, and a separation step of separating the mixture into a cell pellet, which is a mass of the labeled cells, and a residual RI solution containing the radionuclide that was not taken up by the cells. Furthermore, in the method for producing the cell pellet, the diameter of the cells contained in the mixture is 10 μm or more, and the number of cells contained in the mixture is 1.2 × 10⁻⁶. 7 This is more than just cells. As a result, desired cells, such as immune cells of a subject, can be labeled with radioisotopes to form RI-labeled cells in the form of cell pellets, and the visibility of the cell pellets consisting of the RI-labeled cells to be obtained can be sufficiently improved so as not to hinder work, even through a radiation shield or screen. In other words, it becomes possible to protect workers from the radiation of the RI-labeled cells and to work while visually inspecting the cell pellets consisting of the RI-labeled cells.

[0045] According to the cell pellet of the present invention, by administering the RI-labeled cells contained in the cell pellet to a subject such as a patient, the in vivo distribution of the administered immune cells as RI-labeled cells can be evaluated, and the obtained evaluation results of the in vivo distribution can be used as useful information for estimating the effectiveness of cancer treatment by cell immunotherapy. [Examples]

[0046] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to the following examples.

[0047] (Surfactant solution) The surfactant solution is intended to be introduced into the target cells in order to label them (cell labeling). 89This is a surfactant solution that can prevent radioisotopes such as Zr from adhering to or binding to unintended objects. Examples of such unintended objects include containers, the outer surface of cells, and substances other than ligands. In this example, a polysorbate 80 solution was used as such a surfactant solution. This polysorbate 80 solution was prepared as follows.

[0048] In detail, 500 mg of polysorbate 80 (polyoxyethylene sorbitan oleate) was weighed using an electronic balance, and the weighed polysorbate 80 (500 mg) was placed in a 100 mL polyethylene container. Next, ultrapure water was added to this polysorbate 80 to adjust the volume of the mixture of polysorbate 80 and ultrapure water to 50 mL. After that, the mixture was stirred to prepare a polysorbate 80 solution with a polysorbate 80 concentration of 10 mg / mL. The obtained polysorbate 80 solution was stored at room temperature.

[0049] (ligand solution) A ligand solution is a solution of ligands that bind to (i.e., complexize) the radioisotope (RI) so that the RI can pass through the cell membrane. In this example, an oxinate solution was used as the ligand solution. This oxinate solution was prepared as follows.

[0050] In detail, 50 mg of 8-quinolinol (molar mass = 145.16 g / mol) was weighed using an electronic balance, and the weighed 8-quinolinol (50 mg) was placed in a 100 mL polyethylene container. Next, ultrapure water was added to this 8-quinolinol to adjust the volume of the mixture of 8-quinolinol and ultrapure water to 70 mL. Then, this polyethylene container was placed in a 70°C water bath, and the mixture in the polyethylene container was heated and stirred for about 10 minutes. This completely dissolved the 8-quinolinol in the ultrapure water, preparing an 8-quinolinol aqueous solution. The prepared 8-quinolinol aqueous solution was cooled to room temperature.

[0051] Next, 23.83 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) powder was added to the 8-quinolinol aqueous solution in the polyethylene container and stirred. This completely dissolved the HEPES in the 8-quinolinol aqueous solution. To the resulting solution, 10 mL of the polysorbate 80 solution described above was added, and then approximately 5.25 mL of a 10 mol / L sodium hydroxide aqueous solution was added to adjust the pH of the solution to 7.9-8.0. After that, ultrapure water was added to adjust the volume of the solution to 100 mL, and the adjusted solution was stirred to prepare the oxinate solution of this example. When the substances contained in the obtained oxinate solution were measured, the final concentration of oxinate was 0.5 mg / mL, the final concentration of HEPES was 1 mol / L, and the final concentration of polysorbate 80 was 1 mg / mL. The final pH of the oxinate solution was 8.0.

[0052] (RI solution) RI solution is a solution containing radioisotopes used for cell labeling. In this example, zirconium-89-oxinate solution (hereinafter, 89 A Zr solution (abbreviated as Zr solution) was used. 89 The Zr solution was prepared as follows:

[0053] In detail, in a 1.5 mL centrifuge tube, 89 The Zr purified solution (8 μL) and the oxidate solution (44 μL) described above were injected. 89 The Zr purified solution and the oxinate solution were reacted by stirring at 500 rpm for 5 minutes under conditions of a temperature of 25°C. This resulted in the following in this example. 89 A Zr solution was prepared. 89 The reaction between the Zr purified solution and the oxinate solution indicates that 89 The reaction rate between the Zr purified solution and the oxinate solution was confirmed by measuring it using thin-layer chromatography (mobile phase: ethyl acetate). 89 The Zr solution was stored inside a hot cell.

[0054] (Example 1) In Example 1, first, a suspension cell line (THP-1 cells) obtained from a cell bank to simulate immune cells collected from a subject was cultured in a culture medium in a dish. At this time, the cell diameter of the immune cells to be cultured was set to 10 μm or more, and the culture (suspension culture) was continued until the coverage rate of the culture medium (the percentage of the culture dish covered by cells) by the proliferated immune cells reached approximately 95%. As a result, immune cells that had proliferated with a cell diameter of 10 μm or more, that is, cultured cells (suspension cells) with a cell diameter of 10 μm or more were obtained.

[0055] The above-mentioned immune cells were cultured using culture media prepared in each of the two dishes. Then, the entire volume of culture media from both dishes, along with the resulting cultured cells, was transferred to two centrifuge tubes (50 mL each). These two centrifuge tubes were then placed in a centrifuge and centrifuged at 1000 × g for 5 minutes. After this centrifugation, the sides of the caps of the two centrifuge tubes were disinfected with alcohol spray and taken to a clean bench for flame sterilization. Finally, the caps of the two centrifuge tubes were opened, and the supernatant was removed from each using a pre-flame-sterilized aspirator.

[0056] Next, PBS (45 mL) was added to each of the two centrifuge tubes to suspend the cultured cells in the PBS. Then, the two centrifuge tubes were placed back into the centrifuge and centrifuged at 1000 × g for 5 minutes. After this centrifugation, the supernatant was removed from each of the two centrifuge tubes using an aspirator, as described above. PBS (1 mL) was then added to the remaining cultured cells and other residues inside each of the two centrifuge tubes to resuspend the cultured cells in the PBS. This created cell suspensions containing the cultured cells inside each of the two centrifuge tubes.

[0057] At this stage, 10 μL of the cell suspension prepared above was taken as a sample solution, and the collected sample solution was diluted 10-fold. The concentration of viable cultured cells (live cell concentration) in the diluted sample solution (0.1 mL) was measured. As a result, the live cell concentration of the sample solution was 2.93 × 10⁶. 6 The cell count was cells / mL. In other words, the concentration of live cells in the cell suspensions prepared inside each of the two centrifuge tubes was 2.93 × 10⁶. 7 The cell count was cells / mL. Furthermore, the viability of cultured cells in the sample solution was 93.9%.

[0058] Next, the cell suspensions from each of the two centrifuge tubes were combined into a single centrifuge tube (capacity: 50 mL), and PBS was added to adjust the viable cell concentration and volume of the cell suspension in this centrifuge tube to the target cell concentration and volume, respectively. This prepared the cell suspension of Example 1. In Example 1, the target cell concentration was 1.0 × 10⁶. 7 The target cell / mL should be 1 or higher, and the target volume should be 4 mL or higher.

[0059] The concentration of viable cells after diluting the cell suspension prepared above 10-fold was measured and found to be 10.36 × 10⁶. 5 The concentration was cells / mL. That is, the viable cell concentration of the cell suspension in Example 1 was 10.36 × 10⁶ 6 The cell density is cells / mL. Furthermore, the volume of the cell suspension in Example 1 was 4 mL, and the viability of the cultured cells in the cell suspension was 90.1%.

[0060] Next, measure out the cell suspension containing the cultured cells to be labeled (second cell suspension) from the cell suspension prepared above (first cell suspension). At this time, the viable cell concentration of the first cell suspension (= 10.36 × 10) 6 Considering the number of cultured cells in the second cell suspension (cells / mL), the cell count is 1.2 × 10⁶. 7 To obtain a total of at least 1.2 mL of the second cell suspension from the first cell suspension, the number of cells was measured out. The measured second cell suspension was placed in a 50 mL centrifuge tube.

[0061] Next, into the centrifuge tube containing the above-mentioned second cell suspension, 89 Add Zr solution to these second cell suspensions 89 A mixture was prepared with Zr solution. 89 The amount of Zr solution added was 40 μL (= 1.2 mL × 1 / 30), which is 1 / 30 of the volume of the second cell suspension present in the centrifuge tube.

[0062] Subsequently, the centrifuge tube containing the above mixture was placed in a tube heater, and the mixture inside the centrifuge tube was vibrated for 15 minutes at a rotation speed of 200 rpm under a temperature of 37°C. This allowed the cultured cells in the above mixture to be separated from the other cells. 89 Zr (For details 89 The Zr complex is reacted with the cultured cells to form 89 Zr was incorporated into the cultured cells. As a result, the cultured cells 89 Zr-labeled RI-labeled cells were generated in the above mixture. Subsequently, PBS (45 mL) was added to the mixture in the centrifuge tube, thereby suspending the generated RI-labeled cells in the mixture and removing cells that were not taken up by the cultured cells. 89 Zr was suspended in the mixed solution.

[0063] Next, the centrifuge tube containing the above mixture was placed in a centrifuge and centrifuged for 5 minutes at a centrifugal force of 1000 × g. This centrifugal process caused the RI-labeled cells contained in the mixture in the centrifuge tube to settle, forming a cell pellet consisting of the settled RI-labeled cells. At the same time, the mixture was separated from the cell pellet and the above 89 The RI residue (supernatant) containing Zr was separated from the supernatant. The cell pellet in the centrifuge tube was visually inspected through lead glass (line of sight = approximately 1m), and it was possible to see it clearly enough without hindering the work.

[0064] Subsequently, the suction port of a syringe was inserted into the centrifuge tube in which the cell pellet was formed, and the supernatant was removed from the centrifuge tube by the suction action of the syringe. At this time, the cell pellet in the centrifuge tube could be visually observed, so the supernatant could be easily removed without accidentally aspirating and removing the cell pellet. The supernatant thus removed was placed in a 50 mL measuring container, and the radioactivity of the supernatant was measured. As a result, the radiation from the supernatant was 1.257 MBq. Similarly, the radioactivity of the cell pellet was measured. As a result, the radioactivity from the cell pellet was 1.762 MBq. From these measurement results, it was found that in Example 1, the target cultured cells were collected from the total amount of the second cell suspension. 89 58.4% of Zr 89 We were able to confirm that it could be labeled with Zr.

[0065] Subsequently, PBS was injected into the centrifuge tube, thereby separating the cell pellet into multiple RI-labeled cells and dispersing these RI-labeled cells in the PBS. In this way, a solution of the target RI-labeled cells was collected. The measured values ​​and evaluation results for Example 1 are shown in Table 1 below.

[0066] (Comparative Example 1) In Comparative Example 1, the volume of the second cell suspension measured from the first cell suspension containing the target cultured cells was set to 0.6 mL, and the number of cultured cells contained in the measured second cell suspension was set to 0.6 × 10⁶. 7 A cell pellet consisting of RI-labeled cells was formed in the same manner as in Example 1 described above, except that cells were used. The measured values ​​and evaluation results for Comparative Example 1 are shown in Table 1.

[0067] (Comparative Example 2) In Comparative Example 2, the volume of the second cell suspension measured from the first cell suspension containing the target cultured cells was set to 1.0 mL, and the number of cultured cells contained in the measured second cell suspension was set to 1.0 × 10⁶. 7A cell pellet consisting of RI-labeled cells was formed in the same manner as in Example 1 described above, except that cells were used. The measured values ​​and evaluation results for Comparative Example 2 are shown in Table 1.

[0068] [Table 1]

[0069] Table 1 shows the measured values ​​and evaluation results for Example 1 and Comparative Examples 1 and 2 described above. In Table 1, "Cell diameter" is the cell diameter of the cultured cells to be labeled with RI. "Cell concentration" is the concentration of cultured cells (live cell concentration) present in the first cell suspension described above. "Cell volume" is the volume of the second cell suspension measured from the first cell suspension described above. "Cell number" is the number of cultured cells contained in the measured second cell suspension. "Visibility of cell pellet" is the visibility of the formed cell pellet when viewed through lead glass.

[0070] As shown in Table 1, the conditions are that the target cells have a diameter of 10 μm or more, and the number of cells in the cell suspension used for labeling the cells is 1.2 × 10⁶. 7 In Example 1, which satisfied both the condition of having more than 10 cells, the cell pellet consisting of RI-labeled cells could be clearly seen through lead glass. On the other hand, the number of cells in the cell suspension was smaller than in Example 1, at 1.2 × 10⁶. 7 In comparative examples 1 and 2, which did not meet the condition of having more than one cell, the cell pellet consisting of RI-labeled cells could not be visually observed through lead glass.

[0071] It should be noted that the present invention is not limited by the embodiments described above, and configurations that appropriately combine the above-described components are also included in the present invention. Furthermore, all other embodiments, examples, and operational techniques based on the embodiments described above by those skilled in the art are also included in the scope of the present invention. [Explanation of symbols]

[0072] 1 RI labeled cells 2 Cell suspension 3 Mixed liquid 10 cell pellets 11 RI residual liquid 20 centrifuge tubes 30 Aspirator

Claims

1. A preparation step of preparing a cell suspension containing cells in a physiological buffer, A labeling step of labeling the cells contained in a mixture of the cell suspension and a solution of the radionuclide with the radionuclide, A separation step is to separate the mixture into a cell pellet, which is a mass of labeled cells, and an RI residue liquid, which contains the radioactive nuclides that were not taken up by the cells. Includes, The diameter of the cells contained in the mixture is 10 μm or more. The number of cells contained in the aforementioned mixture is 1.2 × 10 7 It is greater than or equal to cells. A method for producing cell pellets, characterized by the following features.

2. The concentration of the cells in the cell suspension is 1.0 × 10 7 cells / mL or higher, A method for producing a cell pellet according to claim 1.

3. The concentration of the cells in the cell suspension is 1.0 × 10 9 cells / mL or less A method for producing a cell pellet according to claim 2.

4. The number of cells contained in the aforementioned mixture is 1.2 × 10 9 cells is below, A method for producing a cell pellet according to any one of the features 1 to 3.

5. The radioactive nuclide is zirconium-89. A method for producing a cell pellet according to any one of the features 1 to 3.

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