Method for preparing syringe for cell administration use, and method for transporting cells using syringe for cell administration use

Abstract

A purpose of the present invention is to provide a method for preparing a cell preparation containing therapeutic cells in an aseptic state into a dosage form capable of being administered to a subject immediately without the need to do a preliminary work in a medical facility. Another purpose of the present invention is to provide a method for transporting a cell preparation from a facility for the preparation of the cell preparation to a medical facility while avoiding the deterioration in the quality of therapeutic cells or the contamination by germs.　Provided are: a method for preparing a cell preparation by filling a suspension into an injection syringe in an aseptic state, in which the suspension comprises therapeutic cells suspended in a non-cryopreserved solution capable of preserving cells stably; and a method for transporting the prepared cell preparation while retaining a high cell survival rate and an aseptic state, the method comprising hermetically sealing the cell preparation, packaging the sealed cell preparation in a constant-temperature container and then transporting the packaged cell preparation.

Description

Method for preparing a syringe for cell administration and method for transporting cells using the syringe for cell administration

[0001] The present invention relates to a method for preparing a syringe for cell administration containing a cell suspension, and further to a method for transporting cells using the syringe for cell administration.

[0002] In recent years, advances in cell research have lowered the technical barriers to regenerative medicine. Cell transplantation, which involves transplanting living cells into patients for treatment, was initially used mainly in bone marrow transplants, where hematopoietic stem cells are transplanted into patients with intractable blood diseases, but its applications are expanding.

[0003] For example, cell-based cell therapy has been increasingly used in the fields of plastic surgery and orthopedics. In these cell therapies, autologous cell administration using mesenchymal stem cells (MSCs) and other stem cells is performed at many medical institutions, including private clinics. The cells used in such treatments are isolated and cultured from tissue collected from the patient, and then formulated into a formulation containing the required number of cells for treatment. The cells are then administered to the patient from whom they were collected.

[0004] Until now, cell therapy has mainly been performed in medical institutions with advanced medical equipment. However, due to the widespread use of regenerative medicine and cell transplantation as described above, even small medical institutions such as private hospitals are now increasingly seeking to provide regenerative medicine and cell transplantation for the treatment of mild diseases and conditions.

[0005] However, such small medical institutions often lack the equipment and technicians required to prepare cell preparations used in cell therapy and other procedures. In such cases, the preparation of cell preparations is generally carried out at an external specialized institution. The cell preparations prepared at the external specialized institution are then transported to the small medical institution and administered to patients for treatment. In order to perform regenerative medicine and cell transplantation at such small medical institutions, the transportation and storage of cell preparations is necessary.

[0006] Currently, cells are typically stored and transported in a frozen state to maintain cell viability. To prepare a cell preparation from cryopreserved cells, the cells must be thawed and then loaded into an instrument for administration. However, as mentioned above, it is difficult for small medical institutions to perform such tasks due to the limited manpower and equipment. Therefore, in order to successfully implement cell therapy in small medical institutions, there is a need for a cell preparation that allows cells to be administered to patients with minimal processing steps.

[0007] Furthermore, because cells in cell preparations are not necessarily stable and are susceptible to environmental influences, cell preparations prepared at external specialized institutions experience a decline in cell quality and viability over time between preparation and administration to patients, as well as vibrations during transport. It is also necessary to prevent the introduction of contaminating bacteria during the preparation process and transport of cell preparations. Therefore, it is extremely important that prepared cells remain in good condition and sterile before being transported to small medical institutions and administered to patients. Therefore, a method is needed that minimizes the deterioration of the quality of cell preparations prepared at external specialized institutions, prepares and transports cell preparations under conditions that prevent the introduction of contaminating bacteria, and enables them to be administered to the intended patients promptly after transport.

[0008] In particular, when administering autologous cells, to ensure that the patient receives autologous cells, it is necessary to clearly identify the lot of the cell product in question at all stages: from the tissue collection stage at the medical institution, to the stage of transporting it to an external specialized institution, to the stage of preparation and culturing of the cell product at the specialized institution, the stage of transporting the cell product, and the stage of handling at the medical institution after it is received. This is all the more true for private clinics with a large number of treatments (cell administrations), such as in the fields of orthopedics and plastic surgery.

[0009] After the cells arrive at and are accepted at a medical institution, preparations for cell administration must be made, such as thawing the cell preparation, loading the cells into an administration device, and adjusting the cell concentration as necessary. However, a cell transportation method that eliminates or minimizes such preparations would be highly desirable for medical institutions.

[0010] Special Publication 2014-513918

[0011] The object of the present invention is to provide a method for preparing a cell preparation containing therapeutic cells under sterile conditions in a form that can be immediately administered to a subject such as a patient, minimizing preparation work, even in small-scale medical institutions, for example.

[0012] A further object of the present invention is to provide a method for transporting the prepared cell preparation from the institution that prepares the cell preparation to, for example, a small medical institution, without deteriorating the quality of the therapeutic cells or causing contamination with germs.

[0013] A further object of the present invention is to provide a method that enables a cell preparation to be reliably administered to a subject, such as a patient, to whom the preparation is to be administered.

[0014] In order to solve the above problems, the inventors conducted extensive research and developed a method for preparing a cell preparation in which a suspension of therapeutic cells in a non-cryopreservative solution capable of keeping the cells stable is filled into an injection syringe under sterile conditions.

[0015] The present inventors have also developed a method for transporting the prepared cell preparation while maintaining a high cell viability by sealing the prepared cell preparation, packaging it in a thermostatic container, and transporting it.

[0016] The inventors have also developed a transportation method that enables the cell preparation to be reliably administered to the subject to be administered by providing cell information and / or information about the subject to be administered to a cell administration syringe filled with the prepared cell preparation.

[0017] Therefore, the present invention has the following features (1) to (15). (1) A method for preparing a cell administration syringe, comprising: a filling step of filling a syringe or a syringe equipped with a suction means with a cell suspension; a sterilized tube attachment step of attaching a sterilized tube to the syringe or the suction means, as necessary; and a removing step of removing gas in the cell administration syringe from the cell suspension via the sterilized tube, all of the steps being performed under sterile conditions. (2) The method described in (1), in which the filling step is performed by aspirating the cell suspension via the suction means attached to the syringe. (3) The method described in (1) or (2), further comprising: a separation step of separating the suction means and the sterilized tube from the cell administration syringe after the removing step; and a sealing means attachment step of attaching a sealing means to seal the site of the cell administration syringe where the suction means is attached. (4) The method according to any one of (1) to (3), wherein the removing step comprises extruding a portion of the cell suspension from the cell administration syringe into the sterilized tube. (5) The method according to any one of (1) to (4), wherein the cell suspension is obtained by suspending cells in a non-cryopreservative solution for a cell preparation. (6) The method according to (5), wherein the non-cryopreservative solution for a cell preparation is a solution containing 0.5 to 12 (w / v) % dextran, and sodium salts, potassium salts, and calcium salts. (7) The method according to any one of (1) to (6), wherein the cells contained in the cell suspension are mesenchymal cells. (8) The method according to (7), wherein the mesenchymal cells include mesenchymal stem cells. (9) A cell transportation method, comprising: a preparation step of preparing a cell administration syringe filled with a cell suspension, a sealing step of sealing the cell administration syringe, a packaging step of packaging the sealed container prepared in the sealing step in a constant temperature container together with a cooling agent, and a transportation step of transporting the packaged constant temperature container to a medical institution at a temperature of 0°C to 37°C, wherein the cell administration syringe is provided with information about the cells and / or information about the subject to be administered. (10) The method according to (9), wherein the cell administration syringe filled with the cell suspension is prepared by the method according to any one of (1) to (8).(11) The method according to (9) or (10), further comprising a storing step of storing the cell administration syringe in a water-absorbing bag before the sealing step. (12) The method according to any one of (9) to (11), wherein the cell administration syringe is placed horizontally in the incubator container and transported in that state. (13) The method according to any one of (9) to (12), wherein the institution that prepares the cell administration syringe and the medical institution are different institutions. (14) A method for administering a cell suspension, wherein, after the incubator container is transported to the medical institution by the method according to any one of (9) to (13), the cell suspension in the cell administration syringe is administered to a subject at the medical institution within four days after the preparation step. (15) The method according to (14), wherein the survival rate of cells in the cell suspension at the time of administration to a subject is 70% or more compared to when the cell suspension was prepared. This specification includes the disclosures of Japanese Patent Application Nos. 2022-056306 and 2022-134738, from which the present application claims priority.

[0018] According to the method of the present invention, a cell preparation can be prepared in the form of a syringe for cell administration containing a suspension of therapeutic cells in a non-cryopreservative solution.

[0019] Furthermore, according to the method of the present invention, the cell preparation can be transported from the cell preparation preparation facility as a package sealed in a sealed container that can maintain a constant temperature. Furthermore, according to the method of the present invention, the prepared cell preparation and its raw material cells can be transported reliably to the destination facility without deterioration of the quality of the cells or contamination with bacteria during transportation.

[0020] Furthermore, according to the method of the present invention, the cell preparation can be reliably administered to a subject such as a patient to whom the preparation is to be administered.

[0021] Photographs showing an overview of the process of filling a suspension of therapeutic cells into an administration syringe. A: Photograph of a syringe with a blunt needle before aspirating the suspension of therapeutic cells. B: Photograph of the syringe after aspirating the suspension of therapeutic cells through the blunt needle. C: Photograph of a sterilized tube to prevent dripping. D: Photograph showing the state in which a sterilized tube to prevent dripping is attached to the blunt needle of a syringe containing a cell suspension, and air is removed. E: Photograph of the syringe with the blunt needle and sterilized tube removed after air has been removed, together with the cap that will be attached to the syringe in the next step. F: Photograph of a syringe with a cap attached. Photographs showing an overview of the process of transporting a syringe filled with a suspension of therapeutic cells from an institution that prepares the therapeutic cells to a medical institution that will administer it. A: Photograph of a syringe with a cap attached. B: Photograph showing the syringe filled with a cell suspension stored in a water-absorbing bag with water-absorbing capabilities. C: A photograph showing a water-absorbing bag containing a syringe placed in a barrier pouch with a sealing function. D: A photograph showing a syringe in a barrier pouch placed sideways in a constant temperature transport package. E: A photograph showing a constant temperature transport package containing a barrier pouch containing a syringe, as well as ice packs and cushioning material to fill any excess space, ready for transport to a medical institution. This shows the change in cell viability over time when different volumes of cell suspension are filled into syringes of different capacities. The relative cell viability (%) on the vertical axis indicates a relative value, with the cell viability at 0 hours after the start of storage set to 100%. This shows the change in cell viability over time when syringes of different capacities are filled with cell suspensions at different numbers of filled cells / volumes of filled liquid, and the syringes are packed in a constant temperature transport package (TACPack (registered trademark)) and transported refrigerated. The relative cell viability (%) on the vertical axis indicates a relative value with the cell viability at 0 hours after the start of storage set to 100%. The graph shows the change in cell recovery rate over time when syringes of different volumes were filled with cell suspensions at different cell numbers / volumes, and the syringes were packed in a constant temperature transport package (TACPack (registered trademark)) and transported under refrigeration. The cell recovery rate (%) on the vertical axis indicates a relative value with the cell recovery rate at 0 hours after the start of storage set to 100%.Photographs showing the morphology of spheroids after adipose-derived MSC spheroid suspensions were filled into vials and syringes under various conditions and then refrigerated with vibration for 24 hours. Photographs showing the morphology of spheroids after adipose-derived MSC spheroid suspensions were filled into vials and syringes under various conditions and then refrigerated with vibration for 24 hours.

[0022] 1. Method for Preparing a Syringe for Cell Administration 1-1. Overview A first aspect of the present invention is a method for preparing a cell preparation for administering therapeutic cells. In the present invention, therapeutic cells are suspended under sterile conditions, and the cell preparation is prepared as a syringe filled with the cell suspension. In the present invention, the number of steps required for administration at a medical institution is minimized. Therefore, for example, at a medical institution administering the cell preparation, the cell suspension prepared in the syringe can be immediately administered to a subject in its original form. In other words, a cell preparation prepared by the method of the present invention does not require procedures such as thawing the cells or loading them into a cell administration device before administration to a subject. Therefore, the method of the present invention significantly reduces the risk of cell quality degradation, bacterial contamination, and operational errors at the medical institution where the cell preparation is administered.

[0023] 1-2. Definitions As used herein, "sterile conditions" refers to conditions under which an object (particularly cells and the interior of a container or device that comes into contact with the cells) can be kept sterile for a desired period of time. Furthermore, "sterile conditions" refers to a state that is substantially free of viable microorganisms (including filamentous fungi, bacteria, etc.) and viruses.

[0024] As used herein, the term "therapeutic cells" refers to cells that are administered to a subject for the purpose of cell therapy.

[0025] As used herein, the term "cell suspension" refers to a solution in which the above-mentioned therapeutic cells are suspended in a cell preservation solution.

[0026] As used herein, the term "syringe" refers to an instrument capable of aspirating and injecting liquids (including suspensions) and gases.

[0027] As used herein, the term "syringe for cell administration" refers to a syringe containing the cell suspension.

[0028] As used herein, the term "suction means" refers to a means attached to the cell administration syringe and used to aspirate the cell suspension into the syringe. Examples of such means include, but are not limited to, injection needles and tubes, and also include cases where the cell suspension is aspirated via a connector or cock.

[0029] As used herein, the term "sterilized tube" refers to a sterilized tube that can be attached to the suction means and that can hold the cell suspension extruded from the cell administration syringe. Examples of such a tube include, but are not limited to, a sterilized winged intravenous needle or an adapter tube, and include any other tube that provides a similar effect. The sterilized tube can also be attached directly to the cell administration syringe to serve as the "suction means."

[0030] As used herein, the term "non-cryopreservation solution for cell preparations" refers to a solution that allows non-cryopreservation of cells, such as a solution containing sodium salts, potassium salts, and calcium salts, or a solution containing 0.5 to 12 (w / v) % dextran, and sodium salts, potassium salts, and calcium salts.

[0031] As used herein, "non-freezing storage" refers to storage under physical conditions such as temperature and pressure at which water does not freeze. For example, at atmospheric pressure, this refers to a temperature at which water does not freeze. A non-freezing temperature refers to, for example, a temperature equal to or higher than 0°C.

[0032] As used herein, the term "cell preparation" refers to a composition containing live cells that can be applied to a subject, or live cells that serve as a source of therapeutic cells.

[0033] As used herein, the term "subject" refers to an individual to whom the cell preparation of the present invention is to be applied. For example, a "subject" is a "patient" in need of treatment or improvement.

[0034] As used herein, the term "patient" refers to a person who has some kind of physical abnormality and is not in a healthy state, such as a person suffering from some kind of disease, as well as a healthy person who receives a cell preparation for cosmetic purposes or to improve or enhance motor function or physical condition. In this specification, this refers to the main subject to whom the administration syringe of the present invention is administered.

[0035] As used herein, the term "mesenchymal cells" includes all cells that constitute mesodermal tissue, including, but not limited to, osteoblasts, adipocytes, muscle cells, chondrocytes, etc. Mesenchymal cells in the present invention also include mesenchymal stromal cells and mesenchymal stem cells.

[0036] As used herein, the term "stem cells" refers to cells that have the ability to differentiate into various cells and the ability to self-replicate. Examples include somatic stem cells and pluripotent stem cells.

[0037] As used herein, "mesenchymal stem cells (MSCs)" refer to cells that meet the following definition and refer to somatic stem cells that have the differentiation potential to differentiate into one or more types of cells belonging to mesodermal tissue. Mesenchymal stem cells as used herein include both mesenchymal stem cells obtained from any tissue and mesenchymal stem cells prepared in vitro. i) They exhibit adhesive properties to plastic when cultured in a standard medium. A standard medium is a basal medium (e.g., αMEM medium) supplemented with serum, serum replacement reagents, or growth factors. ii) They are positive for the surface antigens CD73 and CD90 and negative for CD45 and CD326.

[0038] 1-3. Configuration Each step of the method for preparing a syringe for cell administration of the present invention will be described. Each step of the method of the present invention is carried out under sterile conditions. Essential steps include a filling step, and, if necessary, a sterilized tube attachment step and a removal step.

[0039] The method for preparing a syringe for cell administration of the present invention may further include, as optional steps, a separation step and a sealing means attachment step.

[0040] In the filling step of the present invention, the cell suspension is filled into a syringe or a syringe equipped with a suction means. In a preferred embodiment, the filling is performed by aspirating the cell suspension through a suction means equipped to the syringe.

[0041] This step is carried out under aseptic conditions. Aseptic conditions can be achieved by any known method, and the method used is not particularly limited. Specifically, aseptic conditions can be achieved, for example, by a clean bench, an isolator, a sterile workroom, an access-restricted barrier system, or a combination thereof. Note that all other steps of the method of the first aspect of the present invention are also carried out under aseptic conditions.

[0042] To maintain sterile conditions, all instruments used in the first aspect of the present invention, such as syringes, suction means, and tubes, must be sterilized. The sterilization method used is not particularly limited, as long as it can kill or remove microorganisms in the target object to the extent that the objectives of the present invention are achieved. For example, chemical sterilization using ethanol and sodium hypochlorite, etc.; heat sterilization such as autoclaved steam sterilization and dry heat sterilization; gas sterilization using ozone gas, ethylene oxide gas, and plasma hydrogen peroxide gas, etc.; radiation sterilization using ultraviolet light, gamma rays, and electron beams; and sterile filtration can be used. Sterilization also includes, for example, sterilization. Appropriate sterilization methods and conditions can generally be selected depending on the type of microorganism, the contamination level, and the properties and condition of the target object to be sterilized. For example, the "Guidelines for the Production of Sterile Pharmaceutical Products by Terminal Sterilization" and "Guidelines for the Production of Sterile Pharmaceutical Products by Aseptic Processing" provided by the Ministry of Health, Labor, and Welfare of Japan, and the "WHO Good Manufacturing Practices for Sterile Pharmaceutical Products" provided by the World Health Organization (WHO) may be used as reference.

[0043] The syringe used in the present invention is an instrument capable of aspirating and injecting liquids and gases, and generally has a structure consisting of a cylindrical outer tube and a movable plunger inside. It is sufficient that the syringe is capable of aspirating cell suspensions via an aspirating means and has been sterilized. Preferably, a syringe manufactured for medical use, sterilized, and even more preferably individually packaged, is used.

[0044] The syringe may be made of plastic, glass, metal, or the like, and any of these may be used for the purposes of the present invention.

[0045] The size of the syringe is not particularly limited and is determined depending on the amount of cell suspension to be administered to the subject. For example, a syringe with a capacity of 1x or more, 2x or more, 3x or more, 4x or more, 5x or more, or 10x or more the amount of cell suspension to be administered to the subject per administration can be used. More specifically, syringes with a capacity of 0.5 ml, 1 ml, 2 ml, 5 ml, 10 ml, 20 ml, 30 ml, 50 ml, 100 ml, or more can be used.

[0046] The syringe is equipped with an aspirating means that allows the cell suspension to be aspirated and ejected into the syringe for cell administration. The aspirating means is preferably a collection needle, more preferably an injection needle such as a sharp needle or a blunt needle, and particularly preferably a blunt needle.

[0047] Needles of various sizes (thicknesses) are commercially available, and any size that allows the cells in the cell suspension to be drawn into the syringe without impairing the quality of the cells can be used. The needle size is preferably within the range of 14 gauge (outer diameter 2.0 mm) to 30 gauge (outer diameter 0.3 mm), more preferably 18 gauge (outer diameter 1.2 mm) to 27 gauge (outer diameter 0.4 mm), although needles of sizes outside these ranges can also be used. Like the syringe, the drawing means must be sterile.

[0048] In actual cell therapy, needles of approximately 20-25 gauge are often used to aspirate cell suspensions. When needles within this size range are used, less than 0.09 μL of cell suspension remains in the needle. Even when an 18-gauge needle is used, approximately 0.5 μL of cell suspension remains in the needle. When a syringe is used as the product container to aspirate the cell suspension, more than 90% of the cells can be recovered, and the amount of cell suspension remaining in the needle is not a problem. Based on the above, for example, needles of approximately 20-25 gauge can be used for cell suspensions of single cells, and needles of approximately 16-18 gauge can be used for cell suspensions of spheroid cells.

[0049] The cell suspension is aspirated into the syringe via an aspiration device attached to the syringe. Figure 1A is a photograph of a syringe equipped with an injection needle. The cell suspension can be aspirated into the syringe by pulling the syringe's inner barrel in the direction of the white arrow. Figure 1B is a photograph of a syringe filled with the cell suspension via aspiration. Alternatively, instead of using an injection needle as an aspiration device, a tube can be attached to the tip of the syringe directly or via an adapter, and the cell suspension can be aspirated into the syringe in a manner similar to that of an injection needle. Furthermore, the cell suspension can be aspirated into the syringe via a connector or cock (e.g., a two-way cock, a three-way cock) connected to a container such as a bag filled with the cell suspension. Furthermore, in the present invention, if the cell suspension can be aseptically filled into the syringe without using an aspiration device, such a method can also be employed.

[0050] In the present invention, the cells suspended in the cell suspension are therapeutic cells to be administered to a subject for the purpose of cell therapy, for example. That is, the cell suspension is prepared by suspending the therapeutic cells in a non-freezing preservation solution for cell preparations that is suitable for preserving the cells, particularly in an unfrozen state. Alternatively, the cell suspension may be a suspension of cells that serve as a source of therapeutic cells, as described below.

[0051] The non-cryopreservation solution for cell preparations is not particularly limited as long as it is suitable for preserving cells in an unfrozen state. Examples of suitable non-cryopreservation solutions for cell preparations include solutions containing sodium salts, potassium salts, and calcium salts, more preferably solutions containing 0.5 to 12 (w / v)% dextran and sodium salts, potassium salts, and calcium salts, and even more preferably solutions containing albumin in addition to the above solutions. The concentration of dextran contained in the non-cryopreservation solution for cell preparations is not particularly limited, and may be, for example, 0.5 to 20 (w / v)%, 0.6 to 18 (w / v)%, 0.7 to 16 (w / v)%, 0.8 to 14 (w / v)%, 0.9 to 13 (w / v)%, or 1 to 12 (w / v)%. The albumin concentration is not particularly limited, but may be, for example, 20% (w / v) or less, 15% (w / v) or less, 14% (w / v) or less, 13.5% (w / v) or less, 13% (w / v) or less, 12% (w / v) or less, 11% (w / v) or less, 10.5% (w / v) or less, 10.25% (w / v) or less, or 10.1% (w / v) or less. The concentrations of the sodium salt, potassium salt, and calcium salt are also not particularly limited. Another suitable non-cryopreservation solution for cell preparations is physiological saline containing albumin. In this case, the albumin concentration is not particularly limited, but may be, for example, 0.05-1% (w / v), 0.06-0.5% (w / v), or 0.08-0.12% (w / v).

[0052] The method used to suspend cells in a non-cryopreservation solution is not particularly limited. For example, cells can be suspended by aeration, liquid circulation, or other mechanical agitation. Specific methods include pipetting and tapping.

[0053] The pH of the non-cryopreservation solution for cell preparations is not particularly limited, as long as the cells to be preserved remain viable. Specific pH values ​​are, for example, 3 to 9, 3.5 to 8.5, 4 to 8, 4.5 to 7.5, or 5 to 7.5. The osmotic pressure of the non-cryopreservation solution for cell preparations of this embodiment is also not particularly limited, as long as the cells to be preserved remain viable. For example, the solution may be hypotonic (less than 250 mOsm / L), isotonic (250 to 380 mOsm / L), or hypertonic (greater than 380 mOsm / L).

[0054] The type of therapeutic cells used in the method of the present invention is not particularly limited, and examples thereof include cells derived from biological tissue, cells derived from cells derived from biological tissue, stem cells, cells differentiated from stem cells, and combinations thereof.

[0055] For example, cells derived from biological tissues include epithelial tissue-derived cells, connective tissue-derived cells, muscle tissue-derived cells, nerve tissue-derived cells, or combinations thereof. Furthermore, for example, connective tissue-derived cells and muscle tissue-derived cells can be collectively referred to as mesenchymal cells.

[0056] Mesenchymal cells can be derived from bone marrow, adipose tissue, umbilical cord, placenta, synovium, synovial fluid, dental pulp, heart, and other tissues. Specific examples of mesenchymal cells include skin fibroblasts, osteoblasts, tendon and ligament fibroblasts, adipocytes, chondrocytes, tenocytes, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, mucin-producing cells, and endocrine gland cells (e.g., insulin-producing cells such as β islet cells). Furthermore, in the case of blood, a type of connective tissue, blood cells, unlike other mesenchymal cells, are differentiated from hematopoietic stem cells, but these are also considered mesenchymal cells. Specifically, mesenchymal cells include dendritic cells, monocytes, natural killer (NK) cells, T cells (e.g., alpha-beta (αβ) T cells, gamma-delta (γδ) T cells, cytotoxic T lymphocytes (CTLs), helper T cells, etc.), B cells, macrophages, neutrophils, eosinophils, and the like. Furthermore, these mesenchymal cells may be cells differentiated from pluripotent stem cells, as described below, and specifically may be cardiomyocytes, chondrocytes, nerve cells, etc. derived from iPS cells.

[0057] When stem cells are used, for example, somatic stem cells, pluripotent stem cells, or a combination thereof can be used. Examples of somatic stem cells include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hair follicle stem cells, mammary stem cells, and melanocyte stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells).

[0058] When using stem cells, particularly somatic stem cells, the tissue from which the cells are derived is not particularly limited. For example, in the case of stem cells present in a variety of tissues, such as mesenchymal stem cells, cells derived from any tissue may be used, or a combination of cells derived from multiple types of tissue may be used. Examples of tissues from which mesenchymal stem cells can be derived include the tissues exemplified for mesenchymal cells. Specifically, for example, mesenchymal stem cells may be derived from connective tissue or adipose tissue.

[0059] The therapeutic cells of the present invention may be in a single cell state or a spheroid (cell aggregate) state.

[0060] There are no particular limitations on the target of application of the cell preparation used in the method of the present invention, and it can be applied to, for example, tissues, organs, or individuals of living organisms.

[0061] The cell preparations used in the methods of the present invention may be applied to healthy individuals or individuals suffering from a disease or condition, including, but not limited to, cancer, leukemia, vascular disease, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, neurological disease, burns, chronic inflammation, heart disease such as ischemic cardiomyopathy and dilated cardiomyopathy, immunodeficiency, Crohn's disease, diabetes, arthropathy, facial lipoatrophy, mastectomy, scars, age spots, wrinkles, sagging skin, and the like.

[0062] The application purpose is not particularly limited. For example, it can be used for the improvement, treatment, prevention, and cosmetic surgery of diseases and conditions. Specific examples include regenerative medicine for tissue augmentation for tissue depression and other conditions, immunotherapy such as T cell therapy, NKT cell therapy, and dendritic cell transfer therapy, gene therapy using transfected cells, breast augmentation, wrinkle removal, age spot removal, anti-aging, and other cell transplant therapies. Furthermore, the cell preparation of the present invention can be mixed with fat and used for breast reconstruction in patients who have undergone mastectomy, or for patients with knee osteoarthritis.

[0063] By applying the cell preparation used in the method of the present invention, at least a portion of the applied cells can function in the subject in the same way as endogenous cells.

[0064] 1-3-2. Sterilization Tube Attachment Step In the sterilization tube attachment step of the method of the present invention, a sterilization tube is attached to the suction means.

[0065] The sterilized tube used in the present invention is not particularly limited as long as it is a sterilized tube that can be attached to the suction means attached to the cell administration syringe and that can retain the cell suspension extruded from the cell administration syringe in the removal step described below. Furthermore, if a tube is used as the suction means, it can be used as is.

[0066] Sterile tubing that can be used in the present invention includes sterilized winged intravenous needles, adapter tubing, sterilized catheters, sterilized cannulas, extension tubing, infusion tubing, stomach tubing, and other medical tubing, but sterilized winged intravenous needles are particularly suitable. Figure 1C is a photograph of a winged intravenous needle commercially available from Terumo Corporation that can be used as a sterile tubing.

[0067] 1-3-3. Removal Step In the removal step of the method of the present invention, gas in the cell administration syringe is removed from the cell suspension via the sterilized tube, if necessary.

[0068] This step involves pushing a portion of the cell suspension in the cell administration syringe into a sterile tube along with any gas (e.g., air) present in the cell administration syringe. If gas, such as air, is present in the cell administration syringe, there is a risk that the gas will be introduced into the subject's blood vessels when the cell suspension is administered to the subject. Furthermore, if air enters the subject's blood vessels, there is a risk that the invaded air will cause vascular blockage. Therefore, this step must be performed to remove gas, such as air, from the cell administration syringe. By pointing the outlet of the cell administration syringe upward and pushing out a portion of the cell suspension, the gas, such as air, contained in the cell suspension moves into the sterile tube along with the portion of the cell suspension. While this removal step can be performed at a medical institution after transport before administration to the subject, it is preferable to perform it promptly after the filling step and transport the syringe in a gas-free state.

[0069] Furthermore, the outlet of the sterilized tube is located away from the cell administration syringe, so that the cell administration syringe and the cell suspension extruded from the tube do not come into contact. Therefore, the cell administration syringe is not contaminated by the extruded cell suspension. The means for preventing contamination of the cell administration syringe by the extruded cell suspension are not limited, and if a needle is attached to the syringe, the syringe may be extruded with the needle inserted into an empty sterile bag, or, if the syringe is filled with the cell suspension via a connector or cock, the syringe outlet may be pointed upwards while the connector or cock is still connected (after switching the flow path as necessary if a three-way cock is used), thereby extruding gas, etc.

[0070] Fig. 1D shows a photograph of a syringe containing a cell suspension, with a blunt needle and a sterile tube attached to prevent dripping. By pushing the syringe barrel in the direction of the white arrow, gas such as air can be removed along with a portion of the cell suspension in the syringe.

[0071] 1-3-4. Separation Process and Sealing Means Attachment Process Considering that the cell administration syringe will be transported to a medical institution, there is a risk that the cell suspension will leak out of the syringe during transport if the suction means and sterilized tube remain attached to the cell administration syringe. In the present invention, it is preferable to perform a separation process in which the suction means and sterilized tube are separated from the cell administration syringe, and a sealing means attachment process in which the site of the cell administration syringe where the suction means, such as an injection needle, was attached is sealed. Therefore, the separation process and sealing means attachment process can be performed as optional processes after the separation process.

[0072] In the separation step, the suction means and sterile tubing are separated from the cell administration syringe. Figure 1E is a photograph showing the syringe after the separation step, with the air removed and the blunt needle and sterile tubing removed, along with the cap that will be attached to the syringe in the next step.

[0073] In the sealing means attachment step, a means for sealing the site where the suction means, such as an injection needle, of the administration syringe was attached is attached. By attaching the sealing means to the cell administration syringe, the tip of the syringe where the suction means, such as an injection needle, was attached is sealed. This prevents the cell suspension from leaking from the cell administration syringe and prevents the cell suspension from being contaminated with bacteria, etc.

[0074] The sealing means used here is not particularly limited as long as it can seal the tip of the syringe and keep the cell suspension in the syringe under sterile conditions, and of course the sealing means must be sterile.

[0075] Examples of suitable sealing means include a syringe cap, a luer cap, and a rubber stopper. A variety of syringe caps are commercially available, and materials such as polyethylene, plastic, soft polyvinyl chloride, and silicone rubber can be used without any particular limitation. A sterilized airtight seal or the like can also be used as a sealing means instead of a syringe cap. Figure 1F shows a photograph of a capped syringe for cell administration.

[0076] 2. Cell Transport Method 2-1. Overview A second aspect of the present invention is a cell transport method. In this invention, a cell administration syringe is prepared according to the first aspect, and therapeutic cells contained in the cell administration syringe are administered to a subject, primarily a patient, at a medical institution. For example, the institution that prepares the cell administration syringe and the medical institution are different institutions. Therefore, it is necessary to maintain the prepared cells in good condition from the time of transportation until the cell preparation is administered to the subject, and to ensure the sterility of the cell preparation during transportation.

[0077] Specifically, in the method of this embodiment, a cell administration syringe is prepared, sealed, and the sealed container produced in the sealing process is packaged in a constant-temperature container together with a cooling agent. The packaged constant-temperature container is then transported to a medical institution at a temperature between 0 and 37°C. The constant-temperature container referred to here is sufficient as long as it can insulate the container from the effects of external temperatures to some extent; it does not necessarily have to maintain a constant temperature inside the container. Of course, a container that can control the temperature within a predetermined range is preferable. Alternatively, a low-temperature transport package or cooling agent, as described below, can also be used as appropriate. To accurately administer therapeutic cells to a subject to be administered, cell information and / or information about the subject to be administered is provided to the cell administration syringe. According to the method of this embodiment, the cell administration syringe prepared in the first embodiment can be transported from the specialized institution where the cell administration syringe was prepared to the medical institution where the cell preparation will be administered without the cells in the syringe deteriorating or becoming contaminated with bacteria. The medical institution may be of any size or type, as long as it administers therapeutic cells to a subject. For example, it may be an advanced medical facility such as a university hospital or a general hospital, a medium-sized or small-sized medical facility, or a private clinic or osteopathic clinic. The present invention is particularly suitable for small-scale medical institutions and private clinics that do not have sufficient equipment or management systems, as it minimizes the number of steps required for administration.

[0078] 2-2. Definition The definition of "cell administration syringe" is as described in the first aspect above. As used herein, the term "sealed container" refers to a container that can be sealed to prevent solid and / or gaseous foreign matter from entering the cell administration syringe and to prevent loss of the contents filled in the cell administration syringe.

[0079] As used herein, the term "refrigerant" refers to an agent that enables the "syringe for cell administration" to be kept at a low temperature, or a small bag containing the agent sealed therein. The "refrigerant" is, for example, a bag containing a composition containing water and a highly water-absorbent resin (e.g., sodium polyacrylate) as its main components.

[0080] In this specification, the term "constant temperature transport package" refers to a transport package that has thermal insulation properties that enable the inside of the container to be maintained within a certain temperature range for a certain period of time.

[0081] The definition of "subject" is as stated in the first aspect above. The definition of "patient" is as stated in the first aspect above.

[0082] As used herein, "cell information" refers to information regarding the type, strain, properties, origin, and origin of therapeutic cells contained in a cell administration syringe, and is information useful for identifying the cells.

[0083] As used herein, "subject information" refers to information such as the name, age, sex, and disease name of the subject receiving the therapeutic cell suspension, which is useful for identifying the subject.

[0084] This "cell information" and "specimen information" may be attached directly to the cell administration syringe or to the sealed container that houses the cell administration syringe. There are no particular limitations on the form in which it is attached, and any means such as labels, stickers, printing, or tags can be used. This "cell information" and "specimen information" may also be attached in the form of symbols or barcodes. The specific information corresponding to the attached symbols or barcodes can also be confirmed via email, a database, or the like.

[0085] 2-3. Structure Each step of the cell transport method of the present invention will be described. The method of the present invention includes the essential steps of a preparation step, a sealing step, a packaging step, and a transport step. To accurately administer the cell preparation to the subject, cell information and / or subject information is attached to the cell administration syringe.

[0086] 2-3-1 Preparation Steps The syringe for cell administration filled with the cell suspension is prepared by the method described in the first embodiment, so a detailed description of the preparation steps will be omitted.

[0087] 2-3-2. Sealing Step In order to transport a cell administration syringe filled with a cell suspension without losing the cells in the cell administration syringe or without introducing unwanted bacteria, the cell administration syringe is placed in a sealed container. The sealed container used here is not particularly limited as long as it can achieve the purpose of sealing the cell administration syringe, but examples include barrier pouches, Unipacks, Tupperware, plastic containers with lids such as vials, and glass bottles with lids.

[0088] Although it is not an essential step, when sealing the cell administration syringe in a barrier pouch or the like, it is preferable to store the cell administration syringe in a water-absorbing bag and then place it in a sealed container such as a barrier pouch in case the cell suspension leaks out of the cell administration syringe.

[0089] The water-absorbing bag is not particularly limited as long as it has the function of absorbing leaked liquid. Various products that have water-absorbing properties by containing water-absorbing polymers or the like are commercially available, and the water-absorbing bag most suitable for the cell administration syringe to be transported is selected taking into consideration its water-absorbing properties and size. Examples of water-absorbing bags include "Water Catch (registered trademark)" commercially available from Sanyo Co., Ltd., "Aqua Boy (trademark)" commercially available from Akagi Co., Ltd., water-absorbing bags commercially available from Sugiyamagen Co., Ltd., and "Absorbent Kun (trademark)," a highly water-absorbent polymer commercially available from Ashimori Kogyo Co., Ltd. Note that if a water-absorbing bag for the barrier pouch to be used is commercially available, it is preferable to use it.

[0090] Fig. 2A shows a photograph of a syringe with a cap attached. Fig. 2B shows a photograph of a syringe filled with a cell suspension housed in a water-absorbing bag with a water-absorbing function. Fig. 2C shows a photograph of a water-absorbing bag with a syringe housed in a barrier pouch with a sealing function.

[0091] 2-3-3 Packaging Process A sealed container such as a barrier pouch containing a syringe for cell administration is packed in a package for constant temperature transportation together with a cooling agent.

[0092] The constant temperature transport package used in the present invention is a transport package with insulating and heat-retaining properties that allows the interior of the container to be maintained within a constant temperature range for a certain period of time. Note that "constant temperature" does not necessarily mean that the temperature must be maintained at a constant level; temperature changes are acceptable as long as the temperature is within a set range. The transport package is not particularly limited, but it is preferable to use an insulated container containing an insulating material as the constant temperature transport package. Examples of constant temperature transport packages include, but are not limited to, those containing insulating materials such as vacuum insulation, expanded polystyrene insulation, rigid urethane foam insulation, and glass wool insulation.

[0093] Particularly suitable constant temperature transport packages include TACPack (registered trademark) available from Tamai Kasei Co., Ltd., va-Q-tec (registered trademark) available from va-Q-tec AG, and NeoAce (registered trademark) available from Inoac Corporation. The above-mentioned constant temperature transport packages are capable of maintaining a constant temperature inside the container. As described below, the temperature during transport is maintained between 0°C and 37°C, so when using constant temperature transport packages, it is necessary to select one that meets the specifications required to maintain such a temperature.

[0094] Various ice packs are commercially available, including those packed with a composition containing water and a highly water-absorbent resin (e.g., sodium polyacrylate) as the main components, and any of these can be used for the purposes of the present invention.

[0095] When storing a barrier pouch containing a cell administration syringe in a constant temperature transport package, it is preferable to place the cell administration syringe horizontally. Since cells settle in a cell suspension, storing the cell administration syringe horizontally reduces the bottom area of ​​the syringe when the cells settle compared to when the syringe is stored vertically, thereby minimizing cell deterioration.

[0096] FIG. 2D is a photograph showing the syringe in the barrier pouch placed sideways in a TACPack®, a temperature-controlled transport package.

[0097] Although not essential, it is preferable to fill the remaining space in the constant temperature transportation package with a buffer material after the barrier pouch containing the cell administration syringe has been placed in the constant temperature transportation package. This is because it reduces the damage to the cells caused by impact during transportation. Various materials can be used as the buffer material, including air buffer material (air buffer material), foam buffer, butitol, and packing paper.

[0098] Figure 2E is a photograph showing the state of the packaging for transport to a medical institution, in which a barrier pouch containing a syringe is placed inside a TACPack (registered trademark) constant temperature transport package, as well as cushioning material to fill any excess space.

[0099] The above steps are carried out at the facility where the syringe for cell administration is prepared. Therefore, in the transportation step of the present invention, in order to administer the cell preparation prepared as a syringe for cell administration to a subject, the package prepared in the above steps is transported at a temperature of 0°C to 37°C to the medical institution where the cell preparation will be administered to the subject.

[0100] The temperature during the transportation step is 0 to 37°C, 0 to 30°C, 0 to 25°C, 0 to 20°C, 0 to 17°C, 0 to 15°C, 0 to 14°C, 0 to 13°C, 0 to 12°C, 0 to 11°C, 0 to 10°C, 0.1 to 9°C, 0.2 to 8°C, 0.5 to 7°C, 1 to 6°C, 1.5 to 5°C, or 2 to 5°C, and the temperature is maintained within this range to be most suitable for cell stability. For example, it is preferable to maintain the temperature at around 4°C, as used in the Examples below.

[0101] In the transportation step, the incubator container containing the cell administration syringe is transported to a medical institution, preferably within 24 hours after the completion of the preparation step. After the incubator container containing the cell administration syringe arrives at the medical institution, the cell suspension in the cell administration syringe is administered to a patient at the medical institution. In this respect, the present invention also provides a method for administering a cell suspension. According to the administration method of the present invention, the cell suspension can be administered while maintaining the stability of the cells.

[0102] In the present invention, the means for administering the cell suspension in the cell administration syringe to a patient is not particularly limited. However, a simple method is to connect an injection needle for administration to the cell administration syringe that has been transported and arrived at the medical institution, and then administer the cell suspension to the patient via the injection needle. The injection needle used in this case is not particularly limited, and the needle diameter, length, number of needles, etc. can be selected as desired depending on the disease of the patient and the purpose of administration. Furthermore, the injection needle does not have to be connected directly to the cell administration syringe; it may be connected via a tube or adapter, or it may be connected to an infusion line or catheter for administration.

[0103] To prevent a decrease in cell viability, the time between the end of the preparation process and the administration of the cell suspension to a patient should be short, preferably within 4 days, more preferably within 3 days, even more preferably within 2 days, and most preferably within 1 day. When the cell administration syringe is stored and transported under refrigeration, the viability of the cells in the cell suspension at the time of administration to a subject is 70% or more compared to when the cell suspension was prepared. Under the conditions examined in the following example, the cell viability after 96 hours was 90% or more (Example 2).

[0104] The introduction of unwanted bacteria during transportation should be avoided as much as possible. In the following example, it was demonstrated that sterility was guaranteed when a cell suspension was prepared and transported according to the method of the present invention (Example 4).

[0105] Furthermore, a storage test using a spheroid cell suspension filled into a syringe using the method of the present invention revealed another benefit: the ability to maintain the spheroid shape (Examples 5 and 6). Spheroid cells are sometimes used in cell-based therapies to enhance their therapeutic efficacy. However, the inventors' studies have revealed that, with conventional transport methods, spheroids can collapse due to vibrations during transport, or the set aggregate diameter cannot be maintained. Even in such cases, the method of the present invention has been confirmed to enable transport of a cell suspension of spheroid cells while maintaining the quality, including not only viability but also shape. Furthermore, pluripotent stem cell spheroids, such as iPS cells, produced by suspension culture are not administered directly but are differentiated before use as therapeutic cells. However, when the iPS cell production facility and the facility that differentiates iPS cells into therapeutic cells and prepares cell preparations are different, the method of the present invention can also be used as a suitable means of transporting pluripotent stem cell spheroids, which serve as the source of therapeutic cells.

[0106] The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.

[0107] Example 1: Study of the Volume of the Transport Syringe and the Volume of the Cell Suspension to be Filled (Purpose) Vials and flow tubes are commonly used as containers for storing and transporting frozen cells. Therefore, a preliminary test was first conducted in which prepared cell preparations were placed in flow tubes, placed vertically, and transported under refrigeration without freezing. As a result, a tendency for cell viability to decrease with increasing cell concentration in the container was observed. This result suggested that when cells settle to the bottom of the tube, a large cell mass may deplete nutrients around the cells, increasing the concentration of waste products and resulting in increased cell loss.

[0108] Therefore, the present inventors came up with the idea of ​​using a syringe as a container for a cell preparation. In this example, the cell concentration in the cell suspension to be filled into the syringe and the volume of the syringe to be filled with the cell suspension are examined.

[0109] (Method) 1. Preparation of cell suspension After thawing and washing frozen stored liposuction-derived MSCs, the washing solution was replaced with non-freezing preservative solution for cell preparations and suspended to obtain a cell suspension of 1 × 10 8 The composition of the non-freezing preservation solution for cell preparations used in this example is as shown below.

[0110] Lactated Ringer's solution (Lactec Injection, Otsuka Pharmaceutical Factory (for composition etc., see the package insert, revised December 2016 (9th edition))) + dextran 4 (w / v)% + human serum albumin (HSA) 5 (w / v)%

[0111] 2. Filling of cell suspension The prepared cell suspension was filled into syringes or flow tubes of the following volumes. To fill the syringes, as shown in Figure 1, the cell suspension was aspirated through a blunt needle, a sterile tube was attached, the gas inside the syringe was removed, and the blunt needle and sterile tube were separated and then a cap was attached. (1) 5 mL of cell suspension was filled into a 10 mL syringe and allowed to stand: Level (1) (2) 5 mL of cell suspension was filled into a 5 mL syringe and allowed to stand: Level (2) (3) 2.5 mL of cell suspension was filled into a 5 mL syringe and allowed to stand: Level (3) (4) 1.8 mL of cell suspension was filled into a flow tube and allowed to stand: Level (4) (5) 1.8 mL of cell suspension was filled into a flow tube and transported (Reference example: corresponds to the above preliminary test)

[0112] 3. Measurement of Cell Viability After filling the cell suspension into a syringe or flow tube, the syringe was placed horizontally and the flow tube was placed vertically and left to stand at 4°C (Levels (1) to (4)). For Level (1), cell viability was measured after 48, 72, and 96 hours. For Levels (2), (3), and (4), cell viability was measured after 72 hours. The dead cell concentration and total cell concentration in the cell suspension were measured using a NucleoCounter NC-100 (Chemometec), and cell viability was calculated. The dead cell concentration in this measurement was measured by aspirating the cell suspension into a cassette (model number: 941-0002) containing PI solution, which stains dead cells. In addition, the total cell concentration in this measurement was measured by mixing equal amounts of the cell suspension with Cell Treatment Reagent A100 (product number: 910-0003) and Cell Treatment Reagent B (product number: 910-0002) to stain all cells with PI solution, aspirating them into the cassette described above, and measuring them. The cell number and viability of the adipose-derived MSCs obtained were calculated from the cell concentration measurement values. The calculation formula was as follows:

[0113] Viability (%) of adipose-derived MSCs = 100 - (dead cell concentration (cells / mL) / (total cell concentration (cells / mL) × 3 (dilution ratio at time of measurement)) × 100. Note that cell viability data was normalized with the value after 0 hours set to 100%.

[0114] (Results) The results of measuring the change in cell viability in the cell suspension at each level are shown in Figure 3. As shown in Figure 3, the cell viability in level (1), where the cell suspension was filled into a syringe, gradually decreased over time, but even after 96 hours, 90% of the cell viability was maintained compared to the start of storage.

[0115] In the cases of (2) and (3), where the cell suspension was filled into the syringe, the cell viability remained at 90% after 72 hours compared to the initial storage time. Therefore, it was found that storing the cell suspension in a syringe is preferable in terms of cell viability.

[0116] In addition, in the case of the level (4) where the cell suspension was filled in the flow tube, the cell viability remained at 90% even after 72 hours compared to the start of storage.

[0117] On the other hand, although not a direct comparison, in a test in which a cell suspension was actually transported using a flow tube (Level (5)) (corresponding to the preliminary test in the reference example), the cell viability after 96 hours had decreased to 69% of that at the start of storage.

[0118] Therefore, taking into account the effects of transportation in accordance with actual usage conditions, it was predicted that a syringe would be the most suitable container for filling the cell suspension. Furthermore, it was predicted that the decrease in cell viability during refrigerated transportation could be suppressed by lowering the cell concentration and storing the cell administration syringe sideways to increase the area over which the cells settle.

[0119] Example 2: Changes in cell viability over time during refrigerated transport of a cell product in which a cell suspension was filled into a syringe under various conditions (Objective) To investigate the size of a syringe, which is a candidate container for the cell product, and the volume of the suspension to be filled into it. In this example, a transport test was conducted using a syringe as the container, and the cell viability during transport was evaluated over time.

[0120] (Method) 1. Preparation of cell suspension and filling of cell suspension into syringe After thawing and washing frozen stored lipoaspirate-derived MSCs, a cell suspension was prepared by replacing the washing solution with a non-freezing preservative solution for cell preparations (lactated Ringer's solution + dextran 4 (w / v)% + HSA 1.5 (w / v)%). The prepared cell suspension was filled into a syringe in the same manner as in Example 1.

[0121] The size of the syringe filled with the cell suspension, the number of cells filled / volume of cell suspension, and whether or not it was transported were as follows: (1) 10 × 10 cells in a 10 mL syringe 7 Cells / 5mL filled, transported: Level (1) (2) 10 x 10 cells in a 10mL syringe 7 Filled with cells / 5 mL, no transport: Level (2) (3) 1 × 10 cells in a 10 mL syringe 7 Filled with cells / 5mL, with transport: Level (3) (4) 10 x 10 cells in a 10mL syringe 7 Cells / 10mL filled, with transport: Level (4) (5) 10 x 10 cells in a 2.5mL syringe 7Filled with 2mL cells, transported: Level (5)

[0122] 2. Transportation of syringes filled with cell suspensions Syringes filled with cell suspensions were stored horizontally in a temperature-controlled TACPack (registered trademark) (for 4°C). In levels (1), (3), (4), and (5), the syringes were transported 1.5 times between Kobe and Yokohama at refrigerated temperature (4°C) using Chilled Yu-Pack.

[0123] 3. Measurement of cell viability For levels (1), (2), and (3), cell viability was measured after 48 hours, 72 hours, and 96 hours using a NucleoCounter NC-200. Cell viability was measured by aspirating the cell suspension into a cassette (model number: 941-0012) containing acridine orange solution, which stains nucleated cells, and DAPI solution, which stains dead cells. For levels (4) and (5), cell viability after 72 hours was similarly measured using a NucleoCounter NC-200.

[0124] (Results) The results of measuring the change in cell viability in the cell suspension at each level are shown in Figure 4. As shown in Figure 4, at all levels, the viability tended to decrease over time, but even after 96 hours, 90% or more of the viability at the start of storage was maintained.

[0125] Furthermore, no clear difference in survival rate was observed between the level with transportation (1) and the level without transportation (2). Therefore, it was found that when using a syringe, transportation does not have a significant effect on survival rate.

[0126] Also, 10 x 10 7 Level (1) was a 10 mL syringe filled with 5 mL of cell suspension, and level (2) was a 1 × 10 7 Compared with level (3) where a 10 mL syringe was filled with a cell / 5 mL suspension, level (3) with a lower cell number (low cell concentration) maintained a higher viability.

[0127] 10×10 7 Level (1) was a 10 mL syringe filled with 5 mL of cell suspension, and level (2) was a 10 × 10 7 Level (4) and 10 x 10 cells / 10 mL suspension filled into a 10 mL syringe7 When compared with the standard (5) in which 2 mL of cell suspension was filled into a 2.5 mL syringe, there was no significant difference in viability after 72 hours.

[0128] When a syringe was used as a container for the cell preparation, the cell suspension was filled into it, and the product was transported under refrigeration. In levels (1), (2), and (3), cell viability was maintained at 90% or more of the initial level for 96 hours. In levels (4) and (5), cell viability was also maintained at 90% or more of the initial level for at least 72 hours.

[0129] These results confirmed that, within the experimental conditions of this example, the volume of the preservation solution and the size of the syringe did not significantly affect the viability. Although a lower cell concentration resulted in a higher viability, even at a higher cell concentration, a viability of over 90% was achieved after 72 hours under the experimental conditions of this example.

[0130] Example 3. Changes in cell recovery rate over time during refrigerated transport of a cell product in which a cell suspension was filled into a syringe under various conditions (Objective) To investigate the size of syringes that are candidates for containers to hold the cell product and the volume of suspension to be filled into them. In this example, a transport test was conducted using a syringe as the container, and the cell recovery rate during transport was evaluated over time.

[0131] 1. Preparation of cell suspension and loading of cell suspension into syringe (Method) Frozen and stored lipoaspirate-derived MSCs were thawed and washed, and then the washing solution was replaced with a non-freezing preservative solution for cell preparations (lactated Ringer's solution + dextran 4 (w / v)% + HSA 1.5 (w / v)%), followed by suspension to prepare a cell suspension. The prepared cell suspension was loaded into a syringe in the same manner as in Example 1.

[0132] The size of the syringe filled with the cell suspension, the number of cells filled / volume of cell suspension, and whether or not it was transported were as follows: (1) 10 × 10 cells in a 10 mL syringe 7 Filled with cells / 5mL, with transportation: Level (1) (2) 10 x 10 cells in a 10mL syringe 7 Filled with cells / 5 mL, no transport: Level (2) (3) 1 × 10 cells in a 10 mL syringe 7Filled with cells / 5mL, transported: Level (3) (4) 10 x 10 cells in a 10mL syringe 7 Filled with cells / 10 mL, with transport: Level (4) (5) 10 x 10 cells in a 2.5 mL syringe 7 Filled with 2mL cells, transported: Level (5)

[0133] 2. Transportation of syringes filled with cell suspensions The syringes filled with cell suspensions were stored horizontally in temperature-controlled TACPack (registered trademark) (for 4°C), and levels (1), (3), (4), and (5) were transported 1.5 times between Kobe and Yokohama at refrigerated temperature (4°C) using chilled Yu-Pack.

[0134] 3. Measurement of Cell Recovery Rate For levels (1), (2), and (3), the total cell concentration was measured after 48, 72, and 96 hours using a NucleoCounter NC-200. The total cell concentration was measured by aspirating the cell suspension into a cassette (model number: 941-0012) containing acridine orange solution, which stains nucleated cells, and DAPI solution, which stains dead cells. For levels (4) and (5), the total cell concentration was measured after 72 hours using a NucleoCounter NC-200. The total cell number was calculated from the total cell concentration and liquid volume, and the cell recovery rate, which is the ratio of the number of recovered cells to the number of cells loaded, was calculated.

[0135] (Results) Figure 5 shows the results of measuring the change in cell recovery rate in the cell suspension at each level. As shown in Figure 5, the average cell recovery rates were approximately 98%, 96%, 92%, 92%, and 91% for levels (1), (2), (3), (4), and (5), respectively. Therefore, the cell recovery rate was 90% or higher at all levels. Therefore, when syringes are used as containers for refrigerated transport of product cells, cell loss was less than 10%, which was an extremely good result.

[0136] Example 4: Confirmation of sterility of product after filling into syringe and transporting under refrigeration A medium filling test (process simulation) was conducted to verify that sterility was maintained during the process of filling refrigerated transported cells into a syringe and transporting them in a TACPack (registered trademark). The medium filling test was conducted by conventional methods.

[0137] Syringes (15 syringes: 5 x 10 mL syringes, 5 x 5 mL syringes, 5 x 2.5 mL syringes) filled with medium in the same manner as in Example 1, except that medium was used instead of a cell suspension, were transported from Yokohama to Kobe, and cultured for 14 days. It was confirmed that no bacterial growth occurred in any of the 15 syringes. This confirmed that sterility was ensured in the cell filling process and refrigerated transportation process of the present invention.

[0138] The results of Examples 1 to 4 demonstrate that the use of a cell administration syringe prepared by the method of the present invention as a cell preparation maintains cell viability and is suitable for transporting the cell administration syringe from the institution that prepares it to the medical institution that administers it, without causing contamination by bacteria.

[0139] Example 5. Experimental simulation of refrigerated transportation of adipose-derived MSC spheroids 1. Preparation of spheroid suspension After thawing frozen and stored human adipose-derived MSCs, the spheroids were seeded at a density of 1 x 10 in MEMα (Life Technologies Japan) medium supplemented with 5% hPL, 0.1% gentamicin (Takada Pharmaceutical Co., Ltd.), and 0.1% 0.25 mg / mL amphotericin B (Bristol-Myers Squibb Co., Ltd.). 5 cells / cm 2 Adherent culture was performed at 37°C in a 5% CO2 atmosphere. On the third day of culture, the cells were detached, washed, and collected as single cells. Next, EZSPHERE (registered trademark) SP Microplate 6 well (AGC Technoglass) was used as the culture vessel, and the same medium as the above-mentioned adherent culture was used. The culture volume was 3 mL, and the cell density at the start of culture was 2.7 x 10 6 The collected cells were seeded at 1 x 10 cells / well and cultured in suspension at 37°C in a 5% CO2 atmosphere. After 3 days, the medium was separated by centrifugation to collect spheroids. After washing, the spheroids were transferred to a non-freezing medium for cell preparations (lactated Ringer's solution + dextran 4 (w / v)% + HSA 1.5 (w / v)%) at 1 x 10 cells / well. 6The spheroids were suspended at a concentration of 1000 cells / mL to prepare a spheroid suspension. The cell count was based on the results obtained by dissociating spheroids sampled at the end of the culture into single cells by pipetting with TrypLE™ Select (Life Technologies Japan, Inc.) and counting them using a NucleoCounter NC-200.

[0140] 2. Filling of spheroid suspension The prepared human adipose MSC spheroid suspension was filled into syringes or vials at the following levels. When filling the syringes, the gas was pushed out to ensure no gas remained inside the syringe, and then a Luer cap (Terumo Corporation) was attached. (1) 1 mL of spheroid suspension was filled into a 1 mL syringe: Level (1) (2) 0.5 mL of spheroid suspension was filled into a 1 mL syringe: Level (2) (3) 1 mL of spheroid suspension was filled into a 2 mL vial (Corning): Level (3) (4) 0.5 mL of spheroid suspension was filled into a 2 mL vial (Corning): Level (4)

[0141] 3. Refrigerated Storage of Spheroid Suspensions Under Vibration Conditions After filling, syringes or vials of each level were stored at 4°C for 24 hours while rotating at 10 rpm using a minirotator (AS ONE Corporation) to simulate the vibrations experienced during transportation. The quality of the suspension was then evaluated by observing the cell viability (measured using a NucleoCounter NC-200) and morphology of the cells in each container. Table 1 shows the cell viability for each level after storage, and Figure 6 shows the state of the suspension. The top photograph in Figure 6 shows the state of the spheroids in the suspension at the start of storage, while the bottom four photographs (Level (1), Level (2), Level (3), and Level (4)) show the state of the spheroids in the suspension after storage. As shown in Figure 6, in the suspension filled into vials (levels (3) and (4)), many of the spheroids collapsed, and instead many single cells were observed. This indicated that there was a high possibility that the spheroid shape would not be maintained during transportation, resulting in a decrease in quality. In contrast, surprisingly, no collapse of the spheroids was observed in the suspension filled into syringes (levels (1) and (2)), confirming that transportation was possible while maintaining high quality in terms of both viability and shape retention.

[0142]

[0143] Example 6: Simulated refrigerated transport of human iPS cell spheroids 1. Preparation of spheroid suspension After thawing frozen human iPS cells, iMatrix-511 (Nippi Corporation) was added at 0.5 μg / cm 2 Coated 150cm 2 3000 cells / cm on two dishes 2 Cells were seeded at 100°C and cultured in an adherent culture medium at 37°C in a 5% CO2 atmosphere. StemFit® AK02N (Ajinomoto Co.) was used as the medium. The day of seeding was designated as culture day 0, and the entire medium was replaced on days 1, 4, and 6. The medium volume was 60 mL / dish only during the medium replacement on day 1, and 30 mL / dish otherwise. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM only during cell seeding. On day 7 of culture, cells were detached and suspended in StemFit® AK02N (Ajinomoto Co.) containing a final concentration of 10 μM Y-27632 and 20 μM IWR-1endo (Fujifilm Wako Pure Chemical Industries, Ltd.), and collected as single cells. Next, a single-use bottle (Satake Multimix) was used as the culture vessel, and a VMF reactor (Satake Multimix) was used as the reactor system for controlling the culture. The culture volume was 300 mL, and the cell density at the start of the culture was 1 × 10 5Cells were seeded at a concentration of 1000 cells / mL and culture was initiated. The culture medium used at the time of seeding was StemFit® AK02N (Ajinomoto Co.) supplemented with Y-27632 at a final concentration of 10 μM and IWR-1 endo at a final concentration of 20 μM. During culture, the culture temperature was maintained at 37°C, the gas supply rate was maintained at 0.1 L / min, the carbon dioxide gas supply was automatically controlled to maintain the pH at 7.1-7.2, and the agitation speed was set to 60 mm / sec. The day the culture began was designated culture day 0, and the medium was completely replaced on days 1 and 2. The medium used after the replacement was StemFit® AK02N (Ajinomoto Co., Inc.) supplemented with Y-27632 at a final concentration of 6.9 μM, IWR-1 endo at a final concentration of 20 μM, and LY333531 at a final concentration of 1 μM on the first day of culture, and StemFit® AK02N (Ajinomoto Co., Inc.) supplemented with Y-27632 at a final concentration of 3.0 μM, IWR-1 endo at a final concentration of 20 μM, and LY333531 at a final concentration of 1 μM on the second day of culture. After 3 days, the medium was separated by centrifugation to recover spheroids. 1 x 10 cells were cultured in Ringer's solution (Otsuka Pharmaceutical Co., Ltd.) supplemented with Y-27632 at a final concentration of 10 μM. 7 Spheroids were suspended at a concentration of 1000 cells / mL to prepare a spheroid suspension. The cell count was determined by dissociating spheroids sampled at the end of the culture into single cells using Accutase (Innovative Cell Technology) and pipetting, and then counting the cells using a NucleoCounter NC-200.

[0144] 2. Filling of spheroid suspension The prepared human iPS cell spheroid suspension was filled into syringes or vials at the following levels. When filling the syringes, the gas was pushed out to ensure no gas remained inside the syringe, and then a Luer cap was attached. (1) 1 mL of spheroid suspension was filled into a 1 mL syringe: Level (1) (2) 0.5 mL of spheroid suspension was filled into a 1 mL syringe: Level (2) (3) 1 mL of spheroid suspension was filled into a 1.8 mL vial (Thermo Fisher Scientific): Level (3) (4) 0.5 mL of spheroid suspension was filled into a 1.8 mL vial (Thermo Fisher Scientific): Level (4)

[0145] 3. Refrigerated Storage of Spheroid Suspensions Under Vibration Conditions After filling, syringes or vials of each level were stored at 4°C for 24 hours while rotating at 10 rpm using a mini-rotator (AS ONE Corporation) to simulate the vibrations experienced during transportation. The quality was then evaluated by observing the morphology of the cells in each container. Figure 7 shows the state of the suspensions at each level after storage. The top photograph in Figure 7 shows the state of the spheroids in the suspension at the start of storage, while the bottom four photographs (Levels (1), (2), (3), and (4)) show the state of the spheroids in the suspension after storage. As shown in Figure 7, in the suspensions filled into vials (Levels (3) and (4)), many of the spheroids collapsed, with only a few remaining in a spheroid state. Surprisingly, the morphology of the suspensions filled into syringes (Levels (1) and (2)) remained almost unchanged, confirming that the spheroids could be transported while maintaining their high quality.

[0146] The present invention makes it possible to prepare a cell administration syringe containing a suspension of therapeutic cells without introducing unintended bacteria or contaminants. Furthermore, it makes it possible to transport a cell administration syringe containing a cell suspension from the institution that prepares it to a medical institution that administers the therapeutic cells to a subject without deteriorating the quality of the cells and while maintaining sterility. The inventors have also developed a transportation method that allows therapeutic cells to be reliably administered to the subject by providing the cell administration syringe with cell information and / or information about the subject to be administered. The present invention will be particularly useful in implementing cell therapy in small medical institutions. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for preparing a syringe for cell administration, A filling step in which the cell suspension is filled into a syringe by aspirating the cell suspension through an aspiration means attached to the syringe, A sterile tube attachment step, and The cell suspension includes a removal step of gas from the cell administration syringe via the sterile tube, All of the above steps are carried out under sterile conditions. The above method.

2. A separation step is performed to separate the aspiration means and the sterile tube from the cell administration syringe after the removal step, and The method according to claim 1, further comprising a sealing means attachment step of attaching a sealing means for sealing the aspiration means attachment portion of the cell administration syringe.

3. The method according to claim 1 or 2, wherein the removal step includes pushing a portion of the cell suspension from the cell administration syringe into the sterile tube.

4. The method according to claim 1 or 2, wherein the cell suspension is obtained by suspending cells in a non-freezing storage solution for cell preparations.

5. The aforementioned non-freezing preservation solution for cell preparations contains 0.5 to 12 (w / v)% dextran, The method according to claim 4, wherein the solution comprises a sodium salt, a potassium salt, and a calcium salt.

6. The method according to claim 1 or 2, wherein the cells contained in the cell suspension are mesenchymal cells.

7. The method according to claim 6, wherein the mesenchymal cells include mesenchymal stem cells.

8. A method for transporting cells, Preparation step of preparing a cell administration syringe filled with a cell suspension, A sealing step for sealing the aforementioned cell administration syringe, A packing step in which the sealed container produced in the sealing step is packed together with a coolant in a temperature-controlled container, and a transport step in which the packed temperature-controlled container is transported to a medical institution at a temperature of 0°C to 37°C. Includes, A method of transporting cells, wherein the syringe for cell administration is equipped with cell information and / or information about the subject to be administered the cells.

9. The method according to claim 8, wherein the cell administration syringe filled with the cell suspension is prepared by the method according to claim 1 or 2.

10. The method according to claim 8, further comprising a storage step of storing the cell administration syringe in an absorbent bag before the sealing step.

11. The method according to claim 8, wherein a cell administration syringe is placed horizontally inside the constant temperature container and transported in that state.

12. The method according to claim 8, wherein the institution that prepares the cell administration syringe and the medical institution are different institutions.

13. A method for administering a cell suspension, wherein, after the temperature-controlled container is transported to the medical institution by the method of claim 8, the cell suspension in the cell administration syringe is administered to a subject at the medical institution within 4 days after the preparation step.

14. The method according to claim 13, wherein the viability of cells in the cell suspension at the time of administration to a subject is 70% or more compared to the time of preparation of the cell suspension.