Transplantation device capable of encapsulating cells and use thereof

Abstract

The present application relates to a device for transplantation capable of enclosing cells, a cell medicine in which a transplantation material containing cells is filled in advance in the device for transplantation, and a method for producing the cell medicine, and the like. More specifically, the present application relates to a device for transplantation, a cell medicine in which a transplantation material containing cells is filled in advance in the device for transplantation, and a method for producing the cell medicine, and the like, wherein the device for transplantation is composed of an inner cylinder which is a tubular structure having a fine tube composed of a semi-permeable membrane capable of enclosing the transplantation material containing cells, and an injection portion having an injection port for supplying cells to the fine tube, and an outer cylinder which is a syringe needle or a cannula having a needle tube portion whose inner diameter can accommodate the fine tube, and the inner cylinder is used by being mounted to the outer cylinder.

Description

Technical Field

[0001] Related applications

[0002] This specification contains the contents of the specification of Japanese Patent Application 2023-185161 (filed October 30, 2023), which forms the basis of the priority claim of this application. Technical Field

[0004] The present invention relates to a transplantation apparatus capable of encapsulating cells, a cell-based drug pre-filled with cell-containing transplantation material in the transplantation apparatus, and a method for preparing the cell-based drug. Background Technology

[0005] In regenerative medicine, it is necessary to culture / expand cells in vitro and prepare high-quality cells in sufficient quantities. As a technique for culturing / expanding cells in vitro, a method for culturing / proliferating cells using the lumen of hollow filaments composed of a semi-permeable membrane is known (Patent Document 1). It has been reported that cells that are difficult to proliferate, such as ES cells and iPS cells, can also be cultured in large quantities using hollow filaments (Non-Patent Documents 1 and 2, Patent Document 2). However, in previous reports, methods for supplying cells expanded within hollow filaments to the clinical field in a transplantable state have not been investigated.

[0006] In cell transplantation, methods include application in the form of cell suspensions, application in cell sheets, and application in organoid preparation. Cell suspensions are easy to prepare and can be transplanted with minimal surgical intervention, but controlling the placement of cells at the transplantation site is difficult and cannot cover a wide area. On the other hand, cell sheets can cover a wide area of ​​the transplantation site, but preparation is time-consuming and requires extensive invasive surgical procedures involving large incisions. Organoid preparation requires highly specialized techniques and time.

[0007] The inventors reported that in the transplantation of retinal pigment epithelial cells (RPE), cells can be easily injected under the retina by forming them into band-like aggregates, covering a certain area similarly to a sheet (Patent Document 3). In the transplantation of band-like aggregates, the prepared aggregates need to be retrieved from the device, placed in a culture dish, transported to the transplantation facility, and loaded onto the transplantation device. Therefore, the cell transport range is limited to a walking distance, and loading onto the injection needle requires skilled personnel. Therefore, for practical application, there is a need to develop dosage forms / techniques that facilitate easy transport and loading onto transplantation devices.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: Japanese Patent Application Publication No. 2016-07207

[0011] Patent Document 2: Japanese Patent Application Publication No. 2018-050498

[0012] Patent Document 3: WO2022 / 230977

[0013] Non-patent literature

[0014] Non-patent literature 1: Fujii et al. Cytotechnology. 2020 Apr; 72(2): 227-237

[0015] Non-patent literature 2: Matsushita et al. J Biosci Bioeng. 2019 Oct;128(4):480-486 Summary of the Invention

[0016] The problem the invention aims to solve

[0017] The problem with this invention is to provide means for supplying high-quality cells to the medical field in a transplantable state, or to provide cell preparations that can be used without the need for skilled personnel.

[0018] means for solving problems

[0019] The inventors discovered that by culturing cells in the lumen of a microtube (hollow filament) made of a semipermeable membrane, cells can proliferate and aggregate. The proliferated cells are transported along with the culture container in a state of entering the microtube, and can be easily installed into a transplantation needle for use in the medical field.

[0020] Based on the above knowledge, the present invention provides the following [1] to

[18] .

[0021] [1] A transplantation device consisting of an inner cylinder and an outer cylinder,

[0022] The inner cylinder is a tubular structure comprising: a microtube made of a semi-permeable membrane capable of containing transplantation material containing cells, and an injection port having an injection inlet for supplying cells to the microtube.

[0023] The outer cylinder is an injection needle or cannula, which has a needle tube section, the inner diameter of which is capable of accommodating the microtube.

[0024] The inner cylinder is installed on the outer cylinder for use.

[0025] [2] A transplantation device, which is a tubular structure comprising: a microtube made of a semipermeable membrane capable of containing transplantation material containing cells, and an injection portion having an injection port for supplying cells to the microtube.

[0026] The transplantation device has a structure for installation inside an injection needle or cannula, the injection needle or cannula having a needle tube portion whose inner diameter is capable of accommodating the microtube.

[0027] [3] [1] or [2] describes a transplantation device, wherein the semipermeable membrane is composed of any one or a combination thereof selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene and regenerated cellulose, and derivatives thereof.

[0028] [4] The transplantation device described in any of [1] to [3], wherein the inner diameter of the microtube is 20 μm to 2000 μm.

[0029] [5] The transplantation device described in any of [1] to [4], wherein the pore size of the semipermeable membrane is 0.01 μm to 10 μm.

[0030] The transplantation device described in any of [6] [1] to [5] further comprises one or more of the following (1) to (3):

[0031] (1) The end of the microtube on the opposite side of the injection port can be sealed.

[0032] (2) The injection port can be sealed.

[0033] (3) The injection needle or cannula has a mounting part for mounting to an external surgical device.

[0034] [7] The transplantation device is pre-filled with cell-containing transplantation material and a cell-based drug.

[0035] The transplant device consists of an inner cylinder and an outer cylinder.

[0036] The inner cylinder is a tubular structure comprising: a microtube made of a semi-permeable membrane capable of containing transplantation material containing cells, and an injection port having an injection inlet for supplying cells to the microtube.

[0037] The outer cylinder is an injection needle or cannula, which has a needle tube section, the inner diameter of which is capable of accommodating the microtube.

[0038] The inner cylinder is installed on the outer cylinder for use.

[0039] [8] A cell drug containing cell-derived transplantation material is pre-filled into the transplantation device.

[0040] The transplantation device is a tubular structure comprising: a microtube made of a semipermeable membrane capable of containing transplantation material containing cells, and an injection portion having an injection port for supplying cells to the microtube. The transplantation device has a structure for installation inside an injection needle or cannula, the injection needle or cannula having a needle tube portion whose inner diameter is capable of accommodating the microtube.

[0041] [9] [7] or [8] the cell-based drug, wherein the cell is any one or more of stem cells, progenitor cells, somatic cells, and cells induced by differentiation of stem cells or progenitor cells.

[0042]

[10] [7] to [9] The cell-based drug described in any one of them, wherein the cells comprise retinal pigment epithelial cells.

[0043] The cell-based drug described in any of

[11] [7] to

[10] , wherein at least a portion of the cells constitute an aggregate within the microtube.

[0044]

[12] [7] to

[11] , wherein the semipermeable membrane is composed of any one or a combination thereof selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene and regenerated cellulose, and derivatives thereof.

[0045] The cell drug described in any of

[13] [7] to

[12] , wherein the inner diameter of the microtube is 20 μm to 2000 μm.

[0046] The method for preparing a cell drug described in any of

[14] [7] to

[13] includes the following steps:

[0047] Prepare the transplant device.

[0048] Cell-containing culture medium is injected into the microtube of the tubular structure of the transplantation device, and the tubular structure is placed in the reservoir of a culture container filled with culture medium for culture.

[0049]

[15]

[14] The method described includes the step of sealing the end of the microtube opposite to the injection port after culturing.

[0050] The method described in

[16] ,

[14] or

[15] includes the step of sealing the inlet of the tubular structure after culturing and maintaining it in a reservoir in an anaerobic state.

[0051]

[17] A set of tools, comprising the transplantation apparatus described in any one of [1] to [6], and a culture container,

[0052] The culture container has a reservoir that can be filled with culture medium, and the tubular structure can be configured inside the reservoir for cell culture.

[0053]

[18]

[17] The set of tools described herein, wherein the culture container is capable of holding the tubular structure in an anaerobic state within the reservoir.

[0054] The transplantation device of [1] above uses the transplantation device of [2] as the inner cylinder, and an injection needle or cannula is assembled in the inner cylinder as the outer cylinder. The cell drug of [7] above is assembled in the cell drug of [8] as the injection needle or cannula of the outer cylinder of the transplantation device.

[0055] Therefore, [2], [7], and [8] can also be recorded as follows.

[0056] [2'] The transplantation device comprising the inner cylinder of the transplantation device of [1] is a tubular structure comprising: a microtube made of a semipermeable membrane capable of containing transplantation material containing cells, and an injection section having an injection port for supplying cells to the microtube.

[0057] The transplantation device has a structure for installation inside an injection needle or cannula, the injection needle or cannula having a needle tube portion whose inner diameter is capable of accommodating the microtube.

[0058] [7'] Cellular drugs, wherein the inner cylinder of the transplantation device of [1] is pre-filled with transplantation material containing cells.

[0059] [8'] Cellular drugs, wherein the inner cylinder of the transplantation device of [2] is pre-filled with transplantation material containing cells.

[0060] Invention Effects

[0061] According to the present invention, cells can be proliferated / expanded and aggregated within the lumen of a microtube (hollow filament) composed of a semi-permeable membrane. Since the proliferating cells are transported along with the culture vessel in a state of entry into the microtube, their quality can be maintained for a certain period, and the range of cell transport is expanded compared to the past. Furthermore, since the microtube can be easily installed into a transplantation needle, the skill of a skilled person aspirating cell aggregates from a culture dish or similar vessel and installing them into the transplantation needle is not required. Attached Figure Description

[0062] [ Figure 1 [Illustration] is a diagram showing an example of the inner cylinder (tubular structure) of the transplantation device of the present invention.

[0063] [ Figure 2 [Illustration] is a diagram showing an example of the outer tube (injection needle or cannula) of the transplantation device of the present invention.

[0064] [ Figure 3 [A] is a schematic diagram showing the culture / transport method of the transplantation device of the present invention. A shows a configuration of multiple inner cylinders (tubular structures) in a culture container, and B shows a configuration of one inner cylinder (tubular structure) in a culture container.

[0065] [ Figure 4 [Illustration] is a schematic diagram showing the method of using the transplantation device of the present invention.

[0066] [ Figure 5 [Image caption: Microscopic image of ribbon-like aggregates incubated in a petri dish for 2 days. The freeing of individual retinal pigment epithelial cells from the fragmented aggregates (arrows) tends to be faster than that from the stable aggregates (arrow tips).]

[0067] [ Figure 6 This is a microscopic image taken after the ribbon-like aggregates were placed in a sealed container filled with culture medium and stored under anaerobic conditions for 13 days, then inoculated into a culture dish and cultured for 14 days. The retinal pigment epithelial cells from the stored aggregates moved in the same way as those from the unstored aggregates, and the growth range of each retinal pigment epithelial cell continued to expand during the 14-day culture period.

[0068] [ Figure 7 [A diagram showing the preparation (a), transportation (b), and use (c) of a transplantation device containing retinal pigment epithelial cells.]

[0069] [ Figure 8 Image A is a photograph of the inner tube (tubular structure) of the HepG2 cell line, a human liver cancer cell line. Image B is a microscopic photograph of cells removed from the microtube after culture. The cultured cells form aggregates. Detailed Implementation

[0070] 1. Transplantation device

[0071] 1.1 Transplantation Device I

[0072] In the first embodiment, the transplantation device of the present invention consists of two parts: an inner cylinder and an outer cylinder. Hereinafter, this transplantation device will be referred to as "Transplantation Device I".

[0073] Figure 1 An example of an inner tube is shown (100). The "inner tube" is a tubular structure comprising: a "microtube" (101) made of a semi-permeable membrane capable of containing transplantation material containing cells, and an "injection section" (104) having an injection port (103) for supplying cells to the aforementioned microtube. The injection port may have a sealable structure. The end of the microtube opposite to the injection port may also be sealable (105).

[0074] The "semi-permeable membrane" constituting the inner tube of the microtube is a membrane that is impermeable to cells but allows substances necessary for cell culture / proliferation to pass through the inside of the microtube while allowing unwanted waste to pass through the outside of the microtube. Examples of materials for such semi-permeable membranes include, for example, any one or a combination thereof selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), lactic-glycolic acid copolymer (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene and regenerated cellulose, and derivatives thereof.

[0075] The pore size of the semipermeable membrane is preferably 0.01 μm to 5 μm. By adjusting the pore size of the semipermeable membrane, the permeation of substances inside and outside the microtubes can be controlled, thus maintaining the healthy function of the cells.

[0076] The thickness of the semipermeable membrane is preferably 10 μm to 100 μm, more preferably 10 μm to 50 μm. By adjusting the thickness of the semipermeable membrane, the shape (strength) of the microtube can be maintained while controlling the permeation of substances inside and outside the microtube.

[0077] Microtubes made of semipermeable membranes are, for example, hollow filaments (made of semipermeable membranes).

[0078] The inner diameter of the microtube (hollow filament) is not particularly limited as long as it can encapsulate the cells necessary for transplantation and allow them to proliferate well. For example, the inner diameter of a semipermeable membrane is 20 μm to 2000 μm, preferably 150 μm to 270 μm.

[0079] The length of the microtube (hollow filament) is not particularly limited as long as it can accommodate the cells necessary for transplantation and allow them to proliferate well. It is preferable to be a few mm longer than the length of the needle portion of the outer tube (refer to 404). This is because when the inner tube is installed on the outer tube, the length of the microtube can be adjusted and the closure of the tip can be released by cutting off the tip of the microtube protruding from the needle portion with scissors or the like (404). Furthermore, this is because in the case of subretinal transplantation in ophthalmic surgery, the outer tube may not need to be placed under the retina, and the transplantation of the encapsulated cells can be carried out solely by the invasion of the inner tube.

[0080] The inner diameter and length of the microtube (hollow filament) are appropriately optimized within the aforementioned range, corresponding to the purpose of transplantation and the application site. For example, in ophthalmic surgery, if the diameter of the injection needle used is 25G (0.5mm) and the length is approximately 28mm, then the microtube constituting the inner cylinder, in a manner that allows it to be installed inside the injection needle, has an outer diameter of approximately 70μm to 290μm (31G to 38G) and a length of approximately 33mm to 39mm. In this case, considering the thickness of the semipermeable membrane (10μm to 50μm), the inner diameter of the microtube is approximately 50μm to 270μm.

[0081] At one end of the microtube, there is an "injection section" (104) having an injection port (103) for supplying cells into the aforementioned microtube. The injection port may be a sealable structure, for example, the injection port may be fitted with a plug, cap, or seal that is waterproof and air-barrier (ref 305). As will be described later, since the inner cylinder (tubular structure) can be sealed when placed in the culture container, it is not necessarily required that the inner cylinder itself be sealed.

[0082] The end of the microtube opposite to the injection port (105, 304) can be open or closed. As described above, the closed end can be easily released by cutting off the microtube protruding from the outer tube (needle tube of the injection needle or cannula) after the inner tube is installed on the outer tube (refer to 404).

[0083] The "cells" enclosed within the microtubes are not specifically limited and can be any type of stem cell, progenitor cell, or somatic cell. Examples of stem cells / progenitor cells include pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells); tissue stem cells / progenitor cells such as hematopoietic stem cells / progenitor cells, neural stem cells / progenitor cells, liver stem cells / progenitor cells, pancreatic stem cells / progenitor cells, skin stem cells / progenitor cells, osteochondral stem cells / progenitor cells, and mesenchymal stem cells including adipose stem cells, cardiac stem cells, and dental pulp stem cells. Examples of somatic cells include differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, eye-related cells, adipocytes, and glial cells. Differentiated cells can be induced from pluripotent stem cells or stem cell / progenitor cells.

[0084] Various established ES cell lines can be used as ES cell lines. For example, clinical human ES cell lines provided by the Human ES Cell Research Center of the Institute for Medical Biology, Kyoto University, and human ES cell lines (KhES-1, KhES-1_Crx::Venus, KhES-1_Rx::Venus) provided by RIKEN BRC can be used, but are not limited to these.

[0085] As iPS cells, not only iPS cells induced from the patient's own somatic cells can be used, but also various iPS cell lines can be used. As iPS cell lines, various iPS stored cell lines provided by the Kyoto University iPS Cell Research Foundation, healthy human-derived iPS cell lines provided by RIKEN BRC, KVs09, CLs23, etc., can be used, but are not limited to these.

[0086] There is no particular limitation on the biological species of the cells; selection should be appropriate for the purpose. In the case of human transplantation, primate-derived cells are preferred, especially monkey or human-derived cells, and human-derived cells are even more preferred.

[0087] The transplantation device of the present invention is particularly preferably used for applying cells to the transplantation site using an injection needle or cannula. Examples of such cells include corneal epithelial cells, retinal pigment epithelial cells, neuroretinal cells, conjunctival epithelial cells, limbal epithelial cells, corneal endothelial cells, corneal parenchymal cells, iris parenchymal cells, scleral cells, iris pigment epithelial cells, ciliary body epithelial cells, optic nerve cells, sublimbal fibroblasts, subconjunctival fibroblasts, lacrimal glands, meibomian glands, goblet cells, lens epithelial cells, and eyelid epithelial cells. Preferred eye-related cells are retinal pigment epithelial cells, neuroretinal cells, and lacrimal gland cells; retinal pigment epithelial cells are particularly preferred. In addition to eye-related cells, pancreatic β cells, liver cells, glial cells, and neural progenitor cells are also suitable for the transplantation device of the present invention.

[0088] Figure 2 An example of the outer tube (200) is shown. The "outer tube" is an injection needle or cannula having a needle portion (201) with an inner diameter capable of accommodating the aforementioned microtube (101). The diameter (outer diameter) of the needle portion of the outer tube (injection needle or cannula) corresponds to the purpose of the surgery and the choice of the application site. The tip (202) of the injection needle or cannula can be straight or curved.

[0089] The injection needle or cannula may have a mounting portion for attachment to an external surgical device. Examples of such external surgical devices include syringes, Constellation VFQ, etc.

[0090] The inner and outer cylinders are provided as a kit, and the inner cylinder is attached to the outer cylinder for use in the medical setting (see reference). Figure 4 Therefore, the inner cylinder (tubular structure) has a structure that allows it to be installed inside the outer cylinder (injection needle or cannula) for use.

[0091] 1.2 Transplantation Device II

[0092] In the second embodiment, the transplantation device of the present invention is a tubular structure having the same structure as the inner cylinder (100) described above, and uses a commercially available injection needle or cannula as the outer cylinder, which is installed inside for use (see reference). Figure 1 Hereinafter, this transplantation device will be referred to as "Transplantation Device II".

[0093] The aforementioned transplantation device II is a tubular structure comprising: a microtube (101) made of a semi-permeable membrane capable of containing transplantation material (102) containing cells, and an injection portion (104) having an injection port (103) for supplying cells to the aforementioned microtube. The transplantation device has a structure for use inside an injection needle or cannula (e.g., MedOne0 Poly Tip Cannula 25g / 31g, etc.), which has a needle tube portion capable of accommodating the inner diameter of the aforementioned microtube.

[0094] As for structures used for installation inside injection needles or cannulas, tools known in the art can be used, such as structures where the injection portion of a tubular structure is closely connected and / or fitted to the base of the injection needle or cannula. Furthermore, the structure of the microtube constituting the transplantation device is as described in 1.1 above.

[0095] 2. Cell-mediated drugs

[0096] The present invention provides a cell drug that is pre-filled with cell-containing transplantation material in a transplantation device.

[0097] 2.1 Cell Drug I

[0098] In the first embodiment, cell-containing transplantation material (102) is pre-filled into the transplantation device I. The transplantation device I, as described above, consists of an inner cylinder (100) and an outer cylinder (200). The inner cylinder is a tubular structure that includes a microtube (101) made of a semi-permeable membrane capable of containing cell-containing transplantation material, and an injection portion (104) having an injection port (103) for supplying cells to the microtube. The outer cylinder is an injection needle or cannula having a needle portion (201) capable of accommodating the inner diameter of the microtube. The inner cylinder is installed on the outer cylinder for use.

[0099] 2.2 Cell Drug II

[0100] In the second embodiment, cell-containing transplantation material (102) is pre-filled inside the microtube of the transplantation device II. The transplantation device II, as described above, is a tubular structure comprising: a microtube (101) made of a semi-permeable membrane capable of containing cell-containing transplantation material, and an injection portion (104) having an injection port (103) for supplying cells to the aforementioned microtube. The transplantation device has a structure for installation inside an injection needle or cannula, which has a needle tube portion capable of accommodating the inner diameter of the aforementioned microtube.

[0101] The cells used in the cell-based drug of the present invention are the cells described in 1 as "cells enclosed in microtubes". At least a portion of the cells within the microtubes may form aggregates. In the case of adhesive cells, cells cultured / proliferated within microtubes composed of semi-permeable membranes form aggregates. In the case of retinal pigment epithelial cells, the aggregates formed are similar to the band-like aggregates prepared by the device described in WO2022 / 230977, but also contain smaller flakes. The inventors have confirmed that such flakes, like the band-like aggregates, may also be highly effective as transplantation materials (see experimental examples described later).

[0102] The cell-mediated drugs of the present invention may also contain pharmacologically permissible carriers or media, specifically, sterile water or physiological saline, culture medium, physiological buffers such as PBS, preservatives, surfactants, stabilizers, excipients, preservatives, binders, reducing agents, and isotonic agents. If necessary, cryopreservatives may be added for cryopreservation, and the drugs can be thawed before use.

[0103] 3. Preparation methods of cell-based drugs

[0104] Figure 3 This diagram shows a schematic of the culture vessel used in the preparation of the cell drug. The cell drug of the present invention can be prepared by injecting cell-containing culture medium into a microtube (301) of an inner cylinder (tubular structure), and then culturing the aforementioned tubular structure in a reservoir (303) of a culture vessel (302) filled with culture medium. The number of inner cylinders disposed in the culture vessel is not particularly limited, and can be one or more. If a large number of cell drugs can be readily available at the medical site in the state of being placed in culture vessels, several of them can be used for each surgery.

[0105] Cell culture is performed by placing the aforementioned culture container (302) in a culture apparatus. The culture apparatus may have known tools required for cell culture, such as means for regulating culture temperature, means for regulating oxygen and carbon dioxide concentrations, means for exchanging culture medium, and means for supplying additional components.

[0106] The culture medium and culture conditions can be appropriately determined according to the cells. The cell density within the microtube is 5 x 10⁻⁶. 7 10 cells / mL or higher, preferably 5 x 10 7 Cells / mL ~ 1x10 8 10 cells / mL, more preferably 7.5 x 10 7 Cells / mL ~ 1x10 8 Approximately one cell per mL.

[0107] After cultivation, the end (304) of the microtube opposite to the injection port can be sealed. Sealing methods can include sealing, welding, or using a hollow wire as a blind end.

[0108] The inner cylinder (tubular structure) is preferably sealable when placed inside the culture container. This preserves the quality of the filling transplant material. For example, the inner cylinder (tubular structure) can be sealed by making the culture container a sealable container.

[0109] The culture container is detachable from the culture apparatus and can be removed after cell culture is complete, allowing it to be carried in a tubular configuration. Therefore, cell-based drugs can be provided to medical institutions without freezing, in a state filled with transplantation material.

[0110] 4. Methods for transporting cell-based drugs

[0111] Cellular drugs can be transported in a state where tubular structures are configured within culture vessels. Furthermore, in anaerobic transport, the quality of cells encapsulated within the tubular structures (microtubules) can be maintained for approximately 30 days.

[0112] This specification also discloses a complete set of tools used in the preparation and handling of cell drugs. The aforementioned complete set of tools includes, as essential components, a transplantation device I or a transplantation device II and a culture container (302). As described above, the culture container has a reservoir capable of being filled with culture medium, and a tubular structure constituting the transplantation device can be placed within the reservoir for cell culture. Furthermore, it is preferable that the tubular structure can be maintained in an anaerobic state within the reservoir. Details of the culture container are described in "3. Method for Preparation of Cell Drugs".

[0113] The cell-derived drug (transplantation material) of the present invention can also be frozen while filled within a tubular structure (inner cylinder). In the frozen case, the cultured tubular structure is removed from the culture container and transported in a frozen state. In this case, the cell-derived drug I can be transported as a complete set of tools, consisting of the outer and inner cylinders.

[0114] 5. Methods of using cell-based drugs

[0115] Figure 4 This invention provides an overview of the method of using the cell-based drug. The cell-based drug of the present invention is used by mounting a tubular structure (401) as an inner cylinder onto an injection needle or cannula (402) as an outer cylinder. The outer cylinder is appropriately mounted onto an external surgical device such as an injection cartridge (403). The length of the microtube in the tubular structure is designed to be longer than the length of the needle tube portion of the injection needle or cannula, and during installation, the tip (404) of the microtube in the tubular structure protrudes from the tip of the injection needle or cannula. Therefore, the protruding tip (including the closed portion) of the microtube can be cut off with scissors or the like, pushed out from the injection needle or cannula, and applied to the transplantation site (see reference). Figure 4 ).

[0116] 6. Cellular drugs containing retinal pigment epithelial cells

[0117] As a preferred example of the cell drug of the present invention, a cell drug comprising retinal pigment epithelial cells is described below.

[0118] Retinal pigment epithelial (RPE) cells refer to the epithelial cells and their progenitor cells that make up the retinal pigment epithelium. RPE cells can be isolated from the patient or induced from stem cells such as pluripotent stem cells. The number of RPE cells obtained from the patient is limited, and proliferation and expansion of RPE cells are required for transplantation. In this invention, by culturing / expanding cells in a microtube composed of a semipermeable membrane, RPE cells can be pre-filled into a cell drug for transplantation.

[0119] The culture method for RPE cells is basically in accordance with WO2022 / 230977. As the basal medium, media commonly used in animal cell culture can be used. For example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, or mixtures thereof can be used.

[0120] The culture medium can be serum-containing or serum-free. Serum-free media may contain serum substitutes. Serum substitutes can be commercially available products; for example, Knockout can be used. TM Serum Replacement (KSR), Chemically-defined Lipid concentrated (manufactured by Life Technologies), Glutamax TM (Made by Life Technologies), B27 (Made by Life Technologies), N2 supplement (Made by Life Technologies), ITS supplement (Made by Life Technologies).

[0121] Serum-free culture media may contain appropriate amounts of fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, etc.

[0122] ROCK inhibitors can be added to the culture medium. Examples of ROCK inhibitors include Y-27632 dihydrochloride, Y-27632, Fasudil hydrochloride, Chroman 1, SLx-2119, HSD1590, GSK269962A hydrochloride, Exoenzyme C3, Clostridium botulinum, Ripasudil, Afuresertib, Thiazovin, GSK269962A, RKI-1447, Y-33075, GSK429286A, AT13148, H-1152 dihydrochloride, Y-33075 dihydrochloride, LX7101, SAR407899, ROCK-IN-2, Afuresertib hydrochloride, Hydroxyfasudil, GSK180736A, BDP5290, and SR-3677. The following are recommended supplementary ingredients: CCG-222740, CMPD101, Rho-Kinase-IN-1, SAR407899 hydrochloride, ROCK inhibitor-2, ZINC00881524, H-1152, hydroxyfasudil hydrochloride, fasudil, ROCK2-IN-2, Verosudil, SB-772077B dihydrochloride, GSK-25, CRT0066854 hydrochloride, Ripasudil free base, ROCK-IN-1, etc., with Y-27632 dihydrochloride and Y-27632 being preferred. The concentration of ROCK inhibitor in the culture medium is typically 0 μM to 20 μM, preferably 2 μM to 10 μM. Furthermore, ROCK inhibitor is preferably added during RPE cell aggregation; it is not essential in the post-culture formulation.

[0123] There is no particular limit to the density of cultured RPE cells, usually 2.5 x 10⁻⁶. 3 10 cells / mL or higher, preferably 2.5 x 10⁻⁶. 3 Cells / mL ~ 5 x 10 5 cells / mL, more preferably 1x10⁻¹⁰ 5 Cells / mL ~ 2 x 10 5 Cells / mL.

[0124] There is no particular limitation on the culture time of RPE cells, which is usually 1 to 30 days, preferably 2 to 7 days.

[0125] The culture temperature is, for example, from about 30°C to about 40°C, preferably about 37°C. Furthermore, the CO2 concentration is, for example, from about 1% to about 10%, preferably about 5%.

[0126] For RPE cells cultured / expanded within microtubes, at least a portion of them form aggregates resembling ribbon-like aggregates. Using the transplantation device of this invention, RPE cells can be transported to medical facilities while maintaining their aggregate morphology, and easily fitted into injection needles or cannulas for use.

[0127] Cellular drugs containing RPE cells can be used in the treatment of patients with diseases based on retinal pigment epithelium (RPE) damage, or with retinal pigment epithelium atrophy or injury. Examples of diseases based on RPE damage, or with retinal pigment epithelium atrophy or injury, include, for example, age-related macular degeneration, retinitis pigmentosa, and crystalline retinopathy, as well as retinal pigment epithelium tears, macular dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular holes, degenerative myopia, and traumatic macular diseases.

[0128] Example

[0129] The present invention will be specifically described below through examples, but the present invention is not limited to these examples.

[0130] Experimental Example 1:

[0131] Following the existing report (WO2022 / 230977), band-like aggregates of retinal pigment epithelial cells were prepared. These band-like aggregates were then seeded into culture dishes and cultured for 2 days.

[0132] Culture was performed according to WO2022 / 230977, using RPE maintenance medium containing the ROCK inhibitor (Y-27632). The "RPE maintenance medium" consisted of DMEM-low glucose (Sigma-Aldrich), 30% F-12 (Sigma-Aldrich), 2% L-glutamine solution (Sigma-Aldrich), and 2% B-27. TM Supplement (50X) (Thermo Fisher Scientific Inc.), and gentamicin solution (Sigma-Aldrich).

[0133] The freeing of individual retinal pigment epithelial cells from fragmented aggregates (arrows) tends to be faster than the freeing of cells from stable aggregates (arrow tips). Figure 5 This suggests that even if the band-like aggregates are broken down into smaller pieces, they are still highly likely to be effective as transplant materials.

[0134] Experimental Example 2:

[0135] Following a previous report (WO2022 / 230977), band aggregates of retinal pigment epithelial cells were prepared and placed in containers filled with RPE maintenance medium and sealed. After 13 days of anaerobic storage at 25°C, they were inoculated into culture dishes and cultured at 37°C with 5% CO2 for 14 days. Retinal epithelial cells from both preserved and unpreserved band aggregates showed similar migration patterns, and the growth range of individual retinal pigment epithelial cells continued to expand during the 14-day culture period. Figure 6 This confirms that RPE cells can be preserved in culture medium under anaerobic conditions for at least 13 days and can be maintained / transported under anaerobic and non-dry conditions.

[0136] Example 1:

[0137] Using a semi-permeable membrane permeable to cell culture medium, a tubular structure with the same morphology (33 mm in length, 31 gauge in outer diameter) as the inner tube of the cannula (PolyTip Cannula; MedOne #3218, etc.) used when transplanting band-like aggregates of retinal pigment epithelial (RPE) cells under the retina was prepared. Within its lumen, retinal pigment epithelial cells were cultured to form aggregates (see reference). Figure 7 a) Culture was carried out in accordance with WO2022 / 230977, using RPE maintenance medium containing the ROCK inhibitor (Y-27632).

[0138] For cultured RPE cells, the aforementioned tubular structure was placed in a container filled with culture medium and transported in a sealed manner (see reference). Figure 7 (b) As shown in Experiment 1, the ribbon-like aggregates can be stored in culture medium under anaerobic conditions for at least 13 days and can be transported and provided to hospital facilities both domestically and internationally.

[0139] For the use of drugs containing RPE cells, the aforementioned tubular structure is inserted into an injection needle (28 mm in length, 25 gauge in outer diameter) that is equivalent to the outer tube of a subretinal transplant cannula (PolyTip Cannula; MedOne #3218, etc.), and used in the same manner as the subretinal transplant cannula with an outer tube (PolyTip Cannula; MedOne #3218, etc.) (see reference). Figure 7 c). The outer tube can be either straight or curved at the front, depending on the surgeon's preference.

[0140] The preparation, transportation, and use procedures described above are briefly summarized below.

[0141] preparation:

[0142] 1) Retinal pigment epithelial cells prepared using the same method as WO2022 / 230977 were suspended in RPE maintenance medium to make a concentration of 1 x 10⁻⁶ cells / mL. 8 Cell density per ml.

[0143] 2) Containing 2 x 10 retinal pigment epithelial cells 5 Two μl of cell suspension was injected into a tubular structure made of a semi-permeable membrane with a length of 35 mm and an outer diameter of 31–38.

[0144] 3) Close the end of the aforementioned tubular structure opposite to the injection port.

[0145] 4) Place the aforementioned tubular structure into a container filled with RPE maintenance medium and incubate at 37°C and 5% CO2 for 24–48 hours.

[0146] transportation:

[0147] 1) Fill the tubular structure with RPE maintenance medium to the top and seal it with a waterproof and air-barrier seal.

[0148] 2) Place the closed tubular structure into a container filled with RPE-maintained culture medium and seal it for transport in an anaerobic environment.

[0149] use:

[0150] 1) Open the tubular structure transported in an anaerobic state within a container filled with RPE maintenance medium and insert the outer barrel of a 25G ophthalmic transplantation needle.

[0151] 2) Remove the seal at the top of the tubular structure and install the composite structure of the transplantation needle (outer cylinder) and the tubular structure (inner cylinder) into the ophthalmic transplantation microsyringe.

[0152] 3) Cut open the closed anterior end of the tubular structure, push the RPE cells (aggregates) out of the tubular structure, and transplant them under the retina.

[0153] The combination of the inner tube containing the cells and the outer tube of the injection needle enables the long-term transport of difficult-to-handle cells, such as ribbon-like aggregates, reducing the workload of procedures immediately preceding transplantation in transplantation facilities. This technique can be applied not only to RPE cells but also to other cells that are difficult to transport or that require filling into transplantation devices. Consequently, high-quality cells in a transplantable state can be provided to more medical facilities.

[0154] Example 2:

[0155] Cells (HepG2, a human liver cancer cell line, 5 x 10⁻⁴) were injected from the injection site (104). 7 Cells / mL ~ 1x108 A suspension of cells / mL flows into the microtube (101) of the inner tube (100). Subsequently, the end (105) of the microtube opposite the injection port is sealed with a hemostatic clamp (see reference). Figure 8 A) The microtube (101) is placed into the reservoir (302) of the culture container (300) filled with culture medium (303) along the long axis and the direction of gravity. The culture container is then placed in the culture apparatus for cell culture. After 24 hours of culture, the hemostatic clamp is removed, and the microtube (101, 301) is cut with scissors at a point approximately 1 mm from the connection point between the clamp and the microtube (101) toward the injection port. The culture medium is transported through the injection section (104), and the cell aggregates are removed from the ends (105, 304) of the microtube. The removed cells form aggregates ( Figure 8 B).

[0156] All prior art documents referenced in this specification are incorporated herein by reference.

[0157] Explanation of symbols

[0158] 100: Inner cylinder (tubular structure)

[0159] 101: Microtubes composed of semi-permeable membranes

[0160] 102: Transplantation materials containing cells,

[0161] 103: Injection port

[0162] 104: Injection section

[0163] 105: Closure of the ends of microtubes

[0164] 200: Outer tube (injection needle or cannula)

[0165] 201: Syringe section

[0166] 202: The tip of the injection needle or cannula,

[0167] 300: Culture containers with inner cylinders (tubular structures);

[0168] 301: Inner cylinder (tubular structure)

[0169] 302: Culture container,

[0170] 303: Storage container (culture medium filling)

[0171] 304: Sealing of the ends of microtubes

[0172] 305: Sealing tool for the injection port (plug / seal / cap)

[0173] 400: An outer cylinder (injection needle or cannula) with an inner cylinder (tubular structure).

[0174] 401: Inner cylinder (tubular structure)

[0175] 402: Outer tube (injection needle or cannula)

[0176] 403: Syringe (external surgical device)

[0177] 404: Front end of the inner cylinder (tubular structure)

Claims

1. A transplantation device consisting of an inner cylinder and an outer cylinder, The inner cylinder is a tubular structure comprising: a microtube made of a semi-permeable membrane capable of containing transplantation material containing cells, and an injection port having an injection inlet for supplying cells to the microtube. The outer cylinder is an injection needle or cannula, which has a needle tube section, the inner diameter of which is capable of accommodating the microtube. The inner cylinder is installed on the outer cylinder for use.

2. A transplantation device, which is a tubular structure comprising: a microtube made of a semi-permeable membrane capable of containing transplantation material containing cells, and an injection portion having an injection port for supplying cells to the microtube. The transplantation device has a structure for installation inside an injection needle or cannula, the injection needle or cannula having a needle tube portion whose inner diameter is capable of accommodating the microtube.

3. The transplantation device according to claim 1 or 2, wherein, The semipermeable membrane is composed of any one or a combination thereof selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene and regenerated cellulose, and derivatives thereof.

4. The transplantation device according to claim 1 or 2, wherein, The inner diameter of the microtube is 20μm to 2000μm.

5. The transplantation device according to claim 1 or 2, wherein, The pore size of the semipermeable membrane is 0.01 μm to 10 μm.

6. The transplantation device according to claim 1 or 2, further comprising one or more of the following (1) to (3): (1) The end of the microtube on the opposite side of the injection port can be sealed. (2) The injection port can be sealed. (3) The injection needle or cannula has a mounting part for mounting to an external surgical device.

7. A cell-based drug containing cell-containing transplantation material is pre-filled into the transplantation device. The transplant device consists of an inner cylinder and an outer cylinder. The inner cylinder is a tubular structure comprising: a microtube made of a semi-permeable membrane capable of containing transplantation material containing cells, and an injection port having an injection inlet for supplying cells to the microtube. The outer cylinder is an injection needle or cannula, which has a needle tube section, the inner diameter of which is capable of accommodating the microtube. The inner cylinder is installed on the outer cylinder for use.

8. Cell-based drugs pre-filled with cell-containing transplantation material in the transplantation device. The transplantation device is a tubular structure comprising: a microtube made of a semipermeable membrane capable of containing transplantation material containing cells, and an injection portion having an injection port for supplying cells to the microtube. The transplantation device has a structure for installation inside an injection needle or cannula, the injection needle or cannula having a needle tube portion, the inner diameter of which is capable of accommodating the microtube.

9. The cell-based drug according to claim 7 or 8, wherein, The cell is any one or more of stem cells, progenitor cells, somatic cells, and cells induced from the differentiation of stem cells or progenitor cells.

10. The cell-based drug according to claim 7 or 8, wherein, The cells include retinal pigment epithelial cells.

11. The cell-based drug according to claim 7 or 8, wherein, Within the microtubes, at least a portion of the cells form aggregates.

12. The cell-based drug according to claim 7 or 8, wherein, The semipermeable membrane is composed of any one or a combination thereof selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), lactic acid-glycolic acid copolymer (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acid, polyorthoester, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene and regenerated cellulose, and derivatives thereof.

13. The cell-based drug according to claim 7 or 8, wherein, The inner diameter of the microtube is 20μm to 2000μm.

14. A method for preparing the cell-based drug according to claim 7 or 8, comprising the following steps: Prepare the transplant device. A cell-containing culture medium is injected into the microtube of the tubular structure of the transplantation device, and the tubular structure is placed in a reservoir of a culture container filled with culture medium for culture.

15. The method of claim 14, further comprising the step of sealing the end of the microtube opposite to the injection port after culturing.

16. The method of claim 14, further comprising the step of sealing the inlet of the tubular structure after cultivation, and maintaining it in a reservoir in an anaerobic state.

17. A set of tools comprising the transplantation apparatus as described in claim 1 or 2, and a culture container. The culture container has a reservoir that can be filled with culture medium, and the tubular structure can be configured inside the reservoir for cell culture.

18. The tool kit of claim 17, wherein, The culture container is capable of maintaining the tubular structure in an anaerobic state within the container.

Images