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WO2007032224A1 - Feuille de cellules cultivées et méthode pour sa préparation - Google Patents

Feuille de cellules cultivées et méthode pour sa préparation Download PDF

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Publication number
WO2007032224A1
WO2007032224A1 PCT/JP2006/317512 JP2006317512W WO2007032224A1 WO 2007032224 A1 WO2007032224 A1 WO 2007032224A1 JP 2006317512 W JP2006317512 W JP 2006317512W WO 2007032224 A1 WO2007032224 A1 WO 2007032224A1
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Prior art keywords
cells
cell
thin film
ipe
collagen gel
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English (en)
Japanese (ja)
Inventor
Eiji Kurihara
Noriko Koizumi
Junji Hamuro
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ArBlast Co Ltd
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ArBlast Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to a cultured cell sheet and a method for producing the same. Specifically, the present invention relates to a cultured cell sheet having a cell layer derived from iris pigment epithelial cells or retinal pigment epithelial cells and a method for producing the same.
  • the cultured cell sheet provided by the present invention is used in the treatment of retinal diseases that require reconstruction of the retinal pigment epithelium, such as age-related macular degeneration, retinal pigment degeneration, diabetic retinopathy, myopic retina choroidal atrophy, and retinal pigment tumor. It can be used as a transplant material.
  • retinal diseases that require reconstruction of the retinal pigment epithelium, such as age-related macular degeneration, retinal pigment degeneration, diabetic retinopathy, myopic retina choroidal atrophy, and retinal pigment tumor. It can be used as a transplant material.
  • fibroblast growth factor bFGF
  • bFGF fibroblast growth factor
  • regenerative medicine has been introduced in the field of ophthalmology. For example, attempts have been made to treat corneal epithelial diseases using corneal epithelial stem cells and amniotic membrane (Non-patent Document 1). With the advancement of regenerative medicine, aggressive treatment has been tried for diseases that have not been treated before. On the other hand, for intractable retinal diseases, there are still no established treatments, and many of the conditions are unknown. There are many retinal diseases that become severe once they develop. Although it is not at the stage where clinical results can be evaluated, regenerative medicine using cell transplantation has been developed for retinal chromatic degeneration and age related macular degeneration (AMD), which are representative of intractable retinal diseases. Application is being attempted.
  • AMD age related macular degeneration
  • AMD age-related macular degeneration
  • wet AMD which is said to be common among Japanese, causes severe visual impairment due to the formation of the choroidal neovascular plate in the macular region.
  • vitreous hemorrhage causes sudden visual impairment.
  • Traditionally it was said to be more common among whites, but it often occurs after middle age, and the number of Japanese people is increasing as the population ages.
  • the subretinal neovascularization that causes it is removed together with the vitreous, surgical treatment to move the macular retina, and special compounds and laser light are used. Attempts have been made to eliminate new blood vessels.
  • Non-Patent Document 2 uses IPE as a surrogate cell for retinal pigment epithelium that has been impaired after CNV removal, but its effect is far from clinical application. .
  • RPE retinal pigment epithelium
  • AMD age-related macular degeneration
  • the iris itself is derived from the mesoderm and neuroectoderm, but IPE is derived from the neuroectoderm and is embryologically identical in origin to RPE.
  • IPE which is clinically easy to collect, has also been transplanted into the RPE deficient part as a substitute for the dropped RPE.
  • the cultured IPE shows almost no pigment and it is difficult to confirm the transplanted cells directly. Therefore, when transplanting cultured cells, it is difficult to discuss the relationship between postoperative visual function and transplanted cells.
  • Non-Patent Document 1 Takahiro Nakamura, Shigeru Kinoshita: Position of amniotic membrane in eye regenerative medicine, ophthalmic surgery
  • Non-Patent Document 2 Abe T. et al., Tohoku J Exp Med 191: 7-20,2000
  • Non-Patent Document 3 Peyman GA. Et al., Ophthalmic Surg 22: 102-108, 1991
  • Non-Patent Document 4 Algvere PV. Et al., Graefes Arch Clin Exp Ophthalmol 235: 149-158,
  • Non-Patent Document 5 Weisz JM. Et al, Retina 19: 540-545, 1999
  • Non-Patent Document 6 Abe T. et al., Tohoku J Exp Med 189: 295-305, 1999
  • Non-Patent Document 7 Thumann G. et al., Arch Ophthalmol 118: 1350-1355, 2000
  • Non-Patent Document 8 Del Priore LV. Et al "Am J Ophthalmol 131: 472-480, 2001
  • RPE retinal pigment epithelium
  • an object of the present invention is to provide a transplant material that can be used as an alternative to RPE and that exhibits a high therapeutic effect (good reconstruction of RPE) and a method for producing the same.
  • IPE cells iris pigment epithelial cells
  • RPE cells retinal pigment epithelial cells
  • the substrate for constructing a cell sheet that can be expected to have a high therapeutic effect has the following characteristics: (1) (2) High strength, (3) Good adhesion between cells to be used, and good adhesion between cells to be used and substrate, and (4) Cells seeded on them We thought that it would be required to be able to maintain the functions of Looking at these characteristics and searching for a substrate, we found that a vitrified thin-film collagen gel (collagen gel thin film) was optimal.
  • the collagen gel thin film has a feature that there is almost no swelling at the time of transition from a dry state to a wet state, as compared to a product made from a commonly distributed collagen such as natural collagen or collagen.
  • a cultured cell sheet comprising a vitrified collagen gel thin film having a thickness of about 20 zm or less, and an iris pigment epithelial cell or retinal pigment epithelial cell-derived cell layer formed thereon.
  • the collagen gel thin film has a support for maintaining its shape.
  • the cultured cell sheet according to any one of [1] to [3].
  • the cell layer has a single layer structure
  • the cell density of the cell layer is about 1000-10000 cells / mm 2 ;
  • the planar view shape of the cells constituting the cell layer is a polygonal shape
  • the cell layer has phagocytic ability
  • a method for producing a cultured cell sheet comprising the following steps:
  • vitrified collagen gel thin film having a thickness of about 20 / m or less
  • Substrate consisting of a vitrified collagen gel thin film with a thickness of about 10 ⁇ m or less
  • FIG. 1 Schematic representation of the support used to produce a vitrified collagen gel film
  • FIG. 2 is a stereomicrograph of an IPE cell sheet constructed on a collagen gel thin film.
  • FIG. 3 is a stereomicrograph of an RPE cell sheet constructed on a collagen gel thin film.
  • FIG. 4 is a diagram showing the results (stereomicrograph) of an experiment in which beads were phagocytosed on an IPE cell sheet constructed on a collagen gel thin film.
  • FIG. 5 is a diagram showing the results of an experiment in which beads were phagocytosed on an RPE cell sheet constructed on a collagen gel thin film (photomicrograph).
  • cultured cell sheet refers to a sheet-like construct comprising a cell layer formed by culturing predetermined cells collected from a living body in vitro.
  • cultured IPE sheet refers to a sheet-like construct comprising a cell layer formed by culturing iris pigmented epithelial cells (IPE cells) collected from a living body in vitro
  • cultured RPE sheet refers to a sheet-like construct comprising a cell layer formed by culturing retinal pigment epithelial cells (RPE cells) collected from a living body in vitro.
  • iris pigment epithelial cells IPE cells
  • iris pigment epithelial stem cells ie, includes iris pigment epithelial stem cells.
  • retinal pigment epithelial cell RPE cell
  • irid pigment epithelial cell-derived cell layer refers to a cell layer constructed by culturing iris pigment epithelial cells.
  • cell layer derived from retinal pigment epithelial cells refers to a cell layer constructed by culturing retinal pigment epithelial cells.
  • collagen gel refers to collagen having a network structure containing a large amount of water, and is prepared by gelling a collagen sol.
  • collagen gel dry body means that collagen gel is thinned under vitrified conditions. The substance obtained by drying into a shape is added.
  • collagen gel thin film refers to a sheet-like structure obtained by rehydrating a dried collagen gel.
  • vitrification refers to changing to a hard and transparent substance by drying (Takush i E., Edible eyeballs from fish. Nature 345, 298, 1990).
  • the first aspect of the present invention relates to a cultured cell sheet.
  • the cultured cell sheet of the present invention comprises (1) a vitrified collagen gel thin film having a thickness of about 20 ⁇ m or less, and (2) iris pigmented epithelial cells (IPE cells) or Cell layer derived from retinal pigment epithelial cells (RPE cells).
  • IPE cells iris pigmented epithelial cells
  • RPE cells retinal pigment epithelial cells
  • the cell layer formed on the collagen gel thin film is constructed of cells derived from IPE or RPE. This is confirmed by examining the morphology of cells contained in the cell layer and the presence or absence of expression of markers characteristic of IPE cells (or RPE cells) (eg cytokeratin, RPE65, CRALBP, pP344, Bestro phin). be able to.
  • markers characteristic of IPE cells eg cytokeratin, RPE65, CRALBP, pP344, Bestro phin.
  • the cell layer preferably has some of the following characteristics, particularly preferably all the following characteristics.
  • the cell layer has a single layer structure (single layer structure). This is one of the features that biological IPE and RPE have in common.
  • a cell density of RPE biological is about 3000 to about 5000 cells ZMM 2.
  • the cell density in the cell layer of the present invention is preferably about 1000 to about 100,000 cells Zmm 2 , more preferably about 2000 to about 8000 cells / mm 2 .
  • the planar view shape of the cells constituting the cell layer is a polygonal shape. This is one of the features of RPE cells in the body. By observing this feature, the cell layer of the present invention is expected to exhibit a function similar to that of RPE, similar to the retinal pigment epithelium of a living body.
  • the cell layer of the present invention is expected to exhibit the same function as RPE in a living body.
  • Tight junctions are observed between cells in the cell layer. The presence or absence of tight junctions can be confirmed by examining the expression of tight junction-related protein ZO-1.
  • the cell layer has phagocytic ability. Living RPE cells exert an important function of metabolism of photoreceptors by their phagocytic ability. As the cell layer has phagocytic ability, it is expected to exhibit the same functions as RPE.
  • IPE cells iris pigment epithelial cells
  • RPE cells retinal pigment epithelial cells
  • the collagen gel thin film can be prepared according to the method described in International Publication No. 014774 (WO2005 / 014774A1) or Cell transplantation, Vol. 13, ⁇ .463-473, 2004.
  • the outline of the preparation method is shown below (for details, see the above publication and literature).
  • a collagen aqueous solution for example, type I collagen aqueous solution
  • a predetermined concentration for example, 0.5% (w / v)
  • buffer solution for example, phosphate buffer
  • the container is transferred to a clean bench under conditions of 10 ° C and 40% humidity, and left for 2 days with the container open to dry completely.
  • the collagen gel is vitrified.
  • the dried collagen gel thus obtained is left as it is or after rinsing with PBS or the like and subsequent drying (for example, drying under the same conditions as the above drying treatment), and then aseptic at room temperature.
  • PBS or the like and subsequent drying (for example, drying under the same conditions as the above drying treatment), and then aseptic at room temperature.
  • a collagen gel thin film can be obtained by returning the dried collagen gel to a wet state (rehydration) with an appropriate liquid (for example, a physiological saline, a buffer solution such as PBS, or a culture solution).
  • a vitrified collagen gel thin film is used.
  • a vitrified collagen gel thin film has excellent strength and high transparency. In addition, it can be made extremely thin during use (wet state) with very little swelling during the transition from the dry state to the wet state.
  • a normal collagen sheet that has not been vitrified for example, a type I collagen sheet disclosed in JP-A-2005-229869
  • Such a thick collagen sheet lacks qualification as a substrate for cultured cell sheets that are transplanted under the retina where physical space is scarce.
  • the swelling at the time of transition from the dry state to the wet state is very small. Therefore, the extremely thin state can be obtained at the time of use when used as a substrate for a cultured cell sheet. This is the biggest advantage of the collagen gel thin film.
  • both the IPE cells and the RPE cells adhere well to the vitrified collagen gel thin film, and the adhesion between cells is also improved. It was good. In addition, high cell growth efficiency can be obtained and seeded. It was found that the function of the cells retained was retained.
  • the vitrified collagen gel thin film has many characteristics preferable as a substrate for constructing a cell layer derived from IPE cells or a cell layer derived from RPE cells.
  • a cultured cell sheet (cultured IPE sheet or cultured RPE sheet) obtained using a vitrified collagen gel thin film retains strength suitable for transplantation, and can be expected to have a high therapeutic effect that is easy to handle. It will be a thing.
  • the thickness of the collagen gel thin film used in the present invention is about 20 zm or less.
  • the substrate as a carrier should be as thin as possible.
  • the substrate is required to have sufficient strength to support the cell layer.
  • the thickness of the collagen gel thin film as a substrate is preferably about 5 ⁇ m to 7j3 ⁇ 43 ⁇ 420 ⁇ m, more preferably about 5 ⁇ m to about 15 ⁇ m, and still more preferably about 5 ⁇ m to about 10 ⁇ m.
  • the type and origin of collagen constituting the collagen gel thin film are not particularly limited.
  • Collagen types include type I collagen, type III collagen, type IV collagen, type VIII collagen and the like.
  • a collagen gel thin film is constructed in a state where multiple types of collagen are mixed.
  • the collagen gel thin film is composed of type I collagen (or main component is type I collagen). This is because very good growth of IPE cells and RPE cells is observed on a collagen gel thin film made of type I collagen (see Examples below), and type I collagen is easy to supply stably.
  • Examples of the origin of collagen include horse, ushi, pig, hidge, monkey, chimpanzee, and human. You can also use (recombinant) human collagen prepared by gene recombination technology. Of these, collagen from recombinant human collagen, horse, ushi, or pig is preferred. This is because it is easy to obtain.
  • a physiologically active substance may be added to the collagen gel thin film.
  • Physiologically active substances here include cell growth factor, differentiation-inducing factor, cell adhesion factor, site force in, angiogenesis Inhibitors, analgesic anti-inflammatory agents and the like can be exemplified. Different physiologically active substances may be added in combination.
  • a collagen gel thin film having a compressive fracture strength of 3 ⁇ 4 times to 20 times (about 100 g to about 800 g when measured under the following measurement conditions) as compared to the state before vitrification (collagen gel).
  • a collagen gel thin film that satisfies this condition can be obtained by keeping it for about 40 days or more when it is dried at room temperature. In order to homogenize the gelation of the collagen contained, it is desirable to control the gelling process at 15 ° C or 4 ° C.
  • a collagen gel thin film having an absorbance at 400 nm of 10% to 70% (absorbance of about 0.1 to 0.2) as compared to the state before vitrification (collagen gel) is used.
  • a collagen gel thin film satisfying this condition can be obtained by storing and maintaining after drying for about 40 days or more.
  • the gelation process can be controlled at 15 ° C or 4 ° C.
  • a method performed in this field such as reduced pressure drying or freeze drying can be used.
  • a collagen gel thin film with a support it is preferable to prepare a collagen gel thin film with a support.
  • the support By using the support, the morphology of the collagen gel thin film can be maintained. At the same time, peeling from the container during the production of the thin film becomes easy. As a result, it is possible to ensure and maintain the flatness of the collagen gel thin film at the time of cell seeding or during the following culture. Cell adhesion, proliferation, and organization are improved, and a high-quality cell layer is constructed.
  • the collage gel thin film can be peeled off from the culture vessel relatively easily by grasping the support with tweezers. That is, by using the support, the operability when collecting the prepared cultured cell sheet is improved. Furthermore, since the cultured cell sheet can be collected while maintaining its shape by the support, it can be transferred to a transplantation device without damaging the formed cell layer. However, a highly flat cultured cell sheet suitable for transplantation can also be obtained.
  • the collagen gel thin film with the support can be prepared, for example, by the following procedure.
  • a nylon membrane molded into a donut shape is prepared as a support.
  • collagen sol is prepared and added to the culture dish, gelled, and vitrified by the procedure described above. With the above steps, a collagen gel thin film having a support attached on one side of the outer peripheral portion is completed.
  • the material of the support examples include synthetic fibers (synthetic resins) such as nylon, natural fibers such as cotton, bioabsorbable materials such as polylactic acid, and metals.
  • synthetic fibers synthetic resins
  • the shape of the support is preferably circular (circle, ellipse, square, etc.).
  • the support is used before transplanting the cultured cell sheet to the patient (from the preparation of the cultured cell sheet to immediately before transplantation of the cultured cell sheet). Is removed.
  • IPE cells can be collected routinely from the recipient himself or from the iris of a suitable donor. For example, first, a peripheral iris excision is performed under local anesthesia, and an iris piece is collected. Epithelial cells are separated from the collected iris pieces by trypsinization or manual methods. The separated epithelial cells (IPE cells) are cultured in an appropriate culture medium in which IPE cells can grow as necessary.
  • an appropriate culture medium for example, a commercially available F12 medium supplemented with serum, antibiotics (amphotericin, gentamicin, penicillin, streptomycin, etc.) and the like can be used.
  • antibiotics amphotericin, gentamicin, penicillin, streptomycin, etc.
  • serum to be added to the medium human serum, fetal bovine serum, sheep serum and the like can be used.
  • autologous serum ie, the recipient's own serum
  • autologous serum that eliminates the risk of causing immune rejection.
  • protein components derived from heterologous animals such as commercially available Epi Life TM (Cascade), MCDB153 medium (Nissui Pharmaceutical Co., Ltd.), and media prepared by modifying the amino acid composition of these mediums are completely removed.
  • a serum-free medium containing no serum can also be used. That is, a serum-free culture method may be employed as the culture method in the present invention. In such an embodiment, problems such as rejection of immunity due to contamination of serum-derived components can be avoided.
  • a culture container (culture dish) whose surface is coated with type I collagen, type IV collagen, fibronectin, laminin or the like. This is because adhesion of IPE cells to the surface of the culture container is promoted, and good growth is performed.
  • a culture vessel coated with type I collagen, type IV collagen, or fibronectin see Examples below.
  • the surface of a synthetic polymer or the like is treated with a nanotechnology technique and a material having an interface structure suitable for IPE cell growth is used.
  • the temperature conditions for culturing IPE cells are not particularly limited as long as the IPE cells grow. For example, about 25 ° C to about 45 ° C, preferably about 30 ° C in consideration of the growth efficiency. To about 40 ° C, more preferably about 37 ° C.
  • the culture time (that is, the time of primary culture) for subsequent subculture is, for example, 1 to 60 days, although it varies depending on the state of the cells used.
  • the recipient's own IPE cells when available, it is preferable to use the recipient's own IPE cells. This is because it is possible to produce a cultured IPE sheet that does not cause immune rejection when transplanted, that is, transplantation without immune rejection becomes possible. If the recipient's own IPE cells are not available or difficult to obtain, non-recipient IPE cells can be used, but in this case the donor should be selected in consideration of immunocompatibility. preferable.
  • Subculture cell suspension preparation
  • Subculture can be performed after the IPE cells subjected to culture have proliferated.
  • Subculture can be performed as follows. First, after removing the cell culture medium, the cells are washed with PBS ( ⁇ ) solution, treated with trypsin-EDTA or the like to peel the cells from the surface of the culture container, and the cells are collected in the next step. A culture solution is added to the collected cells to prepare a cell suspension.
  • the cell suspension is seeded in a culture vessel in the same manner as in the primary culture described above and used for culture.
  • Subculture can be performed under the same culture conditions as the above primary culture.
  • the culture time varies depending on the state of the cells used and is, for example, 1 to 60 days.
  • the above subculture can be performed multiple times as necessary. By repeating subculture, the number of cells can be increased and a cell suspension with a high cell density can be prepared. It is preferable to finally prepare a cell suspension having a cell density of about 20,000 cells / ml to 100,000 cells / ml.
  • RPE cells can be collected in a conventional manner from the recipient itself or from the subretinal area of a suitable donor. For example, a scleral incision is first performed under local anesthesia, and the retinal pigment epithelium under the peripheral retina sufficiently separated from the macula is removed. Epithelial cells are separated from the collected retinal pigment epithelium by trypsin treatment or manual method. The separated epithelial cells (RPE cells) are cultured in an appropriate culture medium in which RPE cells can grow as necessary. As the culture medium, for example, a medium obtained by adding serum, antibiotics (amphotericin, gentamicin, penicillin, streptomycin, etc.) to a commercially available DMEM medium can be used.
  • RPE cell passages and cell suspensions can be prepared using the same procedures and conditions as for IPE.
  • Step (1) and step (2) described above are performed independently. Therefore, the power to say that any step can be performed first (or both at the same time). Considering the nature of the material (ie, collagen, cells) handled in each step, the preparation of the collagen gel thin film is usually preceded. And implement.
  • Step of seeding and culturing cells the cells (IPE cells or RPE cells) prepared by the above procedure are seeded on the collagen gel thin film prepared by the above procedure.
  • a culture solution is added to a dried collagen gel prepared in a culture vessel and incubated (for example, 37 ° C, about 5 to 20 minutes).
  • the cell suspension is seeded on the vitrified collagen gel thin film thus obtained.
  • it is preferable to adjust the number of cells to be seeded so that a cell layer having a desired cell density is formed in the finally prepared cultured cell sheet.
  • the culture can be performed under the same conditions as the primary culture described above.
  • the culture time varies depending on the state of the cells used, but is, for example, 1 day to 60 days.
  • the cell culture here is preferably performed in the absence of heterologous animal cells.
  • “in the absence of heterologous animal cells” means that animal cells that are heterologous to the cells are not used as conditions for culturing IPE cells or RPE cells.
  • the conditions are such that cells of animal species other than humans, such as mice and rats, do not exist (coexist) in the culture medium.
  • the primary culture (and subculture as necessary) is also preferably performed in the absence of heterologous animal cells.
  • the collected IPE cells or RPE cells may be seeded directly on the collagen gel thin film and cultured. That is, the culture process (primary culture and subculture of IPE cells or RPE cells) performed before seeding on the collagen gel thin film may be omitted. In this way, the culturing operation can be simplified, and the cell layer can be constructed by a substantially one-step culturing operation.
  • a cultured cell in which a cell layer composed of IPE-derived cells (when IPE cells are used) or RPE-derived cells (when RPE cells are used) is formed on a collagen gel thin film.
  • a sheet is obtained.
  • the cultured cell sheet can be collected by releasing the attachment.
  • the collagen gel thin film is peeled off using tweezers or a knife.
  • the collage gel thin film can be peeled off relatively easily in the culture container by grasping the support with tweezers.
  • the support can maintain the shape of the collagen gel thin film and the cultured cell sheet at the time of peeling.
  • the cultured cell sheet of the present invention is provided, for example, in a state of being stored in a glass or plastic container and immersed in a cell culture medium or a storage solution such as a UW solution.
  • the cultured cell sheet prepared by the above method is a transplant material for the treatment of diseases that require RPE reconstruction, such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, myopic choroidal atrophy, and retinal pigment tumors. It can be used as an alternative to the pigment epithelium.
  • ALD age-related macular degeneration
  • CNV choroidal neovascularization
  • 3 ports are prepared and the vitreous body is excised with a vitreous cutter, and is weak against the retina near the CNV that has been confirmed by a scanning laser ophthalmoscope (S LO) examination before surgery After solidification of diathermy, make a small hole and grasp CNV with an insulator to remove it. After excision of CNV, the subretinal space is washed with a cell transplant needle to form a shallow retinal detachment state. The cultured cell sheet is transplanted into the space thus formed.
  • S LO scanning laser ophthalmoscope
  • Thickness prepared according to the method described in WO 2005/014774 pamphlet A vitrified collagen gel thin film having a thickness of about 20 ⁇ m was obtained from Asahi Techno Glass Co., Ltd. (Chiba, Japan). The outline of the method for preparing the collagen gel thin film is described below.
  • a circular support as shown in FIG. 1 was prepared by cutting a nylon membrane (Amersham # RPN1782B), sterilized, and placed in a hydrophobic polystyrene 24-wall plate.
  • a cell culture solution 0.5% type 1 collagen aqueous solution (Ushi type I collagen, Nitta Gelatin Co., Ltd.) was placed in a sterile conical tube cooled on ice and mixed uniformly.
  • the collagen mixture was placed in a 24-well plate containing the above support, and then gelled by maintaining it in a humidified incubator at 37 ° C in the presence of 5% CO / 95% air for 2 hours.
  • This final concentration 0.25% collagen gel was vitrified by aseptically drying for 2 days in a clean bench under conditions of 10 ° C and 40% humidity with the lid removed. By adding 3 ml of PBS to this, the dried vitrified collagen gel was rehydrated. Rinse several times with 3 ml of PBS. Furthermore, this collagen gel thin film was aseptically dried for 2 days in a clean bench under conditions of 10 ° C and 40% humidity with the lid removed. Thereafter, it was kept aseptically stored at room temperature. In the following experiments, a dried collagen gel that had been stored for 40 days or longer was used.
  • the collagen gel thin film obtained by returning the dried collagen gel to a wet state has very high transparency and strength (for details, see International Publication No. 2005Z014774 pamphlet). In addition, almost no swelling is observed when moving from a dry state to a wet state.
  • a cultured IPE sheet using porcine IPE cells was prepared by the following procedure.
  • the eyeballs were removed from the corpses.
  • the eyeball was immersed in about 20 ml of intraocular perfusate containing penicillin and streptomycin within 10 hours, and the following treatment was performed within 1 hour.
  • the outer part of the cornea was cut about 5 mm with scissors and separated into an anterior ocular tissue containing the cornea and a posterior ocular tissue containing the retina.
  • An eye tissue force was removed using cesi.
  • the iris pigmented tissue could be obtained by thoroughly washing with an intraocular perfusate except for the jelly-like substance contained therein. Subsequent processing is When there was no description, it carried out paying attention to aseptic operation in the clean bench.
  • the iris pigmented epithelial cells are black, and the area of the iris tissue from which the cells have been detached becomes pale and skin-colored. Therefore, cell detachment was continued until the black area could not be visually confirmed from the iris tissue.
  • the solution was aspirated with a pipette and poured into a 15 ml centrifuge tube, and 2 ml of the IPE culture solution was poured into the dish and again poured into a 15 ml centrifuge tube to wash the dish. After another washing operation, centrifugation was performed at 4 ° C and 3000 mm for 2 minutes.
  • IPE medium was used for IPE cell culture. 2-4 To the tube containing the pellet obtained in 2 above, 4 ml of IPE medium was added to suspend the pellet, and the whole solution was added to a 6-well plate (IWAKI) coated with type I collagen. One extra IPE cell is added to one well. Cell culture is incubator with 5% CO and 37 ° C
  • the medium was not changed after the culture, and the plate was left to stand. This is the period when IPE cells adhere to the plate, and the cells were observed daily. It takes about 7-10 days for the cells to adhere to the plate. Thereafter, the IPE medium was changed every 3 days.
  • the medium is exchanged according to the following procedure. First, the IPE culture solution was removed using an lml pipette, and the PBS was removed by washing the lml cache plate, and then washed. When culturing IPE cells, black granules are seen in the medium. This granule is removed by the washing operation. The PBS washing operation is repeated until no granules are observed in the collected PBS.
  • the washing operation is performed 5 times at the first medium change, The medium was changed twice. Thereafter, the IPE culture solution was returned to the lml cache incubator and the culture was continued. The cells became confluent after 2 weeks of culture, and the number of cells reached about 5-7 ⁇ 10 5 cells / well.
  • the tube was transferred to a liquid nitrogen container and left for one week.
  • the tube was transferred to a high-temperature bath preliminarily kept at 37 ° C and thawed immediately.
  • the solution in the tube was transferred to a 15 ml tube, 4 ml of IPE medium was added, and centrifuged at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, add 1 ml of IPE culture solution, suspend, and inoculate the cells on a 24-well plate coated with type I collagen and incubate under conditions of 5% CO and 37 ° C. The culture was carried out. Observation was performed one week later, and cells adhering to the plate were observed.
  • a pellet containing 5 X 10 4 IPE cells was prepared according to 2-4, and 2 ml of IPE culture solution was added and suspended to obtain a cell suspension.
  • the IPE culture solution was placed in a 24-well plate prepared in step 1 to which a dry collagen gel with a thickness of 20 ⁇ m was attached, and incubated at 37 ° C for 15 minutes. The dried body was rehydrated. After adding 1 ml of the above cell suspension onto the wet collagen gel thin film (about 20 ⁇ m thick) obtained as a result, the plate was transferred to an incubator with 5% CO and 37 ° C, and cultured. did. After culturing, the IPE medium was changed every 3 days using the same method as in 2-3. About 2 to 3 days after the start of culture, the cells adhered to the collagen gel thin film and reached confluence about 2 weeks later (Fig. 2).
  • a cultured RPE sheet using porcine RPE cells was prepared by the following procedure.
  • the eyeballs were removed from the corpses.
  • the eyeball was immersed in about 20 ml of intraocular perfusate containing penicillin and streptomycin within 10 hours, and the following treatment was performed within 1 hour.
  • the outer part of the cornea was cut about 5 mm with scissors and separated into anterior ocular tissue containing the cornea and posterior ocular tissue containing the retina.
  • Tissue strength was obtained by removing the vitreous body and retina using cesi, and then removing the scleral retinal pigment membrane and the choroid present on the sclera side. Retinal pigment tissue could be obtained by thorough washing with intraocular perfusate. Subsequent processing was performed with care in aseptic operation in a clean bench unless otherwise stated.
  • the retinal pigment epithelial cells are black, and the area of the retina pigment tissue from which the cells have been detached becomes white. Therefore, cell detachment was continued until the black area from the retinal pigment tissue could not be visually confirmed.
  • the solution was aspirated with a pipette and poured into a 15 ml centrifuge tube, and 2 ml of RPE culture solution was poured into the dish and again poured into a 15 ml centrifuge tube to wash the dish. After another washing operation, centrifugation was performed at 4 ° C and 3000 mm for 2 minutes.
  • RPE medium was used for RPE cell culture unless otherwise stated. 3- In the tube containing the pellet obtained in 2 above, 4 ml of RPE medium was added to suspend the pellet, and the entire solution was added to a 6-well plate (IWAKI) coated with type I collagen. This means that one eye has RPE cells per eye. Cell culture was performed in an incubator with 5% CO and 37 ° C. The medium was not changed after the culture, and the plate was left to stand. This was the time when RPE cells adhered to the plate, and the cells were observed daily. It takes about 7-10 days for the cells to adhere to the plate. Thereafter, the RPE medium was changed every 3 days. The medium was exchanged by the same procedure as 2-3. Cells became confluent after 3 weeks of culture and the cell count reached approximately 3-5 x 10 5 cells / well.
  • RPE cells were preserved in the same manner as 2-5. Cryopreservation One week after RPE culture Attempting to cultivate in lysis, cells observed to adhere to the plate one week after the start of culture. It was.
  • a cultured RPE sheet was prepared in the same manner as in 2- 6. About 2 to 3 days after the start of sheet preparation, the cells adhered to the collagen sheet and reached confluence after about 3 weeks (Fig. 3).
  • the phagocytic ability of the cultured IPE sheet was tested using latex beads.
  • the cultured IPE sheet obtained in 2-6. was used.
  • 1.0 ⁇ 10 9 uncoated latex beads (polyscience) having an average diameter of 1 ⁇ m were placed in an IPE culture solution in which an IPE sheet was immersed.
  • the bead is 1.0 X 10 9 pieces / lml / 1 sheet.
  • a 24-well plate containing a cultured IPE sheet was placed in a 37 ° C incubator and allowed to stand. After 6 hours, the latex bead-containing IPE medium was removed. After adding 1 ml of PBS and shaking the plate, washing was performed by removing PBS.
  • This washing operation was performed until latex beads were no longer observed in the removed PBS, and was usually performed five times.
  • free beads that have not been taken up into the cells are removed from the culture medium.
  • PBS was removed, lml of 100% acetone was added to the well, and the mixture was allowed to stand for 15 minutes. By this operation, the cells are fixed. Acetone was removed, and the well was washed by adding 1 ml of PBS.
  • the washing operation was performed twice. The culture IPE sheet was carefully peeled from the well using cesi, and the culture IPE sheet was placed on a slide glass so that the cell surface was up.
  • the phagocytic ability of the cultured RPE sheet obtained in 3-6 was also evaluated using the same procedure. Granules were observed only in the cultured RPE sheet with beads added, confirming the phagocytic ability (Fig. 5).
  • the pellet was obtained by centrifuging at 4 ° C and 6000 mm for 3 minutes and removing the supernatant. This pellet contains mainly the optic nerve outer segment.
  • the pellet was suspended by adding 500 ⁇ l of pH 8.0 PBS containing 100 mM NaCl, and 1 mg of fluorescent dye SNAEFL succinimidino ester dissolved in 100 ⁇ l of anhydrous dimethylformamide was collected. Fluorescent labels were introduced by standing at room temperature in a dark place for 30 minutes.
  • the culture IPE sheet was carefully peeled off from the tool using sushi, and the culture IPE sheet was placed on a slide glass so that the cell surface was up. 30 ⁇ L of mounting medium (Vector) was placed on the sheet, sealed with a cover glass, and then observed under a fluorescence microscope. Since fluorescence is observed when the optic nerve outer segment is taken up into the cell, phagocytic ability can be evaluated by observing the presence or absence of fluorescence in comparison with an acupuncture sheet without optic nerve outer segment added. As a result, fluorescence was observed only in the cultured ⁇ sheet with the optic nerve outer segment added, confirming that the cultured ⁇ sheet has phagocytic ability.
  • the phagocytic ability of the cultured RPE sheet obtained in 3-6. was also evaluated in the same manner. As a result, fluorescence was observed only in the cultured RPE sheet with the nerve outer segment covered, and the phagocytic ability was confirmed.
  • the iris pigment epithelial cells are black, and the area of the iris tissue from which the cells have been detached becomes a light skin color. Therefore, detachment of the cells is continued until the black area cannot be visually confirmed from the iris tissue.
  • aspirate the solution with a pipette and inject it into a 15 ml centrifuge tube.
  • After removing the supernatant add 5 ml of IPE culture solution, resuspend the pellet, and centrifuge at 4 ° C and 3000 rpm for 2 minutes. Collect the cell pellet by removing the supernatant.
  • IPE medium unless otherwise specified for IPE cell culture. 7— Add lml of IPE medium to the tube containing the pellet obtained in step 2 to suspend the pellet, and add the whole solution to a 24 well plate (IWAKI) coated with type I collagen.
  • IWAKI 24 well plate coated with type I collagen.
  • One tissue worth of IPE cells is added to one tool. Cell culture is performed in an incubator with 5% CO and 37 ° C. After the culture, the medium is not changed, and the plate is left still. This is the period when IP E cells adhere to the plate, and the cells are observed daily. It takes about 7-10 days for the cells to adhere to the plate. After that, change the IPE medium every 3 days. The medium is exchanged according to the following procedure.
  • a scleral incision is performed under local anesthesia for patients with informed consent.
  • a vitrectomy is performed, and about 2 X 2 mm of healthy RPE is taken from the subretinal area sufficiently far from the macula.
  • the excised tissue should be immersed in about 2 ml of intraocular perfusate, and the following treatment should be performed within 1 hour. If there is no other description, perform the subsequent processing with care for aseptic operation in a clean bench.
  • the retinal pigment epithelial cells are black, and the area of the retina pigment tissue from which the cells have been detached becomes white. Therefore, detachment of the cells is continued until no black area can be visually confirmed from the retinal pigment tissue.
  • aspirate the solution with a pipette and inject it into a 15 ml centrifuge tube.
  • RPE cell culture Use RPE medium unless otherwise stated for RPE cell culture.
  • 8- Add lml RPE medium to the tube containing the pellet obtained in step 2 to suspend the pellet, and add the whole solution to a 24-well plate (IWAKI) coated with type I collagen. This means that one tissue of RPE cells has been saved per tool.
  • Cell culture is performed in an incubator with 5% CO and 37 ° C. After the culture, the medium is not changed, and the plate is left still. This is the time when RPE cells adhere to the plate, and the cells are observed daily. It takes about 7-10 days for the cells to adhere to the plate. After that, change the RPE medium every 3 days. Replace the medium in the same way as in 7-3. Cells become confluent about 3-4 weeks after the start of culture.
  • Allogeneic IPE can be collected using an eyeball obtained from an eye bank. After removing the whole eyeball from the corpse, place it in an ocular storage solution such as optisol as soon as possible. Corneal limbal force About 5 mm outside is cut with scissors to separate the anterior ocular tissue including the cornea and the posterior ocular tissue including the retina. Anterior tissue force using cesi Remove the iris tissue except the cornea and lens. Iris pigment tissue can be obtained by thoroughly washing with BSS except for the jelly-like substance contained. Using the iris pigment tissue obtained in this way, prepare a cultured IPE sheet in the same manner as in 7-2 to 7_6.
  • Allogeneic RPE can be collected using an eyeball obtained from an eye bank. After removing the whole eyeball from the cadaver, place it in an ophthalmic preservation solution such as optisol as soon as possible. Corneal limbal force About 5 mm outside is cut with scissors to separate the anterior ocular tissue including the cornea and the posterior ocular tissue including the retina. Remove the posterior ocular tissue force from the vitreous and retina using cesi, then retina pigmented membrane from the sclera And the choroid present on the sclera side is peeled to obtain a retinal pigment tissue. Retinal pigment tissue can be obtained by thorough washing with BSS. Using the retinal pigment tissue thus obtained, a cultured RPE sheet is prepared in the same manner as in 8_2 to 8_6.
  • Transplantation of cultured IPE sheet or cultured RPE sheet can be carried out by the following procedure. First, after placing 2% methylcellulose on the recipient cornea, a contact lens for vitreous surgery is placed and a scleral incision is made at 11 o'clock. After vitrectomy, insert a 36G needle from the scleral incision to make a retinal incision at the lesion site. Artificial retinal detachment is made by injecting a small amount of intraocular perfusate from the retinal incision into the subretinal region. Spread the scleral incision and insert a cultured cell sheet (cultured IPE sheet or cultured RPE sheet) under the retina. After transplanting the cultured cell sheet, laser photocoagulation is performed on the retinal incision, and the lower eye is replaced with air or expandable gas. After transplanting, keep the prone position until air or gas is absorbed.
  • a contact lens for vitreous surgery is placed and a scleral incision is made at 11 o'clock. After
  • Dish coating for IPE primary culture In 2-3, the cells were cultured using a plasma-treated non-coated dish, a type IV collagen dish, a laminin coated dish, a fibronectin coated dish, or a matrigel coated dish instead of the type I collagen coated dish.
  • a plasma-treated non-coated dish a type IV collagen dish
  • a laminin coated dish a laminin coated dish
  • a fibronectin coated dish or a matrigel coated dish instead of the type I collagen coated dish.
  • the optimal carrier type for culture was examined using the number of adherent cells as an index.
  • the number of adherent cells was almost the same as when using type I collagen coated dish.
  • FCS concentration in the culture medium during primary culture was examined.
  • 2 ⁇ 3 and 3 ⁇ 3 culture is performed under the condition that the concentration of FCS in the culture solution is reduced from 10% to 5, 3, 1, or 0%. The existence was observed.
  • adherent cells were present, the number of adherent cells was compared with the FCS 10% condition.
  • the number of adherent cells was the same as the condition of FCS 10% when FCS was 5% or more, and the number of adherent cells was slightly reduced under the condition of FCS 3%, F CS 1% or less Adherent cells were not observed under the conditions.
  • the number of adherent cells was the same as FCS 10% when FCS was 3% or higher, slightly lower when FCS was 1%, and adherent cells were observed when FCS was 0%.
  • the serum concentration in the culture medium during primary culture can be lowered to at least 3% in the case of IPE cells and to at least 1% in the case of RPE cells.
  • a cultured cell sheet was prepared under the condition that the origin of collagen as a component of the substrate (collagen gel thin film) was swine, and the morphology of the cell layer was observed. Specifically, it was observed whether the cells constituting the cell layer had a honeycomb shape. As a result, it was found that even when a substrate made of porcine collagen was used, a culture sheet having a cell layer equivalent to that obtained when a substrate having collagen power derived from ushi was used. In other words, it was clarified that even a substrate prepared using collagen derived from other animal species as a raw material, not only from ushi, is useful for producing cultured cell sheets.
  • the thickness of the substrate was changed from 20 ⁇ m to 10 am, and an attempt was made to produce a cultured cell sheet.
  • both a substrate made of ushi collagen and a substrate made of porcine collagen were examined.
  • a cultured cell sheet equivalent to the thickness of 2 ⁇ could be produced.
  • the obtained cultured cell sheet had sufficient strength and form retention required for the transplant material.
  • the thickness of the cultured cell sheet which has less physical space under the macular part retina to which the cultured cell sheet is transplanted, is 20 zm or less in total. From the above study, it became clear that a 10 ⁇ m-thick substrate could be used, and in fact, a cultured cell sheet with a thickness of 20 ⁇ m or less was successfully constructed.
  • the cultured cell sheet of the present invention can be used as a transplant material for various diseases that require transplantation of retinal pigment epithelium (RPE).
  • the cultured cell sheet of the present invention includes a cell layer having a structure similar to that of living body RPE.
  • the cultured cell sheet of the present invention is formed by culturing and proliferating collected IPE cells or RPE cells in vitro. Therefore, it is possible to produce transplant material based on a small number of IPE cells or RPE cells, and even patients with a reduced number of these cells can make transplant material using their own cells. . This means that a network transplantation that does not cause rejection can be realized.
  • the cultured cell sheet of the present invention can be constructed using autologous cells. If autologous cells are used, it can be said that a cell sheet that exhibits high engraftment and has an excellent therapeutic effect can be constructed.

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Abstract

L’invention concerne une feuille de cellules cultivées de laquelle on attend un effet thérapeutique important. L’invention concerne aussi une méthode pour la préparation de la feuille de cellules cultivées. La feuille de cellules cultivées peut être préparée en cultivant une cellule épithéliale pigmentée d’iris ou une cellule épithéliale pigmentée rétinienne sur une mince couche de gel collagène d’une épaisseur de 20 µm ou moins et qui est préalablement vitrifiée.
PCT/JP2006/317512 2005-09-13 2006-09-05 Feuille de cellules cultivées et méthode pour sa préparation Ceased WO2007032224A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009084507A1 (fr) * 2007-12-28 2009-07-09 Osaka University Procédé de préparation d'un gel de collagène stratifié et gel de collagène stratifié
JP2010046058A (ja) * 2008-07-24 2010-03-04 Two Cells Co Ltd 治療用培養細胞の製造方法
WO2012026531A1 (fr) * 2010-08-25 2012-03-01 独立行政法人農業生物資源研究所 Hydrogel séché, film de vitrigel séché, et procédés pour produire les mêmes
WO2012063925A1 (fr) * 2010-11-12 2012-05-18 独立行政法人農業生物資源研究所 Chambre de culture cellulaire, son procédé de production, modèle de tissu utilisant la chambre de culture cellulaire et son procédé de production
WO2014208778A1 (fr) * 2013-06-28 2014-12-31 国立大学法人東北大学 Film mince nanométrique modelé de support de cellule
EP3015091A4 (fr) * 2013-06-28 2017-03-29 Saga University Préparation de peau artificielle du type timbre
JP2021510810A (ja) * 2018-01-11 2021-04-30 センター フォー アイ リサーチ オーストラリア リミテッドＣｅｎｔｒｅ Ｆｏｒ Ｅｙｅ Ｒｅｓｅａｒｃｈ Ａｕｓｔｒａｌｉａ Ｌｉｍｉｔｅｄ 組織のバイオマーカを定量化するための方法及びシステム
WO2023210609A1 (fr) * 2022-04-27 2023-11-02 ファーマバイオ株式会社 Procédé de production de feuillet cellulaire

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009084507A1 (fr) * 2007-12-28 2009-07-09 Osaka University Procédé de préparation d'un gel de collagène stratifié et gel de collagène stratifié
JP2010046058A (ja) * 2008-07-24 2010-03-04 Two Cells Co Ltd 治療用培養細胞の製造方法
WO2012026531A1 (fr) * 2010-08-25 2012-03-01 独立行政法人農業生物資源研究所 Hydrogel séché, film de vitrigel séché, et procédés pour produire les mêmes
WO2012063925A1 (fr) * 2010-11-12 2012-05-18 独立行政法人農業生物資源研究所 Chambre de culture cellulaire, son procédé de production, modèle de tissu utilisant la chambre de culture cellulaire et son procédé de production
WO2014208778A1 (fr) * 2013-06-28 2014-12-31 国立大学法人東北大学 Film mince nanométrique modelé de support de cellule
EP3015091A4 (fr) * 2013-06-28 2017-03-29 Saga University Préparation de peau artificielle du type timbre
JP2021510810A (ja) * 2018-01-11 2021-04-30 センター フォー アイ リサーチ オーストラリア リミテッドＣｅｎｔｒｅ Ｆｏｒ Ｅｙｅ Ｒｅｓｅａｒｃｈ Ａｕｓｔｒａｌｉａ Ｌｉｍｉｔｅｄ 組織のバイオマーカを定量化するための方法及びシステム
JP7251873B2 (ja) 2018-01-11 2023-04-04 センター フォー アイ リサーチ オーストラリア リミテッド 組織のバイオマーカを定量化するための方法及びシステム
US11963721B2 (en) 2018-01-11 2024-04-23 Centre For Eye Research Australia Limited Method and system for quantifying biomarker of a tissue
WO2023210609A1 (fr) * 2022-04-27 2023-11-02 ファーマバイオ株式会社 Procédé de production de feuillet cellulaire

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