Transplantation device capable of encapsulating cells therein and use thereof

Abstract

The present invention pertains to: a transplantation device capable of encapsulating cells therein; a cell medicine obtained by loading, in advance, a transplantation material containing cells into the transplantation device; a method for producing the cell medicine; etc. More specifically, the present invention pertains to a transplantation device comprising an inner tube and an outer tube. The inner tube is a tubular structural object provided with: a microtube made of a semipermeable membrane that can encapsulate therein a transplantation material containing cells; and an injection part having an injection opening through which cells are supplied into the microtube. The outer tube is an injection needle or cannula provided with a needle tube part having an inner diameter in which the microtube can be accommodated. The inner tube is attached to the outer tube and used. The present invention also pertains to: a cell medicine obtained by loading, in advance, a transplantation material containing cells into the transplantation device; a method for producing the cell medicine; etc.

Description

Transplant device capable of containing cells and its use

[0001] Related Applications: This specification includes the contents described in the specification of Japanese Patent Application No. 2023-185161 (filed October 30, 2023), which is the basis of the priority of this application. Technical Field: The present invention relates to a transplant device capable of containing cells, a cellular medicine in which the transplant device is pre-filled with a transplant material containing cells, and a method for producing the cellular medicine.

[0002] In regenerative medicine, it is necessary to culture and expand cells outside of the body and prepare sufficient quantities of high-quality cells. One known technique for culturing and expanding cells outside of the body is to use the lumen of hollow fibers, which are made up of semipermeable membranes, to culture and grow cells (Patent Document 1). It has been reported that even cells that are difficult to grow, such as ES cells and iPS cells, can be mass-cultured using hollow fibers (Non-Patent Documents 1 and 2, Patent Document 2). However, previous reports have not explored a method for supplying cells expanded inside hollow fibers to clinical settings in a state suitable for transplantation.

[0003] Cell transplantation can be performed using a cell suspension, a cell sheet, or organoids. Cell suspensions are easy to prepare and can be transplanted with minimal surgical intervention, but it is difficult to control the placement of cells at the transplant site and they cannot cover a large area. Cell sheets, on the other hand, can cover a large area, but they take time to prepare and require invasive surgical procedures involving large incisions. Organoid production requires advanced technology and time.

[0004] The inventors have reported that in the transplantation of retinal pigment epithelial cells (RPE), cells formed into string-like aggregates can be easily injected subretinally and can cover a certain area, similar to a sheet (Patent Document 3). Transplantation of string-like aggregates requires retinal cells to be collected from the device, placed in a petri dish, transported to a transplant facility, and loaded onto a transplantation device. Therefore, the transport range of the cells is limited to within walking distance, and loading them onto a transplantation needle requires the skills of an experienced technician. Therefore, for practical application, the development of a formulation and technology that facilitates transport and loading onto a transplantation device was required.

[0005] Patent Publication No. 2016-07207, Patent Publication No. 2018-050498, WO2022 / 230977

[0006] Fujii et al. Cytotechnology. 2020 Apr; 72(2): 227-237Matsushita et al. J Biosci Bioeng. 2019 Oct; 128(4): 480-486

[0007] The object of the present invention is to provide a means for supplying high-quality cells in a state suitable for transplantation to medical settings, or to provide a cell preparation that can be used without the need for skilled artisanal techniques.

[0008] The inventors discovered that by culturing cells in the lumen of microtubes (hollow fibers) made of a semipermeable membrane, the cells can be grown and obtained as aggregates, and that the grown cells can be transported together with the culture vessel while still encased in the microtubes, and can be easily attached to a transplant injection needle for use in medical settings.

[0009] The present invention is based on the above findings and provides the following [1] to

[18] . [1] A transplantation device comprising an inner tube and an outer tube, wherein the inner tube is a tubular structure including a microtube made of a semipermeable membrane capable of containing a transplant material containing cells, and an injection part having an injection port for supplying cells to the microtube, and the outer tube is an injection needle or cannula having a needle part with an inner diameter large enough to accommodate the microtube, the transplantation device being used by attaching the inner tube to the outer tube. [2] A transplantation device having a tubular structure including a microtube made of a semipermeable membrane capable of containing a transplant material containing cells, and an injection part having an injection port for supplying cells to the microtube, the transplantation device being structured to be attached to the inside of an injection needle or cannula having a needle part with an inner diameter large enough to accommodate the microtube. [3] The implantable device according to [1] or [2], wherein the semipermeable membrane is composed of any one or combination 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, polyvinylimidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acids, polyorthoesters, 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 transplant device according to any one of [1] to [3], wherein the inner diameter of the microtubules is 20 μm to 2000 μm. [5] The transplant device according to any one of [1] to [4], wherein the pore size of the semipermeable membrane is 0.01 μm to 10 μm.[6] The transplantation device according to any one of [1] to [5], further comprising one or more of the following (1) to (3): (1) the end of the microtubule opposite to the injection port is sealable, (2) the injection port is sealable, (3) the injection needle or cannula has a portion for attachment to an external surgical device. [7] A cellular medicine in which a transplantation material containing cells is pre-filled in a transplantation device, wherein the transplantation device is composed of an inner tube and an outer tube, the inner tube is a tubular structure comprising microtubules made of a semipermeable membrane capable of containing the transplantation material containing cells, and an injection portion having an injection port for supplying cells to the microtubules, the outer tube is an injection needle or cannula having a needle portion with an inner diameter capable of accommodating the microtubules, and the cellular medicine is used by attaching the inner tube to the outer tube. [8] A cell medicine comprising a transplantation device pre-filled with a transplant material containing cells, wherein the transplantation device is a tubular structure comprising a microtubule made of a semipermeable membrane capable of containing the transplant material containing cells, and an injection part having an injection port for supplying cells to the microtubule, and is structured for use by being attached to the inside of an injection needle or cannula having a needle part with an inner diameter capable of accommodating the microtubule. [9] The cell medicine described in [7] or [8], wherein the cells are any one or more selected from stem cells, progenitor cells, somatic cells, and cells induced to differentiate from stem cells or progenitor cells.

[10] The cell medicine described in any of [7] to [9], wherein the cells include retinal pigment epithelial cells.

[11] The cell medicine described in any of [7] to

[10] , wherein at least a portion of the cells form aggregates within the microtubule.

[12] The cell medicine according to any one of [7] to

[11] , wherein the semipermeable membrane is composed of any one or a combination of materials 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, polyvinylimidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acids, polyorthoesters, polyacetal, polycyanoacrylate, polytetrafluoroethylene (PTFE), biodegradable polyurethane, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene, regenerated cellulose, and derivatives thereof.

[13] The cellular medicine according to any one of [7] to

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

[14] A method for producing a cellular medicine according to any one of [7] to

[13] , comprising the steps of preparing a transplantation device, injecting a culture medium containing cells into the microtubules of a tubular structure of the transplantation device, and placing the tubular structure in a reservoir of a culture vessel filled with the culture medium to perform culture.

[15] The method according to

[14] , comprising the step of sealing the end of the microtubule opposite the inlet after culture.

[16] The method according to

[14] or

[15] , comprising the step of sealing the inlet of the tubular structure after culture and maintaining the cells in an oxygen-free state in the reservoir.

[17] A kit comprising the transplantation device according to any one of [1] to [6] and a culture vessel, wherein the culture vessel has a reservoir that can be filled with a culture medium, and the tubular structure can be placed in the reservoir to perform cell culture.

[18] The kit according to

[17] , wherein the culture vessel is capable of holding the tubular structure in an oxygen-free state within a reservoir.

[0010] The transplant device of [1] above uses the transplant device of [2] as an inner tube, and combines the inner tube with an injection needle or cannula, which is an outer tube. The cellular medicine of [7] above combines the cell medicine of [8] with an injection needle or cannula, which is an outer tube of the transplant device. Therefore, [2], [7], and [8] can also be described as follows. [2'] A transplant device constituting the inner tube of the transplant device of [1], which is a tubular structure comprising a microtube made of a semipermeable membrane capable of containing a transplant material containing cells, and an injection part having an injection port for supplying cells to the microtube, and which has a structure for being attached to the inside of an injection needle or cannula, which has a needle part with an inner diameter large enough to accommodate the microtube. [7'] A cellular medicine obtained by pre-filling the inner tube of the transplant device of [1] with a transplant material containing cells. [8'] A cellular medicine obtained by pre-filling the inner tube of the transplant device of [2] with a transplant material containing cells.

[0011] According to the present invention, cells can be grown and expanded in the lumen of microtubes (hollow fibers) made of a semipermeable membrane, and obtained as aggregates. The grown cells can be transported in a culture vessel while still encased in the microtubes, allowing the quality to be maintained for a certain period of time, thereby expanding the range of cell transport compared to conventional methods. Furthermore, the microtubes can be easily attached to a transplantation needle, eliminating the need for skilled labor to aspirate cell aggregates from a petri dish or the like and attach them to a transplantation needle.

[0012] 1 shows an example of an inner tube (tubular structure) for a transplant device of the present invention. 2 shows an example of an outer tube (injection needle or cannula) for a transplant device of the present invention. 3 shows an outline of a method for culturing and transporting a transplant device of the present invention. 4 shows an embodiment in which multiple inner tubes (tubular structures) are arranged in a culture vessel, and 5 shows an embodiment in which a single inner tube (tubular structure) is arranged in a culture vessel. 6 shows an outline of a method for using a transplant device of the present invention. 7 shows microscopic images of string-like aggregates seeded on a culture dish and cultured for two days. 8 shows that the release of individual retinal pigment epithelial cells from fragmented aggregates (arrows) tended to be faster than the release from solid aggregates (arrowheads). 9 shows microscopic images of string-like aggregates sealed in a container filled with culture medium and stored in an anoxic environment for 13 days, then seeded on a culture dish and cultured for 14 days. 10 shows that retinal pigment epithelial cells migrated from the stored string-like aggregates, just as they did from the unstored string-like aggregates. 11 shows that the engraftment area of ​​individual retinal pigment epithelial cells continued to expand throughout the 14-day culture period. This figure shows an outline of the manufacturing (a), transportation (b), and use (c) of a transplant device containing retinal pigment epithelial cells. A is a photograph of the inner tube (tubular structure) filled with the human hepatoma-derived cell line HepG2. B is a micrograph of cells removed from the microtubules after culturing. The cells have formed aggregates after culturing.

[0013] 1. Implant Device 1.1 Implant Device I In a first embodiment, the implant device of the present invention is composed of two components: an inner tube and an outer tube. Hereinafter, this implant device will be referred to as "implant device I."

[0014] Figure 1 shows an example of an inner tube (100). The "inner tube" is a tubular structure comprising a "microtube" (101) made of a semipermeable membrane capable of containing transplant material containing cells, and an "injection section" (104) having an inlet (103) for supplying cells to the microtube. The inlet may have a sealable structure. The end of the microtube opposite the inlet may be sealable (105).

[0015] The "semipermeable membrane" that makes up the inner tube's microtubules is a membrane that does not allow cells to pass through, but allows substances necessary for cell culture and proliferation to pass into the interior of the microtubules and unnecessary waste products to pass out of the microtubules. Examples of materials for such semipermeable membranes include 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, polyvinylimidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acids, 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, as well as derivatives thereof, or combinations thereof.

[0016] 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 into and out of the microtubules can be controlled, and the cells can be well maintained.

[0017] 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, it is possible to control the permeation of substances into and out of the microtubules and to control the shape maintenance (strength) of the microtubules.

[0018] The microtube made of a semipermeable membrane is, for example, a hollow fiber (made of a semipermeable membrane).

[0019] The inner diameter of the microtubes (hollow fibers) is not particularly limited as long as it can contain the cells necessary for transplantation and allow them to proliferate well. For example, the inner diameter of the semipermeable membrane is 20 μm to 2000 μm, preferably 150 μm to 270 μm.

[0020] The length of the microtubes (hollow fibers) is not particularly limited as long as they contain the cells necessary for transplantation and allow their good proliferation, but it is preferable that they be several millimeters longer than the length of the needle part of the outer tube (see 404). This is because, when using the inner tube attached to the outer tube, the tip of the microtube that protrudes from the needle part can be cut with scissors or the like to adjust the length of the microtube and release the seal at the tip (404). Furthermore, in the case of subretinal transplantation in ophthalmic surgery, the cells contained therein can be transplanted by simply inserting the inner tube without inserting the outer tube under the retina.

[0021] The inner diameter and length of the microtube (hollow fiber) are appropriately optimized within the above-mentioned ranges depending on the purpose of transplantation and the administration site. For example, in the case of ophthalmic surgery, if the diameter of the injection needle used is approximately 25G (0.5 mm) and the length is approximately 28 mm, the microtube constituting the inner tube will have an outer diameter of approximately 70 μm to 290 μm (31 G to 38 G) and a length of approximately 33 mm to 39 mm so that it can be inserted into the injection needle. In this case, taking into account the thickness of the semipermeable membrane (10 μm to 50 μm), the inner diameter of the microtube will be approximately 50 μm to 270 μm.

[0022] At one end of the microtube is an "injection section" (104) having an inlet (103) for supplying cells to the microtube. The inlet may have a sealable structure, for example, the inlet may be fitted with a water-resistant and airtight plug, cap, or seal (see 305). As will be described later, it is sufficient for the inner tube (tubular structure) to be sealed when placed in the culture vessel, so it is not necessarily necessary to seal the inner tube itself.

[0023] The end of the microtube opposite the injection port (105, 304) may be open or may be sealable. As described above, the sealed end can be easily unsealed by cutting the part of the microtube protruding from the outer tube (the needle part of a syringe needle or cannula) with scissors after the inner tube is attached to the outer tube (see 404).

[0024] The "cells" contained within the microtubules are not particularly limited and may be stem cells, progenitor cells, or somatic cells. Examples of stem and progenitor cells include pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells); hematopoietic stem and progenitor cells, neural stem and progenitor cells, hepatic stem and progenitor cells, pancreatic stem and progenitor cells, skin stem and progenitor cells, osteochondral stem and progenitor cells, and tissue stem and progenitor cells such as adipose stem cells, cardiac stem cells, and mesenchymal stem cells, including dental pulp stem cells. Examples of somatic cells include differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, eye-related cells, adipocytes, and glial cells. Differentiated cells may be derived from pluripotent stem cells or stem and progenitor cells.

[0025] ES cells may be any of various established ES cell lines, including, but not limited to, clinical human ES cell lines provided by the Human ES Cell Research Center, Institute of Medical Biology, Kyoto University, and human ES cell lines provided by RIKEN BRC (KhES-1, KhES-1_Crx::Venus, KhES-1_Rx::Venus).

[0026] iPS cells can be derived from the patient's own somatic cells or from various iPS cell lines, including, but not limited to, the various iPS cell stock lines provided by the Kyoto University iPS Cell Research Foundation, and iPS cell lines derived from healthy individuals provided by the RIKEN BRC, such as KVs09 and CLs23.

[0027] The biological species of the cells is not particularly limited and may be appropriately selected depending on the purpose. For human transplantation, primate-derived cells, particularly monkey- or human-derived cells, are preferred, with human-derived cells being more preferred.

[0028] The transplantation device of the present invention is particularly suitable for use with cells administered to a transplantation site using a syringe needle or cannula. Examples of such cells include eye-related cells such as corneal epithelial cells, retinal pigment epithelial cells, neural retinal cells, conjunctival epithelial cells, limbal epithelial cells, corneal endothelial cells, corneal stromal cells, iris stromal cells, scleral cells, iris pigment epithelial cells, ciliary body epithelial cells, optic nerve cells, sublimbal fibroblasts, subconjunctival fibroblasts, lacrimal gland cells, meibomian gland cells, goblet cells, lens epithelial cells, and eyelid epithelial cells. Preferred eye-related cells are retinal pigment epithelial cells, neural retinal cells, and lacrimal gland cells, with retinal pigment epithelial cells being particularly preferred. In addition to eye-related cells, pancreatic beta cells, liver cells, neural glial cells, and neural progenitor cells are also suitable for use with the transplantation device of the present invention.

[0029] FIG. 2 shows an example of an outer tube (200). The "outer tube" is an injection needle or cannula having a needle tube portion (201) with an inner diameter capable of accommodating the microtube (101). The diameter (outer diameter) of the needle tube portion of the outer tube (injection needle or cannula) is selected depending on the purpose of the surgery and the administration site. The tip (202) of the injection needle or cannula may be straight or curved.

[0030] The needle or cannula may have an attachment to an external surgical device, such as a syringe, Constellation VFQ, or the like.

[0031] The inner and outer cylinders are provided as a set (kit), and are used in the medical field by attaching the inner cylinder to the outer cylinder (see Figure 4). Therefore, the inner cylinder (tubular structure) has a structure that allows it to be attached to the inside of the outer cylinder (injection needle or cannula).

[0032] 1.2 Implant Device II In a second embodiment, the implant device of the present invention is a tubular structure having the same configuration as the above-described inner tube (100), and is used by attaching a commercially available injection needle or cannula to the inside of the outer tube (see Figure 1). Hereinafter, this implant device will be referred to as "implant device II."

[0033] The transplantation device II is a tubular structure comprising a microtube (101) made of a semipermeable membrane capable of containing a transplantation material (102) containing cells, and an injection section (104) having an injection port (103) for supplying cells to the microtube, and is configured to be attached to the inside of an injection needle or cannula (e.g., MedOne0 (registered trademark) Poly Tip (registered trademark) Cannula 25g / 31g, etc.) having a needle section with an inner diameter capable of accommodating the microtube.

[0034] The structure for fitting the device inside an injection needle or cannula can be any known means in the art, such as a structure in which the injection portion of the tubular structure is in close contact with and / or engages with the base of the injection needle or cannula. The structure of the microtubes that make up the implantation device is as described in 1.1 above.

[0035] 2. Cellular Medicine The present invention provides a cellular medicine comprising a transplantation device pre-filled with a transplantation material containing cells.

[0036] 2.1 Cellular Medicine I In the first embodiment, the transplant material (102) containing cells is pre-filled into the transplant device I. As described above, the transplant device I is composed of an inner tube (100) and an outer tube (200), the inner tube is a tubular structure having a microtube (101) made of a semipermeable membrane capable of containing the transplant material containing cells, and an injection section (104) having an injection port (103) for supplying cells to the microtube, and the outer tube is an injection needle or cannula having a needle tube section (201) with an inner diameter capable of accommodating the microtube, and is used by attaching the inner tube to the outer tube.

[0037] 2.2 Cellular Medicine II In the second embodiment, a transplant material (102) containing cells is pre-filled inside the microtubules of the transplant device II. As described above, the transplant device II is a tubular structure comprising a microtubule (101) made of a semipermeable membrane capable of containing the transplant material containing cells, and an injection part (104) having an injection port (103) for supplying cells to the microtubule, and is structured to be attached to the inside of an injection needle or cannula having a needle part with an inner diameter large enough to accommodate the microtubule.

[0038] The cells used in the cell medicine of the present invention are the cells described in 1 as "cells encapsulated in microtubules." At least a portion of the cells in the microtubules may form aggregates. In the case of adhesive cells, cells cultured and grown in microtubules made of a semipermeable membrane form aggregates. In the case of retinal pigment epithelial cells, the aggregates formed are similar to the string-like aggregates produced by the device described in WO2022 / 230977, but also contain smaller pieces. The inventors have confirmed that these pieces may also be highly effective as transplant materials, similar to the string-like aggregates (see the test examples described below).

[0039] The cellular medicine of the present invention may contain a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, culture medium, physiological buffer solution such as PBS, preservatives, surfactants, stabilizers, excipients, antiseptics, binders, reducing agents, and isotonicity agents. If necessary, a cryopreservative may be added to the medicine, and the medicine may be frozen and stored, and then thawed before use.

[0040] 3. Manufacturing Method of Cellular Medicine Figure 3 shows an outline of a culture vessel used in the manufacture of cellular medicine. The cellular medicine of the present invention can be manufactured by injecting a culture medium containing cells into the fine tubes (301) of an inner tube (tubular structure), placing the tubular structure in a reservoir (303) of a culture vessel (302) filled with culture medium, and culturing the cells. There is no particular limit to the number of inner tubes arranged in the culture vessel, and one or more may be used. If many cellular medicines can be kept on hand in a medical setting in the form of culture vessels, several of them can be used for each surgery.

[0041] The culture vessel (302) is set in a culture device to culture the cells. The culture device may have known means necessary for cell culture, such as a means for adjusting the culture temperature, a means for adjusting the oxygen and carbon dioxide concentrations, a means for exchanging the culture medium, and a means for supplying additional components.

[0042] The culture medium and culture conditions are determined appropriately depending on the cells. The cell density in the microtubules is 5x10 7 cells / mL or more, preferably 5x10 7 cells / mL ~ 1x10 8cells / mL, more preferably 7.5x10 7 cells / mL ~ 1x10 8 Approximately cells / mL.

[0043] After the incubation, the end (304) of the microtube opposite to the injection port may be sealed by sealing, welding, or by using a hollow fiber with a blind end.

[0044] It is preferable that the inner tube (tubular structure) be sealable while it is placed in the culture vessel. This maintains the quality of the filled transplant material. For example, the inner tube (tubular structure) can be sealed by making the culture vessel a sealable container.

[0045] The culture vessel is detachable from the culture device, and after cell culture is complete, it can be removed from the culture device and carried with the tubular structure still in place. This allows the cell medicine to be provided to medical institutions in a state filled with transplant material without being frozen.

[0046] 4. Method for transporting cellular medicines Cell medicines can be transported by placing the tubular structures in a culture vessel. When transported in an oxygen-free environment, the quality of the cells contained in the tubular structures (microtubules) can be maintained for approximately 30 days.

[0047] This specification also discloses a kit for use in the production and delivery of a cellular medicine. The kit essentially comprises a transplantation device I or a transplantation device II and a culture vessel (302). As described above, the culture vessel has a reservoir that can be filled with a culture medium, and is capable of culturing cells by placing a tubular structure that constitutes the transplantation device in the reservoir. Furthermore, it is preferable that the tubular structure can be maintained in an oxygen-free state within the reservoir. Other details of the culture vessel are as described in "3. Method for producing a cellular medicine."

[0048] The cellular medicine (transplant material) of the present invention can also be frozen while filled in a tubular structure (inner tube). When freezing, the tubular structure after culture is removed from the culture vessel and transported in a frozen state. In this case, the cellular medicine I may be transported in a kit consisting of the outer tube and inner tube.

[0049] 5. Method of Use of Cellular Medicine Figure 4 shows an outline of the method of use of the cellular medicine. The cellular medicine of the present invention is used by attaching the inner tube, a tubular structure (401), to the outer tube, a syringe needle or cannula (402). The outer tube is appropriately attached to an external surgical device such as a syringe barrel (403). The length of the microtubules in the tubular structure is designed to be longer than the length of the needle portion of the syringe needle or cannula, and when attached, the tip (404) of the microtubules in the tubular structure protrudes from the tip of the syringe needle or cannula. Therefore, the protruding tip of the microtubule (including the sealing portion) can be cut using scissors or the like, pushed out from the syringe needle or cannula, and applied to the transplant site (see Figure 4).

[0050] 6. Cell Drug Comprising Retinal Pigment Epithelial Cells As a suitable example of the cell drug of the present invention, a cell drug comprising retinal pigment epithelial cells is described below.

[0051] Retinal pigment epithelial (RPE) cells refer to the epithelial cells that make up the retinal pigment epithelium and their progenitor cells. RPE cells can be isolated from patients or induced from stem cells such as pluripotent stem cells. Because the number of RPE cells obtainable from patients is limited, they must be expanded and proliferated before transplantation. In this invention, cells are cultured and expanded in microtubules made of a semipermeable membrane, allowing for the production of a cell medicine in which RPE cells are pre-loaded into a transplant device.

[0052] The method for culturing RPE cells essentially follows WO2022 / 230977. Media commonly used for culturing animal cells can be used as the basal medium. 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's medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof can be used as the basal medium.

[0053] The medium may be a serum-containing medium or a serum-free medium. The serum-free medium may contain a serum substitute. The serum substitute may be a commercially available product, for example, Knockout TM Serum Replacement (KSR), Chemically-defined Lipid concentrate (manufactured by Life Technologies), Glutamax TM (Life Technologies), B27 (Life Technologies), N2 supplement (Life Technologies), and ITS supplement (Life Technologies) can be used.

[0054] The serum-free medium may contain, as appropriate, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

[0055] A ROCK inhibitor may be added to the 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, Thiazovivin, GSK269962A, RKI-1447, Y-33075, GSK429286A, AT13148, H-1152 dihydrochloride, Y-33075 dihydrochloride, LX7101, SAR407899, ROCK-IN-2, and Afuresertib. Examples of ROCK inhibitors include hydroxyfasudil hydrochloride, hydroxyfasudil, GSK180736A, BDP5290, SR-3677, 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, and ROCK-IN-1, and preferred are Y-27632 dihydrochloride and Y-27632. The concentration of the ROCK inhibitor contained in the medium is usually 0 μM to 20 μM, preferably 2 μM to 10 μM. Although it is preferable to add a ROCK inhibitor when aggregating RPE cells, the addition of a ROCK inhibitor is not essential for the preparation after culture.

[0056] The density of the cultured RPE cells is not particularly limited, but is usually 2.5 x 10 3 cells / mL or more, preferably 2.5x10 3 cells / mL ~ 5x10 5 cells / mL, more preferably 1x10 5 cells / mL ~ 2x10 5 cells / mL.

[0057] The culturing time for RPE cells is not particularly limited, but is usually 1 to 30 days, preferably 2 to 7 days.

[0058] The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0059] RPE cells cultured and expanded in microtubules form aggregates, at least some of which resemble string-like aggregates. By using the transplantation device of the present invention, RPE cells can be transported to a medical institution while maintaining their aggregate morphology, and can be easily attached to an injection needle or cannula for use.

[0060] Cellular medicines containing RPE cells can be used to treat patients with diseases caused by damage to the retinal pigment epithelium or diseases accompanied by atrophy or damage of the retinal pigment epithelium. Examples of diseases caused by damage to the retinal pigment epithelium or diseases accompanied by atrophy or damage of the retinal pigment epithelium include ophthalmic diseases such as age-related macular degeneration, retinitis pigmentosa, and related diseases such as crystalline retinopathy, retinal pigment epithelial tears, macular dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular hole, degenerative myopia, and traumatic macular disease.

[0061] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0062] Test Example 1: String-like aggregates of retinal pigment epithelial cells were prepared according to a previous report (WO2022 / 230977). The string-like aggregates were seeded on a culture dish and cultured for 2 days.

[0063] Culture was performed using RPE maintenance medium containing ROCK inhibitor (Y-27632) as described in WO2022 / 230977. 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. TMSupplement (50X) (Thermo Fisher Scientific Inc.), and Gentamicin solution (Sigma-Aldrich).

[0064] The release of individual RPE cells from fragmented aggregates (arrows) tended to be faster than that from solid aggregates (arrowheads) (Fig. 5), suggesting that even if string-like aggregates are broken down into small pieces, they may still be effective as transplantation materials.

[0065] Test Example 2: Following a previous report (WO2022 / 230977), string-like aggregates of retinal pigment epithelial cells were prepared, placed in a container filled with RPE maintenance medium, sealed, and stored at 25°C under anoxic conditions for 13 days. They were then seeded onto culture dishes and cultured for 14 days at 37°C and 5% CO2. Retinal epithelial cells migrated from the stored string-like aggregates in the same way as unpreserved string-like aggregates, and the engraftment area of ​​individual retinal pigment epithelial cells continued to expand throughout the 14-day culture period (Figure 6). These results confirmed that RPE cells can be stored in anoxic conditions in culture medium for at least 13 days and can be maintained and transported under anoxic, non-dried conditions.

[0066] Example 1: A tubular structure (length 33 mm, outer diameter 31 gauge) similar in shape to the inner tube of a cannula (PolyTip Cannula; MedOne #3218, etc.) used for subretinal transplantation of string-like aggregates of retinal pigment epithelial (RPE) cells was fabricated using a semipermeable membrane permeable to cell culture medium, and RPE cells were cultured within the lumen to form aggregates (see Figure 7a). Culture was performed using RPE maintenance medium containing a ROCK inhibitor (Y-27632) as described in WO2022 / 230977.

[0067] After culturing, the tubular structures are placed in a container filled with culture medium and transported in a sealed state (see Figure 7b). As shown in Test Example 1, the string-like aggregates can be preserved in culture medium in an oxygen-free environment for at least 13 days, and are thought to be suitable for transport and provision to hospitals both in Japan and overseas.

[0068] To administer the medicine containing RPE cells, the tubular structure is inserted into a syringe needle (28 mm long, 25 gauge outer diameter) that corresponds to the outer barrel of a subretinal transplant cannula (PolyTip Cannula; MedOne #3218, etc.), and used in the same way as a subretinal transplant cannula with an outer barrel (PolyTip Cannula; MedOne #3218, etc.) (see Figure 7c). The outer barrel can be straight or bent, depending on the surgeon's preference.

[0069] The following is a brief summary of the above-mentioned manufacturing, transportation, and use procedures. Manufacturing: 1) Retinal pigment epithelial cells prepared in the same manner as in WO2022 / 230977 were cultured at 1x10 8 2) Retinal pigment epithelial cells 2x10 5 2 μl of the cell suspension containing the cells is injected into a 35 mm long tubular structure made of a semipermeable membrane with an outer diameter of 31-38 gauge. 3) The end of the tubular structure opposite the injection port is closed. 4) The tubular structure is placed in a container filled with RPE maintenance medium and cultured at 37°C in 5% CO2 for 24-48 hours.

[0070] Transportation: 1) Fill the tubular construct with RPE maintenance medium up to the top and close it with a waterproof and airtight seal. 2) Place the closed tubular construct in a container filled with RPE maintenance medium, seal it, and transport it in an oxygen-free environment.

[0071] Usage: 1) The tubular structure, which has been shipped in an anoxic state in a container filled with RPE maintenance medium, is unpacked and inserted into the outer barrel of a 25G ophthalmic transplantation needle. 2) The seal on the top of the tubular structure is removed, and the composite structure consisting of the transplantation needle (outer barrel) and the tubular structure (inner barrel) is attached to a microsyringe for ophthalmic transplantation. 3) The closed tip of the tubular structure is cut, and the RPE cells (aggregates) are pushed out of the tubular structure and transplanted under the retina.

[0072] The combination of a cell-containing inner tube and an outer needle enables long-term transport of cells that are difficult to handle, such as string-like aggregates, and reduces the number of steps required before transplantation at transplant centers. This method can be applied not only to RPE cells but also to other cells that are difficult to transport or load into transplant devices. This will enable the provision of high-quality cells in a state suitable for transplantation to more medical centers.

[0073] Example 2: Cells (human hepatoma-derived cell line HepG2, 5x10 7 cells / mL ~ 1x10 8 A suspension of 100 cells / mL was flowed into the microtubules (101) of the inner tube (100). The end (105) of the microtubule opposite the injection port was then sealed with a hemostatic clip (see Figure 8A). The microtubules (101) were then placed in the reservoir (302) of the culture vessel (300) filled with culture medium (303) along their longitudinal axis and the direction of gravity. The culture vessel was then placed in a culture device and cells were cultured. After 24 hours of culture, the hemostatic clip was removed, and the microtubules (101, 301) were cut open with scissors approximately 1 mm from the connection point between the clip and the microtubule (101) toward the injection port. Cell aggregates were then extracted from the ends (105, 304) of the microtubules by pumping medium through the injection port (104). The extracted cells had formed aggregates (Figure 8B).

[0074] All prior art documents cited herein are hereby incorporated by reference.

[0075] 100: Inner tube (tubular structure), 101: Microtube made of semipermeable membrane, 102: Transplant material containing cells, 103: Inlet port, 104: Injection part, 105: Sealing of end of microtube, 200: Outer tube (injection needle or cannula), 201: Needle tube part, 202: Tip of injection needle or cannula, 300: Culture vessel in which inner tube (tubular structure) is placed, 301: Inner tube (tubular structure), 302: Culture vessel, 303: Reservoir (filled with culture medium), 304: Sealing of end of microtube, 305: Sealing means for inlet port (stopper, seal, cap), 400: Outer tube (injection needle or cannula) in which inner tube (tubular structure) is set, 401: Inner tube (tubular structure), 402: Outer tube (injection needle or cannula), 403: syringe barrel (external surgical device), 404: tip of inner tube (tubular structure)

Claims

1. A transplant device comprising an inner tube and an outer tube, wherein the inner tube is a tubular structure having a microtube made of a semipermeable membrane capable of containing transplant material including cells, and an injection section having an injection port for supplying cells to the microtube, and the outer tube is an injection needle or cannula having a needle section with an inner diameter capable of accommodating the microtube, and the transplant device is used by attaching the inner tube to the outer tube.

2. A transplant device having a tubular structure comprising a microtube made of a semipermeable membrane capable of containing transplant material including cells, and an injection section having an injection port for supplying cells to the microtube, the transplant device being configured for use by being attached to the inside of an injection needle or cannula having a needle section with an inner diameter capable of accommodating the microtube.

3. The implantable device of claim 1 or 2, wherein the semipermeable membrane is composed of any one or combination of materials selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), polyhydroxy acid, polycaprolactone, polycarbonate, polyamide, polyethylene, polyurethane, polyarylate, polysulfone, polyethersulfone, polyester, polystyrene, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polyvinyl fluoride, polyvinylimidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, polytetrafluoroethylene (PTFE), biodegradable polyurethanes, 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 implantable device according to claim 1 or 2, wherein the inner diameter of the microtubules is between 20 μm and 2000 μm.

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

6. The implantable device of claim 1 or 2, further comprising one or more of the following (1) to (3): (1) the end of the microtubule opposite the injection port is sealable; (2) the injection port is sealable; (3) the injection needle or cannula has an attachment portion for attachment to an external surgical device.

7. A cellular medicine in which a transplantation device is pre-filled with transplantation material containing cells, said transplantation device being composed of an inner tube and an outer tube, said inner tube being a tubular structure comprising a microtube made of a semipermeable membrane capable of containing the transplantation material containing cells, and an injection section having an injection port for supplying cells to said microtube, said outer tube being an injection needle or cannula having a needle section with an inner diameter capable of accommodating said microtube, and said cellular medicine being used by attaching said inner tube to said outer tube.

8. A cellular medicine in which a transplantation device is pre-filled with a transplantation material containing cells, said transplantation device being a tubular structure comprising a microtube made of a semipermeable membrane capable of containing the transplantation material containing cells, and an injection section having an injection port for supplying cells to said microtube, said cellular medicine having a structure for use by being attached to the inside of an injection needle or cannula having a needle section with an inner diameter capable of accommodating said microtube.

9. The cell medicine according to claim 7 or 8, wherein the cells are one or more selected from stem cells, progenitor cells, somatic cells, and cells induced to differentiate from stem cells or progenitor cells.

10. The cell medicine according to claim 7 or 8, wherein the cells comprise retinal pigment epithelial cells.

11. The cell medicine described in claim 7 or 8, wherein at least a portion of the cells form aggregates within the microtubules.

12. The cell medicine according to claim 7 or 8, wherein the semipermeable membrane is composed of any one or combination 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, polyvinylimidazole, chlorosulfonated polyolefin, polyethylene oxide, polyphosphazene, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, polytetrafluoroethylene (PTFE), biodegradable polyurethanes, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile polyacrylate, ethylene-vinyl acetate polymer, acyl-substituted cellulose acetate, polymethyl methacrylate, polypropylene, and regenerated cellulose, as well as derivatives thereof.

13. The cell medicine according to claim 7 or 8, wherein the inner diameter of the microtubules is 20 μm to 2000 μm.

14. A method for producing a cell medicine as described in claim 7 or 8, comprising the steps of preparing a transplantation device, injecting a culture medium containing cells into the fine tubes of a tubular structure of the transplantation device, and placing the tubular structure in a reservoir of a culture vessel filled with culture medium to carry out culture.

15. The method according to claim 14, further comprising the step of sealing the end of the microtubule opposite the injection port after incubation.

16. The method according to claim 14, further comprising the step of sealing the inlet of the tubular structure after culturing and maintaining the structure in an oxygen-free state within the reservoir.

17. A kit comprising the transplant device according to claim 1 or 2 and a culture vessel, the culture vessel having a reservoir that can be filled with a culture medium, and the tubular structure portion being disposed in the reservoir for cell culture.

18. The kit according to claim 17, wherein the culture vessel is capable of holding the tubular structure in an oxygen-free condition within a reservoir.

Images