A complex comprising cell aggregates including the neuroretina and a matrix, and a method for producing the same.

Abstract

The purpose of the present invention is to provide a complex containing two or more neural retina-containing cell aggregates and a matrix, and a method for manufacturing the same. The complex according to the present invention contains two or more cell aggregates, which contain pluripotent stem cell-derived neural retinas, and a matrix, where the two or more cell aggregates are disposed inside the matrix. The method for manufacturing the complex according to the present invention is a method for manufacturing a complex in which two or more neural retina-containing cell aggregates are disposed inside a matrix, the method including: (1) a first step for producing the two or more neural retina cell aggregates from pluripotent stem cells, and (2) a second step for making the matrix into a gel by causing the two or more cell aggregates to come into contact with the matrix or a matrix precursor in a prescribed arrangement.

Description

[Technical Field]

[0001] The present invention relates to a complex comprising cell aggregates including a neuroretina and a matrix, and a method for producing the same. [Background technology]

[0002] In connection with retinal transplantation therapy for diseases based on retinal tissue damage, such as retinitis pigmentosa, research into methods for producing neuroretina from pluripotent stem cells is actively being conducted. As a method for producing neuroretina from pluripotent stem cells, for example, a method is known in which aggregates of pluripotent stem cells are cultured in suspension in a culture medium containing a BMP signaling pathway activator to obtain neuroretina (Patent Documents 1, 2 and Non-Patent Document 1).

[0003] On the other hand, techniques utilizing gelatin in transplantation have been investigated. For example, methods include solidifying living retinal tissue with gelatin to improve the ease of cutting out the photoreceptor cell layer (Non-Patent Literature 2), embedding living retinal tissue with temperature-sensitive gelatin (Non-Patent Literature 3), and embedding human fetal-derived retinal tissue with gelatin to protect fragile fetal tissue (Non-Patent Literature 4).

[0004] Furthermore, fibrin gels are administered to humans for the purpose of tissue repair through tissue adhesion and closure. One example is Volheer® Tissue Adhesion Gel. In addition, a laminate of fibrin gel and sheet-like cell cultures has been disclosed in which fibrin gel is used to increase the strength of transplant tissue (Patent Document 3). Furthermore, there have been reports on the transplantation of retinal progenitor cells embedded in fibrin gel (Non-Patent Document 5).

[0005] However, the use of matrices such as gelatin or fibrin gel for the purpose of adhering two or more neuroretinal tissues is not known to date. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2015 / 025967 [Patent Document 2] International Publication No. 2016 / 063986 [Patent Document 3] Japanese Patent Publication No. 2016-52271 [Non-patent literature]

[0007] [Non-Patent Document 1] Atsushi Kuwahara et al., “Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue”, Nature Communications, 6, Article number:6286 (2015) [Non-Patent Document 2] Taylor et al., IOVS 1989 Aug; Volume 30, Issue 8 [Non-Patent Document 3] M'Barek et al., Sci. Transl. Med.2017 Dec; Volume 9, Issue 421 [Non-Patent Document 4] Seiler et al., Prog Retin Eye Res.2012Nov;31(6):661-687 [Non-Patent Document 5] Tamer AE Ahmed et al., Frontiersin Bioengineering and Biotechnology, Biomaterials, February 2015, Volume2, Article 85, 1-11 [Overview of the project] [Problems that the invention aims to solve]

[0008] As a method for formulating transplanted tissues for regenerative medicine applications, there has been a long-felt need for a method of easily bonding a plurality of tissues together. Accordingly, an object of the present invention is to provide a complex comprising cell aggregates containing two or more neural retinas. [Means for Solving the Problem]

[0009] The present inventors studied a method for forming a complex by bonding or embedding cell aggregates containing two or more neural retinas using some kind of "glue". The foreign material "glue" interferes with engraftment of the complex after transplantation, and can also be a foreign substance to the living body. For this reason, the present inventors considered that a matrix, which is a substance that strongly bonds even in a small amount and is degraded in vivo, is preferable as the "glue".

[0010] As a result of intensive studies, the present inventors succeeded in producing a complex of cell aggregates containing two or more neural retinas and a matrix by using fibrin gel or gelatin as a "glue" and embedding cell aggregates containing two or more neural retinas. In this method, embedding cell aggregates containing two or more neural retinas in a matrix enables suction and discharge with a syringe without disrupting the matrix gel.

[0011] That is, the present invention relates to the following inventions. [1] A complex comprising: cell aggregates containing neural retina derived from two or more pluripotent stem cells; and a matrix, wherein the two or more cell aggregates are arranged in the matrix. [2] The complex according to [1], wherein the neural retina is a neural retina sheet for transplantation having the following characteristics (1) to (10): (1) it is derived from pluripotent stem cells, (2) it has a three-dimensional structure, (3) it comprises a neural retinal layer having a plurality of layer structures including a photoreceptor layer and an inner layer, (4) the photoreceptor layer comprises one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptors, (5) The inner layer contains one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells and bipolar cells, horizontal cells and Müller glial cells, (6) The surface of the neuroretinal layer has an apical surface, (7) The inner layer is located inside the photoreceptor layer that is located along the apical surface, (8) The area of ​​the neuroretinal layer is 50% or more of the total surface area of ​​the neuroretinal sheet for transplantation, (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neuroretinal layer, (10) Expression of neuroretinal cell-related genes is observed in the above-mentioned neuroretinal sheet for transplantation, and expression of non-neuroretinal cell-related genes is not observed, and the non-neuroretinal cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eyeball-related tissue marker genes. [3] The complex according to [1] or [2], wherein the cell aggregates have a major axis of 600 μm to 2500 μm, and two or more of the cell aggregates are arranged in rows in the matrix. [4] The complex according to any one of [1] to [3], wherein the matrix is ​​fibrin gel or gelatin. [5] A method for producing a complex in which two or more cell aggregates containing neuroretina are arranged in a matrix, (1) The first step involves creating two or more neuroretinal cell aggregates from pluripotent stem cells, (2) A second step of bringing two or more cell aggregates into contact with a matrix or matrix precursor in a predetermined arrangement, and then gelling the matrix, A manufacturing method that includes this. [6] The manufacturing method according to [5], which includes arranging 2 to 20 cell aggregates in a row in the second step. [7] The manufacturing method according to [5] or [6], wherein the matrix is ​​a fibrin gel, and in the second step, two or more cell aggregates are arranged in a row, the two or more arranged cell aggregates are brought into contact with either a fibrinogen solution or a thrombin solution, and then into contact with the other of the fibrinogen solution or thrombin solution, thereby reacting the fibrinogen and thrombin and causing a gel. [8] The manufacturing method according to [5] or [6], wherein the matrix is ​​gelatin. [9] A pharmaceutical composition comprising the complex described in any of [1] to [3].

[10] A method for treating a disease based on damage to retinal cells or retinal tissue, or damage to retinal tissue, comprising transplanting a complex described in any of [1] to [3] into a subject requiring transplantation. [Effects of the Invention]

[0012] According to the present invention, a complex comprising cell aggregates containing two or more neuroretinas and a matrix can be provided, thereby facilitating the simultaneous transplantation of cell aggregates containing multiple neuroretinas (NRs). [Brief explanation of the drawing]

[0013] [Figure 1] In Example 1, the process of encapsulating multiple (8-13) neuroretinal implants (Caps) in a fibrin gel (composite) and the aspiration and dispensing procedures using Surflo were examined. The images show bright-field stereomicroscope images, fluorescence stereomicroscope images, and bright-field + fluorescence stereomicroscope images. [Figure 2] In Example 2, the process of encapsulating multiple (8-10) implantable neuroretinal caps in gelatin (composite) and the aspiration and dispensing procedures using a 1 ml tip with a wide tip are shown in bright-field stereomicroscope images, fluorescence stereomicroscope images, and bright-field + fluorescence stereomicroscope images. [Figure 3]This is a fluorescence microscope image showing the results of immunostaining with Crx and Chx10 on cell aggregates containing a neuroretina for transplantation, as shown in Reference Example 1. [Figure 4] This is a fluorescence microscope image showing the results of immunostaining with Rx and Recoverin on cell aggregates containing a neuroretina for transplantation, as shown in Reference Example 1. [Figure 5] This is a conceptual diagram showing how to fabricate caps and rings from typical cell aggregates. [Figure 6] This is a conceptual diagram illustrating the fabrication of caps and rings from cell aggregates of various shapes. The areas shown in black and gray represent unintended tissues. [Figure 7] The height, major axis, and minor axis of the graft in Reference Example 3 are shown, along with images of a typical graft and a schematic diagram of the graft. [Figure 8] This is a confocal fluorescence microscope image showing the results of immunostaining of the graft with Crx, Chx10, Rx, and Recoverin in Reference Example 4. [Figure 9A] The results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Reference Example 5 are shown. [Figure 9B] The results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Reference Example 5 are shown. [Figure 10] This image shows the results of observing the engraftment after transplantation using a fluorescence microscope, following quantitative PCR analysis of RNA extracted from the ring in Reference Example 6, and subsequent transplantation of the graft (cap) under the rat retina. [Figure 11] This is a fluorescence microscope image showing the results of immunostaining performed on a cap and ring prepared from a single cell aggregate in Reference Example 7. [Figure 12] This is a fluorescence microscope image showing the results of immunostaining performed on a cap prepared from a single cell aggregate in Reference Example 8. [Modes for carrying out the invention]

[0014] [Definition] "Stem cells" refer to undifferentiated cells that possess the ability to differentiate and proliferate (especially self-renewal). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability. Pluripotent stem cells are those that can be cultured in vitro and possess the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues (pluripotency). Multipotent stem cells are those that have the ability to differentiate into multiple types of tissues and cells, though not all of them. Unipotent stem cells are those that have the ability to differentiate into specific tissues or cells.

[0015] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ cells, tissue-derived stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells created from germ cells (e.g., testes) are also included in the category of pluripotent stem cells.

[0016] Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a culture medium containing bFGF. Methods for producing embryonic stem cells are described, for example, in WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from designated institutions, and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Human ES cells (derived from KhES-1, Non-Patent Literature 1) genetically modified to possess the human embryonic stem cell genes Rx::Venus, Rx::AcGFP, and Crx::Venus reporter genes are available from the RIKEN Institute.

[0017] "Induced pluripotent stem cells" are somatic cells that have been reprogrammed using known methods to induce pluripotency.

[0018] Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126(4), pp. 663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007 and possess pluripotency and self-renewal ability similar to embryonic stem cells (Cell, 2007, 131(5), pp. 861-872; Science, 2007, 318(5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26(1), pp. 101-106).

[0019] Induced pluripotent stem cells are specifically cells in which pluripotency is induced by reprogramming differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells by expressing one of several combinations of genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, and Esrrb. Preferred reprogramming factor combinations include (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466).

[0020] In addition to the method of producing induced pluripotent stem cells through direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds, etc. (Science, 2013, 341, pp. 651-654).

[0021] Furthermore, it is possible to obtain induced pluripotent stem cell lines. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, established at Kyoto University, are available from Kyoto University and iPS Academia Japan, Inc. As induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells, established at Kyoto University, are available from Kyoto University.

[0022] In this specification, pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0023] In this specification, pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells) (for example, embryonic stem cells established from an embryo within 14 days of fertilization).

[0024] Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture by methods well known to those skilled in the art.

[0025] "Retinal tissue" refers to the tissue in a living retina in which one or more types of retinal cells that make up each retinal layer exist in a certain order, while "neural retina" refers to retinal tissue that includes the inner neural retina layer, which does not include the retinal pigment epithelium layer, as described later.

[0026] "Retinal cells" refer to the cells that make up each retinal layer in a living retina, or their progenitor cells. Retinal cells include, but are not limited to, photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelium (RPE) cells, ciliary body, their progenitor cells (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, etc.), and retinal progenitor cells. Among the retinal cells, the cells that constitute the neuroretinal layer (also called neuroretinal cells or neuroretina-related cells) specifically include photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, and their precursor cells (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, etc.). In other words, neuroretina-related cells do not include retinal pigment epithelial cells or ciliary body cells.

[0027] "Mature retinal cells" refers to cells that may be found in the retinal tissue of an adult human, specifically differentiated cells such as photoreceptor cells (rod and cone cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod and cone cells), Müller glial cells, retinal pigment epithelial (RPE) cells, and ciliary cells. "Immature retinal cells" refers to progenitor cells whose differentiation into mature retinal cells is predetermined (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, retinal progenitor cells, etc.).

[0028] Photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, Müller glial cell progenitor cells, and retinal pigment epithelial cell progenitor cells are progenitor cells whose differentiation into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cells, respectively, has been determined.

[0029] "Retinal progenitor cells" refer to progenitor cells that can differentiate into any immature retinal cell type, such as photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, Müller glial cells, and retinal pigment epithelial progenitor cells, and ultimately differentiate into any mature retinal cell type, such as photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.

[0030] Photoreceptor cells are cells found in the photoreceptor layer of the retina in living organisms, and their role is to absorb light stimuli and convert them into electrical signals. There are two types of photoreceptor cells: cones, which function in bright light, and rods, which function in dim light (these are called cone photoreceptor cells and rod photoreceptor cells, respectively). Cone photoreceptor cells include S-cone photoreceptor cells that express S-opsin and receive blue light, L-cone photoreceptor cells that express L-opsin and receive red light, and M-cone photoreceptor cells that express M-opsin and receive green light. Photoreceptor cells differentiate and mature from photoreceptor progenitor cells. Whether a cell is a photoreceptor cell or a photoreceptor progenitor cell can be easily confirmed by a person skilled in the art, for example, by the expression of cell markers described later (Crx and Blimp1 expressed in photoreceptor progenitor cells, Recoverin expressed in photoreceptor cells, rhodopsin, S-Opsin, and M / L-Opsin expressed in mature photoreceptor cells, etc.), the formation of outer segment structures, etc. In one embodiment, photoreceptor progenitor cells are Crx-positive cells, and photoreceptor cells are rhodopsin, S-Opsin, and M / L-Opsin-positive cells. In one embodiment, rod photoreceptor cells are NRL and Rhodopsin-positive cells. In one embodiment, S cone photoreceptor cells are S-opsin-positive cells, L cone photoreceptor cells are L-opsin-positive cells, and M cone photoreceptor cells are M-opsin-positive cells. That is, the term "photoreceptor cell" as used herein is a concept that includes photoreceptor progenitor cells and mature photoreceptor cells.

[0031] The neuroretina preferably contains 10% or more, and more preferably 20% or more, photoreceptor cells or photoreceptor progenitor cells.

[0032] The neuroretina preferably contains 10% or more neuroretinal progenitor cells, more preferably 20% or more. Furthermore, the neuroretina preferably contains 10% or more photoreceptor progenitor cells. In one embodiment, the neuroretina also contains 3% or more mature photoreceptor cells.

[0033] The presence of neuroretinal cells can be confirmed by the presence or absence of expression of neuroretinal cell-related genes (hereinafter sometimes referred to as "neuroretinal cell markers" or "neuroretinal markers"). The presence or absence of expression of neuroretinal cell markers, or the proportion of neuroretinal cell marker-positive cells in a cell population or tissue, can be easily confirmed by those skilled in the art. Examples include antibody-based methods, nucleic acid primer-based methods, and sequencing reaction methods. As an antibody-based method, the expression of the neuroretinal cell marker protein can be confirmed, for example, by flow cytometry or immunostaining using commercially available antibodies, by dividing the number of cells positive for a specific neuroretinal cell marker by the total number of cells. As an antibody-based method, the expression of the neuroretinal cell marker RNA can be confirmed, for example, by PCR, semi-quantitative PCR, or quantitative PCR (e.g., real-time PCR). As an sequencing reaction method, the expression of the neuroretinal cell marker RNA can be confirmed, for example, by a nucleic acid sequencer (e.g., next-generation sequencer).

[0034] Examples of neuroretinal cell markers include Rx (also known as Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6, and Chx10 (also known as Vsx2) expressed in neuroretinal progenitor cells, and Crx and Blimp1 expressed in photoreceptor progenitor cells. Other examples include Chx10, which is strongly expressed in bipolar cells; PKCα, Goα, VSX1, and L7, which are expressed in bipolar cells; TuJ1 and Brn3, which are expressed in retinal ganglion cells; Calretinin and HPC-1, which are expressed in amacrine cells; Calbindin, which is expressed in horizontal cells; Recoverin, which is expressed in photoreceptor cells and photoreceptor progenitor cells; Rhodopsin, which is expressed in rod cells; Nrl, which is expressed in rod photoreceptor cells and rod photoreceptor progenitor cells; S-opsin and LM-opsin, which are expressed in cone photoreceptor cells; RXR-γ, which is expressed in cone cells, cone photoreceptor progenitor cells, and ganglion cells; TRβ2, OTX2, and OC2, which are expressed in cone photoreceptor cells or their progenitor cells that appear in the early stages of differentiation among cone photoreceptor cells; and Pax6, which is commonly expressed in horizontal cells, amacrine cells, and ganglion cells.

[0035] A "positive cell" refers to a cell that expresses a specific marker on its surface or inside the cell. For example, a "Chx10 positive cell" refers to a cell that expresses the Chx10 protein.

[0036] "Retinal pigment epithelial cells" refer to epithelial cells located outside the neural retina in a living retina. Whether or not cells are retinal pigment epithelial cells can be easily confirmed by those skilled in the art, for example, by the expression of cell markers (RPE65, MITF, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (dark brown), tight junctions between cells, and characteristic polygonal or cobblestone-like cell morphology. Whether or not cells have the function of retinal pigment epithelial cells can be easily confirmed by the secretion capacity of cytokines such as VEGF and PEDF. In one embodiment, retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.

[0037] The term "retinal layer" refers to the various layers that make up the retina, and specifically includes the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane.

[0038] The term "neuroretinal layer" refers to the various layers that make up the neuroretina, specifically including the photoreceptor cell layer, outer limiting membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. The "photoreceptor cell layer" refers to the outermost layer of the neuroretina, which contains a large number of one or more types of cells selected from the group consisting of photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), photoreceptor progenitor cells, and retinal progenitor cells. The layers other than the photoreceptor cell layer are called inner layers. Which retinal layer each cell belongs to can be confirmed by known methods, such as the presence or absence or degree of expression of cell markers.

[0039] In retinal tissue at a stage where the proportion of photoreceptor cells or photoreceptor progenitor cells is low, the layer containing proliferating neuroretinal progenitor cells is called the "neuroblastic layer," and both an inner neuroblastic layer and an outer neuroblastic layer exist. As is well known to those skilled in the art, this can be determined by the difference in color intensity (the outer neuroblastic layer is lighter and the inner neuroblastic layer is darker) under a bright-field microscope.

[0040] The term "ciliary body" includes the developmental and adult "ciliary body," "ciliary margin," and "ciliary body." Markers for the ciliary body include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The "ciliary marginal zone (CMZ)" is, for example, the tissue located at the boundary between the neuroretina and the retinal pigment epithelium in the living retina, and is a region that contains retinal tissue stem cells (retinal stem cells). The ciliary margin is also called the ciliary margin or retinal margin, and the ciliary margin, ciliary margin, and retinal margin are equivalent tissues. The ciliary margin is known to play an important role in the supply of retinal progenitor cells and differentiated cells to retinal tissue, and in maintaining the structure of retinal tissue. Examples of marker genes for the ciliary body periphery include the Rdh10 gene (positive), the Otx1 gene (positive), and the Zic1 gene (positive). A "ciliary body periphery-like structure" refers to a structure that resembles the ciliary body periphery.

[0041] A "cell aggregate" is defined as any structure formed by the adhesion of multiple cells, without any particular limitations. For example, it refers to a mass formed by the aggregation of cells dispersed in a medium such as a culture medium, or a mass of cells formed through cell division. Cell aggregates also include those that form specific tissues.

[0042] A "sphere-like cell aggregate" refers to a cell aggregate that has a nearly spherical, three-dimensional shape. A nearly spherical, three-dimensional shape is a shape with a three-dimensional structure, and when projected onto a two-dimensional plane, it may appear as a spherical shape that is circular or elliptical, or a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by the overlapping of 2 to 4 circular or elliptical shapes when projected onto a two-dimensional plane). In one embodiment, the core of the aggregate has a vesicular layered structure, and under a bright-field microscope, it is observed that the central part is dark and the outer edge is bright.

[0043] "Epithelial tissue" is tissue formed when cells completely cover the surface of the body, tubular lumens (such as the digestive tract), and body cavities (such as the pericardial cavity). The cells that make up epithelial tissue are called epithelial cells. Epithelial cells have polarity in the apical-basal direction. Epithelial cells can form strong bonds with each other through adherent junctions and / or tight junctions, creating layers of cells. Epithelial tissue is tissue formed by one to more than ten layers of these cell layers stacked on top of each other. Tissues that can form epithelial tissue include fetal and / or adult retinal tissue, brain and spinal cord tissue, ocular tissue, and nerve tissue. Neuroretina as used herein is also epithelial tissue. "Epithelial structure" refers to structures characteristic of epithelial tissue, such as the apical surface or basement membrane.

[0044] In this specification, epithelial structures containing nerve tissue are referred to as neuroepithelium. In particular, epithelial structures containing the neuroretina are referred to as neuroretinal epithelium.

[0045] "Continuous epithelial tissue" refers to tissue that has a continuous epithelial structure. A continuous epithelial structure means that the epithelial tissue is continuous. For example, continuous epithelial tissue means that 10 cells to 10 cells are connected tangentially to the epithelial tissue. 7 Cells, preferably 30 to 10 in the tangential direction 7 cells, more preferably 10 2 cells ~10 7This refers to the arrangement of cells. Continuous epithelial structures do not have structures where the apical surface is divided, as seen in rosette-like structures. In one embodiment, the number of cells per unit area of ​​a cross-section of retinal tissue having a continuous epithelial structure is, for example, when evaluating the number of cell nuclei in a frozen section of about 10 μm thickness, 100 μm 2 The number of cells per cell is 10 to 900, preferably 30 to 300, more preferably 50 to 250, and even more preferably 75 to 160.

[0046] For example, continuous epithelium formed in retinal tissue is formed on the surface of the retinal tissue in a manner that is generally parallel and continuous with the photoreceptor layer (outer granular layer), among the layers that make up the neuroretinal layer, where the apical surface is located, and which has an apical surface characteristic of epithelial tissue. For example, in the case of a cell aggregate containing retinal tissue made from pluripotent stem cells, an apical surface is formed on the surface of the aggregate, and continuous neuroepithelium is formed in which 10 or more cells, preferably 30 or more cells, more preferably 100 or more cells, and even more preferably 400 or more cells, of photoreceptor cells or photoreceptor progenitor cells are arranged in regular, continuous rows tangentially to the surface. Neuroretina containing such continuous neuroepithelium is neuroretinal epithelium containing continuous epithelium.

[0047] In one embodiment, epithelial tissue undergoes polarization to form an "apical surface" and a "basement membrane." The "basement membrane" is the basal layer produced by epithelial cells, containing a large amount of laminin and type IV collagen, and having a thickness of 50-100 nm. The "apical surface" is the surface (surface surface) formed on the opposite side of the "basement membrane." In one embodiment, in retinal tissue where the developmental stage has progressed to the point where photoreceptor cells or photoreceptor progenitor cells can be observed, the "apical surface" is the surface that is in contact with the photoreceptor layer (outer granular layer) where photoreceptor cells and photoreceptor progenitor cells exist, after the formation of the outer limiting membrane. Furthermore, such an apical surface can be identified by immunohistochemical staining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical-PKC (hereinafter abbreviated as "aPKC"), E-cadherin, N-cadherin).

[0048] Whether retinal tissue contains continuous epithelium can be confirmed by the continuity of the apical surface of the retinal tissue (i.e., its undisrupted morphology). The continuity of the apical surface can be determined, for example, by immunostaining apical surface markers (e.g., aPKC, E-cadherin, N-cadherin) or markers of photoreceptor cells or photoreceptor progenitor cells located on the apical side (e.g., Crx or recabin), and analyzing the positional relationship between the apical surface, the photoreceptor layer, and each retinal layer in the acquired images. Retinal layers other than the apical surface and the photoreceptor layer (outer granular layer) can be identified by immunostaining with DAPI staining, PI staining, Hoechst staining, or marker proteins localized in the cell nucleus (e.g., Rx, Chx10, Ki67, Crx, etc.).

[0049] Specifically, the continuity of the apical surface can be identified by the continuous presence of cells that express both cellular markers on the apical surface side, i.e., photoreceptor cell markers or photoreceptor progenitor cell markers and markers that can stain the cell nucleus.

[0050] [Complex] The complex of the present invention comprises cell aggregates containing two or more pluripotent stem cell-derived neuroretinas and a matrix, wherein the two or more cell aggregates are arranged in the matrix. The cell aggregates containing neuroretinas, the matrix, and the complex containing them will be described in detail below.

[0051] (1) Cell aggregates including the neuroretina In one embodiment, the cell aggregate containing the neuroretina is a cell aggregate (sphere-like cell aggregate) as described below. In one embodiment, the cell aggregate containing the neuroretina is a sheet-like retinal tissue excised from the sphere-like cell aggregate. The sheet-like retinal tissue is preferably a sheet-like neuroretina containing continuous epithelium of the neuroretina (hereinafter also referred to as a neuroretinal sheet). In one embodiment, the cell aggregate containing the neuroretina is a neuroretina for transplantation, preferably a neuroretinal sheet for transplantation. The neuroretina for transplantation or neuroretinal sheet for transplantation is a human neuroretina suitable for human transplantation, and more preferably consists only of neuroretina.

[0052] In the neuroretina, a neuroretinal layer is formed that includes at least a photoreceptor layer, and the photoreceptor layer contains at least one type of cell selected from the group consisting of photoreceptor cells, photoreceptor progenitor cells, and retinal progenitor cells. The photoreceptor layer is formed at least on the outermost part of the cell aggregate, and photoreceptor cells or photoreceptor progenitor cells may also be present inside the cell aggregate, or a photoreceptor layer may be formed inside as well. The photoreceptor cells, etc., are present in a continuous, tangential direction to the surface of the cell aggregate, i.e., they are adherent to one another, and the continuous presence of the photoreceptor cells, etc., in a tangential direction to the surface of the cell aggregate forms a photoreceptor layer containing the photoreceptor cells, etc. Note that the tangential direction refers to the direction tangential to the surface of the cell aggregate, i.e., the direction in which the photoreceptor cells, etc., are arranged in the photoreceptor layer, and is parallel or transverse to the neuroretina.

[0053] The neuroretina is derived from pluripotent stem cells, preferably from human pluripotent stem cells. The human pluripotent stem cells are preferably human embryonic stem cells or human induced pluripotent stem cells, and more preferably human induced pluripotent stem cells. Furthermore, the neuroretina is preferably a neuroretina for transplantation.

[0054] One embodiment of retinal tissue described herein includes an apical surface and a basal surface, where the apical surface forms an apical surface containing the neuroretinal layer, which is epithelial tissue, through adhesive junctions between cells, and the basal surface forms a basal surface adjacent to the inner layer of the neuroretina. Such retinal tissue can be called neuroretinal epithelium. Preferably, such retinal tissue is a neuroretina sheet, which is a sheet-like retinal tissue. The apical surface has a smooth shape with little change in curvature, while the basal surface has an irregular shape with large changes in curvature. In one embodiment, for example, the change in curvature of the retinal tissue surface may be close to the change in curvature of an ellipse (for example, a major axis of 1 to 10 relative to a minor axis of 1) (this can also be described as a continuous change in curvature). In another embodiment, for example, the change in curvature of the basal surface of the retinal tissue may be close to a rapid change in curvature that fluctuates between positive and negative values, like the teeth of a saw (this can also be described as a rapid change in curvature).

[0055] (Method for producing cell aggregates) Cell aggregates containing neuroretina are derived from pluripotent stem cells and can be obtained specifically by differentiating pluripotent stem cells. Methods disclosed in WO2011 / 055855, WO2013 / 077425, WO2015 / 025967, WO2016 / 063985, WO2016 / 063986, WO2017 / 183732, PLoS One. 2010 Jan 20;5(1):e8763., Stem Cells. 2011 Aug;29(8):1206-18., Proc Natl Acad Sci USA. 2014 Jun 10;111(23):8518-23, and Nat Commun. 2014 Jun 10;5:4047 are examples of methods for differentiating neuroretina, but are not particularly limited.

[0056] Furthermore, the method described in Bryce T. McLelland et al., IOVS, May 2018, Vol. 59, No. 6, p2586, can be cited as a method for producing retinal tissue. Additionally, the methods described in the following literature can be cited as methods for producing retinal tissue. Nakano, T. et al. Cell Stem Cell 10,771-785 (2012). Kawahara A. et al. Nature Communications,6, p.6286(2015). Kuwahara A, Yamasaki S, et al. Sci Rep.2019 Dec 12;9(1):18936. Lamba, D. A., Gust, J. & Reh, TACell Stem Cell 4, 73-79, (2009). Zhu, J., Cifuentes, H., Reynolds, J. &Lamba, DA Cell Stem Cell 20,374-384.e375(2017) Meyer, JS et al. Stem Cells 29,1206-1218, (2011). Zhong, X. et al. Nat Commun 5,doi:10.1038 / ncomms5047 (2014). Boucherie, C., et al. Stem Cells 31,408-414, doi:10.1002 / stem.1268 (2013). Gonzalez-Cordero, A. et al. Nat Biotechnol31, 741-747, (2013). Mellough, CB et al. Stem Cells 33,2416-2430, (2015). Hallam, D. et al. Stem Cells,doi:10.1002 / stem.2883 (2018). Reichman, S. et al.PNAS 111, 8518-8523,(2014). Gagliardi, G. et al. Stem Cell Reports 11,665-680, (2018). Tucker, BA, et al. Stem Cells Transl Med2, 16-24, (2013). Wahlin, KJ et al. Sci Rep 7, 766,(2017). DiStefano, T. et al. Stem Cell Reports 10,300-313, (2018).

[0057] In one specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A), (B), (C), and (D). (A) A step of culturing pluripotent stem cells in a culture medium for pluripotent stem cells in the absence of feeder cells. (B) A step in which cells obtained in step (A) are cultured in suspension to form cell aggregates. (C) A step of further suspension culturing the cell aggregates obtained in step (B) in a culture medium containing a BMP signaling pathway agent, and (D) A step in which the cell aggregates obtained in step (C) are cultured in suspension and allowed to mature. Step (A) may further include a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator. Furthermore, step (B) may include a sonic hedgehog signaling pathway activator and / or a Wnt signaling pathway inhibitor, as described later.

[0058] This method is also disclosed in WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732, for example. For more details, please refer to WO2015 / 025967, WO2016 / 063985, WO2017 / 183732, WO2019 / 017492, WO2019 / 054514, WO2019 / 054515, etc.

[0059] The "culture medium for pluripotent stem cells" used in process (A) is a culture medium capable of culturing pluripotent stem cells under feeder-free conditions, and such a medium includes a medium containing undifferentiated maintenance factors.

[0060] In this specification, the undifferentiated maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of undifferentiated maintenance factors commonly used by those skilled in the art include FGF signaling pathway activators, TGFβ family signaling pathway activators, and insulin. Specifically, examples of FGF signaling pathway activators include fibroblast growth factor (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ family signaling pathway activators include TGFβ signaling pathway activators and Nodal / Activin signaling pathway activators. Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling pathway activators include Nodal, ActivinA, and ActivinB. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the culture medium in step (A) preferably contains bFGF as the undifferentiated maintenance factor.

[0061] The concentration of the undifferentiated maintenance factor in the culture medium used in step (A) is a concentration capable of maintaining the undifferentiated state of the pluripotent stem cells being cultured, and can be appropriately set by those skilled in the art. For example, specifically, when bFGF is used as the undifferentiated maintenance factor in the absence of feeder cells, its concentration is usually about 4 ng to 500 ng / mL, preferably about 10 ng to 200 ng / mL, and more preferably about 30 ng to 150 ng / mL.

[0062] Many synthetic culture media have been developed and are commercially available for use under feeder-free conditions for pluripotent stem cell culture. An example is Essential 8 medium (manufactured by Life Technologies). Essential 8 medium is DMEM / F12 medium containing, as an additive, L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and a TGFβ family signaling pathway activator (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Other commercially available feeder-free media include S-medium (DS Pharma Biomedical), StemPro (Life Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14), mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), TeSR-E8 (STEMCELL Technologies), or StemFit (Ajinomoto Co., Inc.). By using these media in step (A), the present invention can be easily carried out. By using these media, it is possible to culture pluripotent stem cells under feeder-free conditions. The media used in step (A) is, as an example, a serum-free medium that does not contain any BMP signaling pathway activators, Wnt signaling pathway activators, or Wnt signaling pathway inhibitors.

[0063] Unless otherwise specified, the culture medium used for preparing cell aggregates including the neuroretina, i.e., the culture medium used in steps (B), (C), and (D) above, can be a basal medium for cell proliferation (also called a basal medium). The basal medium for cell proliferation is not particularly limited as long as it can be used to culture cells, and any commercially available basal medium for cell proliferation can be used as appropriate. Specifically, examples of media that can be used to culture animal cells include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, MEM medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, Leibovitz's L-15 medium, or mixtures thereof. In addition, a medium supplemented with N2 medium may be used.

[0064] In this specification, TGFβ family signaling pathway inhibitors refer to substances that inhibit the signaling pathway transmitted by the TGFβ family, i.e., the Smad family. Specifically, examples include TGFβ signaling pathway inhibitors (e.g., SB431542, LY-364947, SB505124, A-83-01, etc.), Nodal / Activin signaling pathway inhibitors (e.g., SB431542, A-83-01, etc.), and BMP signaling pathway inhibitors (e.g., LDN193189, Dorsomorphin, etc.). These substances are commercially available.

[0065] In this specification, a Sonic Hedgehog (hereinafter sometimes referred to as "Shh") signaling pathway activator is a substance that can enhance signal transduction mediated by Shh. Examples of Shh signaling pathway activators include SHH, partial peptides of SHH (e.g., Sonic Hedgehog N-Terminus (Shh-N), recombinant Human Sonic Hedgehog (C24II) N-Terminus (SHH-C24II), recombinant Mouse Sonic Hedgehog (C25II) N-Terminus (SHH-C25II)), Hedgehog family proteins other than Shh (e.g., Hh, IHH, DHH, EHH, TwHH), PMA (Purmorphamine), SAG (Smoothened Agonist), etc.

[0066] In step (A) above, the concentrations of the TGFβ family signaling pathway inhibitor and the Sonic Hedgehog signaling pathway activator should be such that differentiation into retinal cells can be induced. For example, SB431542 is usually used at a concentration of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is usually used at a concentration of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is usually used at a concentration of 1 to 2000 nM, preferably 10 to 300 nM. SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM. PMA is usually used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM.

[0067] In the culture of pluripotent stem cells under feeder-free conditions in step (A), an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold in place of feeder cells. Examples of matrices that can be used as a scaffold include laminin (Nat Biotechnol 28,611-615,(2010)), laminin fragments (Nat Commun 3,1236,(2012)), basement membrane preparations (Nat Biotechnol 19,971-974,(2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin.

[0068] The culture time of pluripotent stem cells in step (A) is not particularly limited as long as the effect of improving the quality of the cell aggregates formed in step (B) can be achieved when cultured in the presence of a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator (e.g., 100 nM to 700 nM), but is usually 0.5 to 144 hours. In one embodiment, it is preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours).

[0069] The culture medium used in step (B) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding contamination with chemically undetermined components, a serum-free medium is preferably used. To avoid the complexity of preparation, for example, a serum-free medium to which an appropriate amount of a commercially available serum substitute such as KSR has been added can be used. The amount of KSR added to the serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%.

[0070] In forming aggregates, first, dispersed cells are prepared by dispersing the cells obtained in step (A). The "dispersed cells" obtained by the dispersion operation include, for example, a state in which 70% (preferably 80% or more) or more are single cells and 30% or less (preferably 20% or less) are clusters of 2 to 50 cells. Dispersed cells are in a state in which cell-to-cell adhesion (e.g., surface adhesion) is almost completely eliminated.

[0071] A suspension of dispersed cells is seeded into a culture vessel, and the dispersed cells are cultured under non-adherent conditions with respect to the culture vessel, thereby aggregating a plurality of cells to form an aggregate. In one embodiment, when a fixed number of dispersed stem cells are placed into each well of a multi-well plate (U-bottom, V-bottom) such as a 96-well plate and the plate is subjected to static culture, the cells rapidly aggregate to form one aggregate in each well (the SFEBq method). When cells are cultured in suspension using a 96-well plate, about 1×10 3 to about 1×10 5 cells (preferably about 3×10 3 to about 5×10 4 cells, about 4×10 3 to about 2×10 4 cells) is added to the wells, and the plate is left standing to form aggregates.

[0072] In one embodiment, the medium used in step (B) comprises a Sonic Hedgehog signaling pathway agonist. That is, as a specific embodiment, cell aggregates containing neural retina can be prepared by a method comprising the following steps (A), (B) and (C): (A) a step of culturing pluripotent stem cells in the absence of feeder cells, in a medium containing an undifferentiation maintenance factor, which may optionally contain a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway agonist, (B) a step of forming cell aggregates by suspension-culturing the cells obtained in step (A) in a medium containing a Sonic Hedgehog signaling pathway agonist, (C) a step of further suspension-culturing the cell aggregates obtained in step (B) in a medium containing a BMP signaling pathway agonist. In step (B), the sonic hedgehog signaling pathway activators described above can be used at the concentrations described above (e.g., 10 nM to 300 nM). Preferably, the sonic hedgehog signaling pathway activators are included in the culture medium from the start of suspension culture. ROCK inhibitors (e.g., Y-27632) may be added to the culture medium. The culture time is, for example, 12 hours to 6 days. In one example, the culture medium used in step (B) is a medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, Wnt signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0073] In one embodiment, the cell aggregates in step (B) are at a differentiation stage that express pluripotency markers. Specifically, this is a differentiation stage in which one or more markers selected from Oct3 / 4, Sox2, Klf4, Nanog, Sall4, lin28, Esrrb, and Esrrb can be detected.

[0074] In this specification, a BMP signaling pathway activator is a substance that can enhance the signaling pathway mediated by BMPs. Examples of BMP signaling pathway activators include BMP proteins such as BMP2, BMP4, or BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, or BMP partial peptides. BMP2, BMP4, and BMP7 proteins are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries.

[0075] Examples of media used in step (C) include serum-free media or serum media (preferably serum-free media) to which a BMP signaling pathway activator has been added. Serum-free media and serum media can be prepared as described above. In one example, the media used in step (C) is a media that does not contain one or more (preferably all) selected from the group consisting of Wnt signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators. In another example, the media used in step (C) is a media that does not contain a Sonic Hedgehog signaling pathway activator. In yet another example, the media used in step (C) may contain a Wnt signaling pathway activator.

[0076] The concentration of the BMP signaling pathway activator should be such that it can induce differentiation into retinal cells. For example, in the case of human BMP4 protein, it should be added to the culture medium at a concentration of approximately 0.01 nM to approximately 1 μM, preferably approximately 0.1 nM to approximately 100 nM, more preferably approximately 1 nM to approximately 10 nM, and even more preferably approximately 1.5 nM (55 ng / mL).

[0077] The BMP signaling pathway activator may be added approximately 24 hours after the start of suspension culture in step (A), or it may be added to the culture medium within a few days after the start of suspension culture (for example, within 15 days). Preferably, the BMP signaling pathway activator is added to the culture medium between day 1 and day 15 after the start of suspension culture, more preferably between day 1 and day 9, and most preferably on day 3.

[0078] As a specific embodiment, for example, on days 1 to 9, preferably 1 to 3, after the start of suspension culture in step (B), part or all of the culture medium is replaced with a medium containing BMP4, and the medium is prepared so that the final concentration of BMP4 is about 1 to 10 nM. The culture can then be performed in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, and more preferably 2 to 5 days. In this case, to maintain the same concentration of BMP4, part or all of the culture medium may be replaced with a medium containing BMP4 once or twice. Alternatively, the concentration of BMP4 may be gradually reduced. For example, the concentration of BMP signaling pathway activators (BMP4) may be maintained from day 2 to 10 after the start of suspension culture in step (B), and then the concentration of BMP signaling pathway activators (BMP4) may be gradually reduced from day 6 to 20 after the start of suspension culture in step (B).

[0079] The culture conditions in steps (A) to (C) above, such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0080] By varying the culture period in step (C) above, it is possible to produce retinal cells at various stages of differentiation as retinal cells contained in cell aggregates. In other words, it is possible to produce retinal cells in cell aggregates containing immature retinal cells (e.g., retinal progenitor cells, photoreceptor progenitor cells) and mature retinal cells (e.g., photoreceptor cells) in various proportions. By extending the culture period in step (C), the proportion of mature retinal cells can be increased.

[0081] Steps (B) and / or (C) described above may also be carried out using the method disclosed in WO2017 / 183732. That is, in steps (B) and / or (C), the cells may be suspended in a medium further containing a Wnt signaling pathway inhibitor to form cell aggregates.

[0082] The Wnt signaling pathway inhibitor used in step (B) and / or step (C) is not particularly limited as long as it can suppress Wnt-mediated signaling, and may be any protein, nucleic acid, small molecule compound, etc. The Wnt-mediated signal is transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and LRP5 / 6 (low-density lipoprotein receptor-related protein 5 / 6). Examples of Wnt signaling pathway inhibitors include substances that directly act on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus protein, etc.), and substances that inhibit physiological activity resulting from signal transduction by Wnt receptors [CKI-7(N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476(4-[4-(2 Examples include, but are not limited to, low molecular weight compounds such as, 3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide, IWR-1-endo(IWR1e)(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl]-N-8-quinolinyl-benzamide), and IWP-2(N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide). One or more of these may be included as Wnt signaling pathway inhibitors. CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc., are known Wnt signaling pathway inhibitors and are readily available commercially. IWR1e is preferably used as the Wnt signaling pathway inhibitor.

[0083] The concentration of the Wnt signaling pathway inhibitor in step (B) should be such that it can induce the formation of good cell aggregates. For example, in the case of IWR-1-endo, it should be added to the culture medium at a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When using a Wnt signaling pathway inhibitor other than IWR-1-endo, it is desirable to use it at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo.

[0084] In step (B), it is preferable to add the Wnt signaling pathway inhibitor to the culture medium as early as possible. The Wnt signaling pathway inhibitor is usually added to the culture medium within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, from the start of suspension culture in step (B), and even more preferably at the start of suspension culture in step (B). Specifically, for example, this can be done by adding a basal medium to which the Wnt signaling pathway inhibitor has been added, or by replacing part or all of the basal medium. The period during which the cells obtained in step (A) are exposed to the Wnt signaling pathway inhibitor in step (B) is not particularly limited, but preferably, after being added to the culture medium at the start of suspension culture in step (B), it is allowed to act until the end of step (B) (immediately before the addition of the BMP signaling pathway inhibitor). Even more preferably, as described later, the cells are continuously exposed to the Wnt signaling pathway inhibitor even after the end of step (B) (i.e., during the period of step (C)). In one embodiment, as will be described later, the Wnt signaling pathway inhibitor may be allowed to act continuously even after the completion of step (B) (i.e., during the period of step (C)) until retinal tissue is formed.

[0085] In step (C), any of the aforementioned Wnt signaling pathway inhibitors can be used as the Wnt signaling pathway inhibitor, but preferably, the same type of Wnt signaling pathway inhibitor used in step (B) is used in step (C).

[0086] The concentration of the Wnt signaling pathway inhibitor in step (C) should be such that it can induce retinal progenitor cells and retinal tissue. For example, in the case of IWR-1-endo, it should be added to the culture medium at a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When using a Wnt signaling pathway inhibitor other than IWR-1-endo, it is desirable to use it at a concentration that shows Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the culture medium in step (C) is preferably 50 to 150, more preferably 80 to 120, and even more preferably 90 to 110, when the concentration of the Wnt signaling pathway inhibitor in the culture medium in step (B) is set to 100, and it is more preferable that it is equivalent to the concentration of the Wnt signaling pathway inhibitor in the culture medium in step (B).

[0087] The timing of adding the Wnt signaling pathway inhibitor to the culture medium is not particularly limited as long as aggregate formation including retinal cells or retinal tissue can be achieved, but the earlier the better. Preferably, the Wnt signaling pathway inhibitor is added to the culture medium at the start of step (C). More preferably, after the Wnt signaling pathway inhibitor is added in step (B), it is continuously included in the culture medium in step (C) as well (i.e., from the start of step (B)). Even more preferably, after the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B), it is continuously included in the culture medium in step (C) as well. For example, a BMP signaling pathway activator (e.g., BMP4) may be added to the culture obtained in step (B) (a suspension of aggregates in the culture medium containing the Wnt signaling pathway inhibitor).

[0088] The duration for which the Wnt signaling pathway inhibitor is applied is not particularly limited, but is preferably 2 to 30 days, more preferably 6 to 20 days, 8 to 18 days, 10 to 18 days, or 10 to 17 days (e.g., 10 days), starting from the time of the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B). In another embodiment, the duration for which the Wnt signaling pathway inhibitor is applied is preferably 3 to 15 days (e.g., 5, 6, or 7 days), more preferably 6 to 10 days (e.g., 6 days), starting from the time of the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B).

[0089] The cell aggregates obtained by the method described above can also be cultured for approximately 2 to 4 days in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor (step (D)), and then cultured for approximately 30 to 200 days (30 to 150 days, 50 to 120 days, 60 to 90 days) in serum-free medium or serum medium that does not contain the Wnt signaling pathway activator and the FGF signaling pathway inhibitor (step (E)) to produce a neuroretina containing a ciliary body periphery-like structure.

[0090] In one embodiment, a neuroretina containing a ciliary body periphery-like structure can be produced from cell aggregates obtained in steps (A) to (C) at 6 to 30 days and 10 to 20 days (10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th day) after the start of suspension culture in step (B) by steps (D) and (E).

[0091] The Wnt signaling pathway activator is not particularly limited as long as it can enhance Wnt-mediated signal transduction. Specific examples of Wnt signaling pathway activators include GSK3β inhibitors (e.g., 6-Bromoindirubin-3'-oxime (BIO), CHIR99021, Kenpaullone). For example, in the case of CHIR99021, the concentration can range from about 0.1 μM to about 100 μM, preferably from about 1 μM to about 30 μM.

[0092] The FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit FGF-mediated signal transduction. Examples of FGF signaling pathway inhibitors include SU-5402, AZD4547, and BGJ398. For example, SU-5402 is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 5 μM.

[0093] In step (D), the culture medium used is, for example, a culture medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, Wnt signaling pathway inhibitors, SHH signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0094] Some or all of the above steps (E) can be performed using a continuous epithelial tissue maintenance medium disclosed in WO2019 / 017492. That is, the continuous epithelial structure of the neuroretina can be maintained by culturing using a continuous epithelial tissue maintenance medium. As an example, a medium prepared by adding B27 supplement (e.g., Thermo Fisher Scientific, 21103049) to Neurobasal medium can be used as a continuous epithelial tissue maintenance medium.

[0095] In step (E) above, it is preferable to gradually replace the culture medium with a medium for maintaining continuous epithelial tissue in order to achieve both differentiation and / or maturation of retinal cells (especially photoreceptor cells) and maintenance of the continuous epithelial structure. For example, the cells can be cultured for the first 10 to 30 days using a basal medium for cell proliferation (e.g., DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine), for the next 10 to 40 days using a mixed medium of basal medium for cell proliferation and continuous epithelial tissue maintenance (a medium prepared by mixing DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine with Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine in a 1:3 ratio), and for the next 20 to 140 days using a continuous epithelial tissue maintenance medium (e.g., Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine).

[0096] In any part or all of the above step (E), regardless of whether a basal medium for cell proliferation, a medium for maintaining continuous epithelial tissue, or a mixture thereof is used, the medium may further contain a thyroid hormone signaling pathway activator. For details of the culture method using a medium containing a thyroid hormone signaling pathway activator, refer to WO2019 / 054514. By culturing in a medium containing a thyroid hormone signaling pathway activator, it becomes possible to produce cell aggregates containing neuroretina in which the proportion of bipolar cells, amacrine cells, ganglion cells, or horizontal cells contained in the neuroretina is low, and the proportion of photoreceptor progenitor cells is increased.

[0097] In this specification, a thyroid hormone signaling pathway agonist is a substance that can enhance signal transduction mediated by thyroid hormones, and is not particularly limited as long as it can enhance the thyroid hormone signaling pathway. Examples of thyroid hormone signaling pathway agonists include triiodothyronine (hereinafter sometimes abbreviated as T3), thyroxine (hereinafter sometimes abbreviated as T4), and thyroid hormone receptor (preferably TRβ receptor) agonists.

[0098] Furthermore, as thyroid hormone receptor agonists well known to those skilled in the art, see International Publication No. 97 / 21993, International Publication No. 2004 / 066929, International Publication No. 2004 / 093799, International Publication No. 2000 / 039077, International Publication No. 2001 / 098256, International Publication No. 2003 / 018515, International Publication No. 2003 / 084915, International Publication No. 2002 / 094319, International Publication No. 2003 / 064369, and Japanese Patent Publication No. 20 Examples of compounds such as diphenylmethane derivatives, diaryl ether derivatives, pyridazine derivatives, pyridine derivatives, or indole derivatives described in Japanese Patent Publication No. 02-053564, Japanese Patent Publication No. 2002-370978, Japanese Patent Publication No. 2000-256190, International Publication Brochure No. 2007 / 132475, International Publication Brochure No. 2007 / 009913, International Publication Brochure No. 2003 / 094845, International Publication Brochure No. 2002 / 051805, or International Publication Brochure No. 2010 / 122980.

[0099] When T3 is used as a thyroid hormone signaling pathway agonist, it can be added to the culture medium in a range of, for example, 0.1 to 1000 nM. Preferably, concentrations that have thyroid hormone signaling-enhancing activity equivalent to T3 concentrations of around 1 to 500 nM; more preferably 10 to 100 nM; even more preferably 30 to 90 nM; and even more preferably around 60 nM are used. When T4 is used as a thyroid hormone signaling pathway agonist, it can be added to the culture medium in a range of, for example, 1 nM to 500 μM. Preferably, the range is 50 nM to 50 μM; more preferably 500 nM to 5 μM. When other thyroid hormone receptor agonists are used, any concentration that shows activity comparable to that of T3 or T4 at the above concentrations is acceptable.

[0100] The culture medium used in step (E) may optionally contain L-glutamine, taurine, serum, etc. In one example, the culture medium used in step (E) is a culture medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, FGF signaling pathway inhibitors, Wnt signaling pathway activators, Wnt signaling pathway inhibitors, SHH signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0101] As a specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A) to (E): (A) A step of culturing pluripotent stem cells in the absence of feeder cells in a medium that contains undifferentiated maintenance factors and may optionally contain TGFβ family signaling pathway inhibitors and / or Sonic Hedgehog signaling pathway activators. (B) A step of forming cell aggregates by suspension culture of the cells obtained in step (A) in a medium which may contain a Wnt signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator. (C) A step in which the cell aggregates obtained in step (B) are further cultured in suspension in a culture medium containing a BMP signaling pathway agent. (D) A step of culturing the cell aggregates obtained in step (C) in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor for a period of 2 to 4 days, and (E) A step in which the cell aggregates obtained in step (D) are cultured for approximately 30 to 200 days in a serum-free medium or serum medium that does not contain Wnt signaling pathway activators and FGF signaling pathway inhibitors, but may contain thyroid hormone signaling pathway activators.

[0102] As a specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A) to (E): (A) A step of culturing pluripotent stem cells for 12 to 48 hours in a medium containing undifferentiated maintenance factors and TGFβ family signaling pathway inhibitors and / or Sonic Hedgehog signaling pathway activators, in the absence of feeder cells. (B) A step in which cells obtained in step (A) are cultured in suspension for 12 hours to 72 days (24 hours to 48 hours) in a medium containing a Wnt signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator to form cell aggregates. (C) The cell aggregates obtained in step (B) are further cultured in suspension for 8 to 15 days (10 to 13 days) in a medium containing a BMP signaling pathway activator. (D) A step of culturing the cell aggregates obtained in step (C) in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor for 2 to 4 days, and (E) A step in which the cell aggregates obtained in step (D) are cultured for about 10 to 200 days in a serum-free medium or serum medium that does not contain Wnt signaling pathway activators and FGF signaling pathway inhibitors, but may contain thyroid hormone signaling pathway activators.

[0103] Step (E) may include culturing the cells in a basal medium for cell proliferation for 10 to 30 days, then culturing them in a mixed medium of the basal medium for cell proliferation and a continuous epithelial tissue maintenance medium containing a thyroid hormone signaling pathway activator for 10 to 40 days, and further culturing them in a continuous epithelial tissue maintenance medium containing a thyroid hormone signaling pathway activator for 20 to 140 days. In one embodiment, step (E) includes culturing for 20 to 60 days (30 to 50 days) in the presence of a thyroid hormone signaling pathway activator. In one embodiment, the incubation period from step (B) to step (E) is 70 to 100 days (80 to 90 days).

[0104] The above-described methods can be used to produce cell aggregates containing neuroretina, but are not limited to these methods. In one embodiment, cell aggregates containing neuroretina can also be obtained as a mixture of cell aggregates. In another embodiment, for example, one cell aggregate can be produced in each well of a 96-well plate, and cell aggregates containing neuroretina can be obtained one at a time.

[0105] The neuroretina according to the present invention comprises a neuroretinal layer including a photoreceptor cell layer and an inner layer, and the photoreceptor cell layer contains one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, and has an epithelial tissue structure, having an apical surface and a base. The epithelial tissue structure is preferably a continuous epithelial structure.

[0106] In one embodiment, the neuroretina according to the present invention can be obtained by cutting it out from a cell aggregate of the neuroretina using tweezers, a knife, scissors, etc. A neuroretinal sheet can be obtained by cutting it out from a sphere-like cell aggregate of the neuroretina. A neuroretinal sheet refers to a multilayered sheet-like structure that maintains the layered structure of the neuroretinal layer described above.

[0107] In one embodiment, a cell aggregate containing a neuroretina may contain multiple overlapping neuroretinas (see, for example, conceptual diagrams (1) and (2) in Figure 6). In one embodiment, the cell aggregate containing a neuroretina includes at least a first epithelial tissue and a second epithelial tissue, the first epithelial tissue including a human neuroretina, and the second epithelial tissue having a continuity of tangent slopes on a surface different from that of the first epithelial tissue, and may include cells other than retinal cells and / or retinal pigment epithelial cells. Examples of cells other than retinal cells included in the second epithelial tissue include cells of ocular-related tissue and cerebrospinal tissue, and examples of ocular-related tissue include retinal pigment epithelial cells and peripheral ciliary body structures. The neuroretina according to the present invention can be obtained by visually cutting the neuroretina from the aggregate so as not to include the second epithelial tissue. The neuroretina according to the present invention may include the region on the first epithelial tissue furthest from the second epithelial tissue in the cell aggregate, particularly the region near the center of the first epithelial tissue. In one embodiment, the neuroretina according to the present invention may include a region near the center of continuous epithelial tissue.

[0108] In one embodiment, a cell aggregate containing a neuroretina comprises a first epithelial tissue (target epithelial tissue) containing a neuroretina for transplantation, and a second epithelial tissue (non-target epithelial tissue) having a continuity of tangent slopes on the surface different from that of the first epithelial tissue and containing non-neuroretinal cells. Here, the first epithelial tissue substantially does not contain non-neuroretinal cells (non-target cells) and represents epithelial tissue from which a neuroretina for transplantation can be excised. On the other hand, the second epithelial tissue may contain a neuroretina, but is an epithelial tissue that is unsuitable for excising a neuroretina for transplantation because it contains non-target cells. In another embodiment, a cell aggregate containing a neuroretina comprises only the first epithelial tissue (target epithelial tissue) containing a neuroretina for transplantation and does not contain the non-target epithelial tissue.

[0109] The neuroretina for transplantation includes at least a photoreceptor layer, which is formed at least on the outermost part of the cell aggregates, and may also contain photoreceptor cells or photoreceptor progenitor cells on the inside, or a photoreceptor layer may be formed on the inside. The photoreceptor cells are continuous in the tangential direction to the surface of the cell aggregates, i.e., they are adherent to one another, and the continuous presence of the photoreceptor cells in the tangential direction to the surface of the cell aggregates forms a photoreceptor layer containing the photoreceptor cells. The tangential direction refers to the direction tangential to the surface of the cell aggregates, i.e., the direction in which the photoreceptor cells are arranged in the photoreceptor layer, and is parallel or transverse to the neuroretina. Furthermore, the tangential inclination on the surface of epithelial tissue refers to the direction in which cells are arranged when each cell in the epithelial tissue is arranged in a certain direction, and is parallel or transverse to the epithelial tissue (or epithelial sheet).

[0110] Cell aggregates used to prepare a neuroretina for transplantation may contain non-intended tissues other than the neuroretina. Such non-intended tissues include epithelial tissues other than the neuroretina, i.e., second epithelial tissues including non-intended epithelial tissues. Examples of second epithelial tissues include ocular tissues and cerebrospinal tissues. Ocular tissues refer to tissues surrounding the ocular tissues of the non-neuroretina and include retinal pigment epithelial cells, ciliary body (e.g., periphery of the ciliary body), lens, and cornea. Cerebrospinal tissues refer to the nerve tissues of the brain and spinal cord and include the forebrain, telencephalon, cerebrum, diencephalon, hypothalamus, midbrain, hindbrain, cerebellum, and spinal cord. In one embodiment, cerebrospinal tissues may also include the pituitary gland.

[0111] Examples of cell aggregates containing primary and secondary epithelial tissues include the conceptual diagrams in Figure 5 and Figures 6 (3) and (5). The conceptual diagram in Figure 5 shows an example of a cell aggregate in which a portion of the neuroretina, which is the primary epithelial tissue, contains ophthalmoplastic tissue (retinal pigment epithelial cells, ciliary body) (black area in Figure 5) as the secondary epithelial tissue. Conceptual diagram (3) in Figure 6 shows an example of a cell aggregate in which multiple neuroretinas overlap (e.g., conceptual diagrams (1) and (2) in Figure 6), and further contains ophthalmoplastic tissue (retinal pigment epithelial cells, ciliary body) (black area in conceptual diagram (3) in Figure 6) as the secondary epithelial tissue. Conceptual diagram (5) in Figure 6 shows an example of a cell aggregate in which cerebrospinal tissue (cerebrum, etc.) (gray area in conceptual diagram (5) in Figure 6) is present as the secondary epithelial tissue. As shown in conceptual diagram (4) in Figure 6, unintended tissue may be included inside the cell aggregate containing the neuroretina for transplantation. In this case, it does not meet the definition of "having a continuity of tangent slope on a surface different from the continuity of tangent slope on the surface of the first epithelial tissue," and therefore does not qualify as the second epithelial tissue. It is preferable to select the neuroretina for transplantation and the sample for quality evaluation from cell aggregates that do not contain non-target tissue on the inside.

[0112] <Extraction process> When excising a neuroretina for transplantation from a cell aggregate, it is desirable to use the presence of marker-positive cells for photoreceptor cells or photoreceptor progenitor cells as an indicator. Furthermore, it is desirable to use the absence of marker-positive cells of non-target cells as an indicator.

[0113] To obtain a neuroretina for transplantation that contains photoreceptor cells or photoreceptor progenitor cell marker-positive cells, and in which the number of marker-positive cells in non-target cells is below the standard (or in which non-target cell markers are negative), it is desirable to evaluate the quality of the neuroretina for transplantation in advance.

[0114] For example, the quality of a neuroretina for transplantation can be evaluated by sampling some or all of a cell aggregate containing a neuroretina with an epithelial structure derived from pluripotent stem cells as a quality evaluation sample (hereinafter referred to as the "extraction step"), and detecting markers expressed in the extracted sample by a method well known to those skilled in the art. Sampling some of a cell aggregate as a quality evaluation sample means selecting some (one or more) or all of a cell aggregate from a group of cell aggregates, and isolating a portion of the selected cell aggregate as an evaluation sample using tweezers, scissors, and / or a knife (e.g., dissecting). Sampling all of a cell aggregate as a quality evaluation sample means selecting some (one or more) cell aggregates from a group of cell aggregates, and separately picking up all of the selected one or more cell aggregates as a quality evaluation sample. When selecting one or more cell aggregates from a group of cell aggregates, random sampling is preferable. In this specification, when a portion of a cell aggregate is extracted as a quality evaluation sample, the cell aggregate is referred to as "a cell aggregate containing a quality evaluation sample," and when the entire cell aggregate is extracted as a quality evaluation sample, the cell aggregate is referred to as "a cell aggregate of a quality evaluation sample."

[0115] In one embodiment, the quality evaluation sample is a portion of a cell aggregate containing a neuroretina with an epithelial structure derived from pluripotent stem cells. By extracting a portion of the cell aggregate as a quality evaluation sample, there is an advantage in that the entire cell aggregate is not destroyed, and the neuroretina contained in the remaining portion can be used for transplantation. That is, if the quality evaluation sample, which is a portion of the cell aggregate, is judged to be acceptable by the judgment process described later, the neuroretina with an epithelial structure in the cell aggregate containing the quality evaluation sample can be used as a neuroretina for transplantation and can be used for transplantation.

[0116] In cell aggregates, areas exhibiting a continuous epithelial structure and appearing as two distinct layers—an outer neuroblastic layer and an inner neuroblastic layer—can be identified as the neuroretina. On the other hand, ocular tissues, particularly retinal pigment epithelial cells, which constitute a second epithelial tissue, appear black under the naked eye or microscope, making them easily distinguishable from the neuroretina by those skilled in the art. Furthermore, cerebrospinal fluid tissues, which also constitute a second epithelial tissue, can be easily distinguished from the neuroretina by those skilled in the art, focusing on morphological characteristics such as the absence of a continuous epithelial structure on the surface of the cell aggregate, the absence of morphological features specific to the neuroretina, and / or a duller color. Therefore, even in cell aggregates containing a second epithelial tissue, those skilled in the art can isolate neuroretina for transplantation and samples for quality evaluation from the first epithelial tissue containing the neuroretina.

[0117] As described above, in one embodiment, a quality evaluation sample is set and extracted based on a certain positional relationship with the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted. That is, by setting the neuroretina to be transplanted or a candidate thereof, the region to be cut out as a quality evaluation sample can be determined. Here, the neuroretina to be transplanted (also called a graft or cap) and its candidates can be identified in one embodiment by their position within the cell aggregate described above (e.g., near the center of the epithelial tissue (continuous epithelial tissue), and if there is a second epithelial tissue, further, the region on the first epithelial tissue furthest from the second epithelial tissue), and the size described later in (neuroretina sheet to be transplanted). Therefore, a person skilled in the art can set a neuroretina having these characteristics as the neuroretina to be transplanted or a candidate thereof. When extracting a neuroretina for transplantation and a quality evaluation sample from the same cell aggregate, a person skilled in the art can define the quality evaluation sample (also called a ring) as a region that is at least partially continuous with or adjacent to the neuroretina for transplantation as defined above, and as the narrowest possible region within which quality evaluation is possible. When extracting a portion of one or more cell aggregates from the same lot of the cell aggregate as a quality evaluation sample, a person skilled in the art can define the quality evaluation sample as the portion of the neuroretina for transplantation or a candidate thereof from the cell aggregate as defined above. In this case, the size of the sample cut out for quality evaluation may be the size described later (neuroretina for transplantation sheet), or it may be even smaller. Therefore, the quality evaluation sample can be defined and extracted based on its position relative to the neuroretina for transplantation or a candidate thereof and the size described above.

[0118] <Detection Process> The method for evaluating the quality of a neuroretina for transplantation according to the present invention includes detecting the expression of neuroretinal cell-related genes and non-neurotinic cell-related genes (off-target cell-related genes) in a quality evaluation sample (detection step). It is preferable that the detection step quantitatively detects the expression levels of the genes. The off-target cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eyeball-related tissue marker genes.

[0119] (Neuroretinal cell-related genes) Neuroretinal cell-related genes (target cell-related genes) refer to genes expressed by neuroretinal cells. Preferred neuroretinal cell-related genes include photoreceptor cells (rod and cone cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod and cone bipolar cells), Müller glial cells, or their progenitor cells, neuroretinal progenitor cells, etc., which are expressed at higher levels compared to non-target cells. Examples of neuroretinal cell-related genes include the neuroretinal cell markers listed above, with RAX, Chx10, SIX3, SIX6, RCVRN, CRX, NRL, and NESTIN being preferred. The GenBank IDs for the neuroretinal cell markers are shown in Table 1 below. [Table 1]

[0120] The neuroretinal cell-related genes are preferably those listed in Table 1, but are not limited to these. Other neuroretinal cell-related genes include Rax2, Vsx1, Blimp1, RXRG, S-opsin, M / L-opsin, Rhodopsin, Brn3, and L7.

[0121] (Non-neuroretinal cell-related genes) This can be identified by detecting off-target cells induced as by-products during the manufacturing process of cell aggregates, including neuroretina, as pharmaceutical raw materials, and genes expressed in cells or tissues from which such off-target cells may be produced as by-products (hereinafter referred to as non-neuroretinal cell-related genes or off-target cell-related genes).

[0122] In one embodiment, non-neuroretinal cell-related genes (non-target cell-related genes) include brain and spinal cord tissue marker genes and eyeball-related tissue marker genes. In one embodiment, undifferentiated iPS cell marker genes may also be included as non-neuroretinal cell-related genes.

[0123] In one embodiment, the brain and spinal cord tissue marker gene may be one or more genes selected from the group consisting of telencephalon marker genes, diencephalon / midbrain marker genes, and spinal cord marker genes. The diencephalon / midbrain marker gene may be one or more genes selected from the group consisting of diencephalon marker genes, midbrain marker genes, and hypothalamic marker genes related to the hypothalamus, which is part of the diencephalon.

[0124] In one embodiment, the eye-related tissue marker gene may be one or more genes selected from the group consisting of optic stalk marker genes, ciliary body marker genes, lens marker genes, and retinal pigment epithelial marker genes.

[0125] Telencephalon marker genes refer to genes expressed in the telencephalon. Telencephalon marker genes may include one or more genes selected from the group consisting of FoxG1 (also known as Bf1), Emx2, Dlx2, Dlx1, and Dlx5. The GenBank IDs of telencephalon marker genes are shown in Table 2 below. [Table 2]

[0126] The telencephalon marker genes are preferably those listed in Table 2, but are not limited to these. Other telencephalon marker genes include Emx1, LHX2, LHX6, LHX7, and Gsh2.

[0127] The term "diencephalon / midbrain marker gene" refers to a gene expressed in the diencephalon and / or midbrain. A diencephalon / midbrain marker gene may include one or more genes selected from the group consisting of OTX1, OTX2, and DMBX1. The GenBank IDs for the diencephalon / midbrain marker genes are shown in Table 3 below. The diencephalon / midbrain marker gene may also include hypothalamic markers, which are a region of the diencephalon and are described later. Specifically, a diencephalon / midbrain marker gene may include one or more genes selected from the group consisting of OTX1, OTX2, OTX2, DMBX1, Rx, Nkx2.1, OTP, FGFR2, EFNA5, and GAD1. [Table 3]

[0128] Hypothalamic marker genes refer to genes expressed in the hypothalamus. Hypothalamic marker genes may include one or more genes selected from the group consisting of Rx, Nkx2.1, Dmbx1, OTP, gad1, FGFR2, and EFNA5. The GenBank IDs for hypothalamic marker genes are shown in Table 4 below. [Table 4]

[0129] Spinal cord marker genes refer to genes expressed in the spinal cord. Spinal cord marker genes may include one or more genes selected from the group consisting of HoxB2, HoxA5, HOXC5, HOXD1, HOXD3, and HOXD4. The GenBank IDs of spinal cord marker genes are shown in Table 5 below. [Table 5]

[0130] The spinal cord marker genes are preferably those listed in Table 5, but are not limited to these. Other examples of spinal cord marker genes include gene groups that form Hox clusters.

[0131] On the other hand, in one embodiment, when retinoids (e.g., retinoic acid, retinal, retinol, all-trans-retinoic acid, 11-cis-retinoic acid) are used in the manufacturing process, HOX gene expression (e.g., HOXC5, HOXA5, and HOXB2) may be observed even when high-quality retinal tissue is produced. HOX gene expression is thought to be regulated by retinoic acid signaling, and HOX gene expression increases to an extent that does not affect the differentiation induction of retinal tissue. The effect of this retinoic acid signaling is thought to be due to the promotion of posteriorization along the anterior-posterior axis. Therefore, when retinoids are used in the manufacturing process (especially when retinoids are used after the start of differentiation induction into the retina), HOX genes (e.g., HOXC5, HOXA5, and HOXB2) can be excluded from the genes targeted for quality evaluation, or the quality of the neuroretina for transplantation can be judged as good even if the expression of these genes is observed.

[0132] An Optic Stalk marker gene refers to a gene expressed in the Optic Stalk. An Optic Stalk marker gene may include one or more genes selected from the group consisting of GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A. The GenBank IDs of Optic Stalk marker genes are shown in Table 6 below. [Table 6]

[0133] Lens marker genes refer to genes expressed in the lens of the eye. Lens marker genes may include one or more genes selected from the group consisting of CRYAA and CRYBA1. The GenBank IDs of lens marker genes are shown in Table 7 below. [Table 7]

[0134] Ciliary body marker genes refer to genes expressed in the ciliary body, the periphery of the ciliary body, and / or the ciliary body. Ciliary body marker genes may include one or more genes selected from the group consisting of Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The GenBank IDs of ciliary body marker genes are shown in Table 8 below. [Table 8]

[0135] Retinal pigment epithelial marker genes refer to genes expressed in retinal pigment epithelial cells. Retinal pigment epithelial marker genes include the retinal pigment epithelial markers listed above and may include one or more genes selected from the group consisting of MITF, TTR, and BEST1. The GenBank IDs of retinal pigment epithelial marker genes are shown in Table 9 below. [Table 9]

[0136] In one embodiment, the off-target cell-related gene may further include an undifferentiated pluripotent stem cell marker gene.

[0137] The undifferentiated pluripotent stem cell marker gene may include one or more genes selected from the group consisting of Oct3 / 4, Nanog, and lin28. Preferably, the undifferentiated pluripotent stem cell marker gene is one or more genes selected from the group consisting of Oct3 / 4, Nanog, and lin28. The GenBank IDs of the undifferentiated pluripotent stem cell marker genes are shown in Table 10 below. [Table 10]

[0138] (Detection method) In one embodiment, the detection of expression of neuroretinal cell-related genes and non-neuroretinal off-target cell-related genes is not particularly limited, but examples include Western blotting, immunohistochemistry, flow cytometry / flow cytometry (FACS®, BD Inc., etc.), Northern blotting, electrophoresis, PCR (preferably quantitative PCR (qPCR) and / or real-time PCR), gene tip analysis, and next-generation sequencing. Of these, quantitative PCR is useful in terms of quantitative accuracy, detection sensitivity, result stability, and speed. Furthermore, by applying equipment used for single-cell quantitative PCR (e.g., Biomark HD (Fluidigm Inc.), etc.) to normal quantitative PCR, it is possible to evaluate multiple quality assessment samples in a short time.

[0139] In one embodiment, the expression levels of neuroretinal cell-related genes and non-neuroretinal cell-related genes in two or more quality evaluation samples may be simultaneously detected by quantitative PCR. This quantitative PCR may be performed, for example, by a method including the following steps (1) to (5). Specific methods are well known to those skilled in the art. (1) Prepare a channel plate having channels connecting the independent sample wells in the sample well group and the independent primer wells in each of the primer well groups, a solution containing nucleic acids obtained from two or more of the above quality evaluation samples (sample solution), and a solution containing one or more primers specific to one or more of the above neuroretinal cell-related genes or the above non-neuroretinal cell-related genes (primer solution). (2) In the sample well group, add the above sample solution to each quality evaluation sample so that there is 1 sample solution per sample well. (3) Add the primer solution to one or more primer wells in the above group of one or more primer wells so that they form different groups of primer wells. (4) Mixing the nucleic acid and the primer separately via the above-mentioned channel, (5) Perform quantitative PCR using the mixture obtained in (4).

[0140] Furthermore, flow cytometry analysis using a flow cytometer capable of detecting the proportion of expression cells is also useful. In recent years, improvements in detection speed have led to the availability of high-throughput flow cytometers (such as FACS®) capable of evaluating a large number of samples. Therefore, the use of high-throughput flow cytometers is also useful for detecting the expression of neuroretinal cell-related genes and non-neuroretinal off-target cell-related genes. Such high-throughput flow cytometers can be commercially available (e.g., MACSQuant® Analyzers: manufactured by Miltenyi Biotec).

[0141] <Judgment process> A neuroretinal sheet for transplantation, excised from a cell aggregate, can be determined to be suitable for transplantation, i.e., suitable for transplantation, if it shows expression of neuroretinal cell-related genes and no expression of non-neuroretinal cell-related genes (determination step).

[0142] The detection of neuroretinal cell-related gene expression means that, in a gene expression detection method, the expression of neuroretinal cell-related gene is detected at a level that is substantially detectable by the method (e.g., above the detection limit). Conversely, the absence of non-neuroretinal cell-related gene expression means that, in a gene expression detection method, the expression of non-neuroretinal cell-related gene cannot be substantially detected by the method (e.g., below the detection limit). Substantial detectability means that the gene is detected to a degree that goes beyond the point at which it cannot be said that the gene is substantially functional. A person skilled in the art can appropriately set this depending on the gene and the detection method. For example, in the case of a quantitative gene expression detection method, ranges of over 0% to 10% and over 0% to 5% can be judged as not being substantially detectable (i.e., the expression of the gene cannot be detected) based on the detection limit of the gene expression.

[0143] In one embodiment, it is preferable to determine that the material is suitable for use as a neuroretina for transplantation if the following criteria 1 and 2 are met in the quantitative PCR method. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 10 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes is 5 or greater.

[0144] The Threshold Cycle (Ct) value refers to the number of cycles at which a constant amount of amplification product is reached in the region where gene amplification by PCR occurs exponentially. Since the Ct value is inversely correlated with the initial amount of gene, it is used to calculate the initial copy number of gene. In one embodiment, the "2^Ct value (2 to the power of Ct)" is inversely proportional to the initial amount of gene and is therefore used to calculate the initial copy number of gene. Specifically, a sample containing twice the initial amount of gene will have a Ct value one cycle earlier than a sample containing only half the number of copies of gene before amplification. A constant amount of amplification product can be set by anyone skilled in the art, as long as it falls within the region where gene amplification by PCR occurs exponentially.

[0145] An internal standard gene refers to a gene whose expression level differs little between samples. Suitable internal standard genes include those well known to those skilled in the art, such as 18S ribosomal RNA, β-actin, HPRT, α-tubulin, transferrin receptor, ubiquitin, and GAPDH, but GAPDH is preferred.

[0146] The Ct value is inversely correlated with the initial amount of the gene and therefore depends on the gene expression level within the cell. That is, when the concentration of the nucleic acid-containing solution is constant, the Ct value will differ depending on the internal standard gene used, and the difference between the Ct value of a specific gene and the Ct value of the internal standard gene (ΔCt value) is affected by the internal standard gene used. Unless otherwise specified, the ΔCt values ​​in this specification are based on the values ​​obtained when GAPDH is used as the internal standard gene.

[0147] When using an internal standard gene other than GAPDH as the internal standard gene, the ΔCt values ​​of Criterion 1 and Criterion 2 can be corrected by comparing the expression levels of GAPDH and the internal standard gene other than GAPDH.

[0148] In one embodiment, when β-actin is used as the internal standard gene, in the present invention's method, the Ct value of GAPDH is approximately 1 lower than that of β-actin, meaning that the absolute amount of RNA in GAPDH is about twice the absolute amount of RNA in β-actin. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 9 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes can be 4 or greater.

[0149] In one embodiment, when HPRT is used as the internal standard gene, in the present invention's method, the Ct value of GAPDH is approximately 7 lower than that of HPRT, meaning that the absolute amount of RNA in GAPDH is 2 times the absolute amount of RNA in HPRT. 7 Since it is approximately (128 times), Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 3 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes can be -2 or greater.

[0150] The neuroretinal cell-related genes can be any of the genes mentioned above. There are multiple genes that can be considered neuroretinal cell-related. That is, even when extracted from the same neuroretinal cell, the Ct value of reference value 1 may differ depending on the type of neuroretinal cell-related gene. A person skilled in the art can determine whether a neuroretinal cell-related gene is being expressed for each gene by setting a ΔCt value that can be determined from publicly known information such as the expression site and expression level of the neuroretinal cell-related gene.

[0151] For example, with respect to the Chx10 gene, if GAPDH is used as the internal standard, the ΔCt value may be 20 or less, preferably 15 or less, and more preferably 10 or less.

[0152] For example, with respect to the Recoverin gene, if GAPDH is used as the internal standard, the ΔCt value may be 16 or less, preferably 11 or less, and more preferably 6 or less.

[0153] Generally, the difference (ΔCt value) between the Ct value of a neuroretinal cell-related gene and the Ct value of an internal standard gene (e.g., GAPDH) may be, for example, 25 or less, 20 or less, 15 or less, or 10 or less. The difference between the Ct value of a neuroretinal cell-related gene and the Ct value of an internal standard gene may be, for example, -10 or greater, -5 or greater, 0 or greater, or 5 or greater.

[0154] Non-neuroretinal cell-related genes can be any of the genes mentioned above. There are multiple genes that can be considered non-neuroretinal cell-related. That is, even when extracted from the same non-retinal cell, the Ct value will differ depending on the non-neuroretinal cell-related gene. A person skilled in the art can determine that a neuroretinal cell-related gene is being expressed for each gene based on publicly known information such as the expression site and expression level of the non-neuroretinal cell-related gene. For example, for the PAX2 gene, if GAPDH is used as the internal standard, the ΔCt value may be 5 or higher. For the HOXB2 gene, if GAPDH is used as the internal standard, the ΔCt value may be 5 or higher. Generally, the difference between the Ct value of a non-neuroretinal cell-related gene and the Ct value of the internal standard gene may be 30 or less, 25 or less, or 20 or less. Also, the difference between the Ct value of a non-neuroretinal cell-related gene and the Ct value of the internal standard gene may be, for example, 0 or more, 3 or more, or 5 or more.

[0155] In one embodiment, the neuroretina according to the present invention is (1) Derived from pluripotent stem cells, (2) Having a three-dimensional structure, (3) A neuroretinal layer having a multilayer structure including a photoreceptor cell layer and an inner layer, (4) The photoreceptor layer includes one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, (5) The inner layer contains one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells and bipolar cells, (6) The surface of the neuroretinal layer has an apical surface, (7) Inside the photoreceptor layer located along the apical surface, (8) The area of ​​the neuroretinal layer is 50% or more of the total surface area of ​​the neuroretinal sheet for transplantation. (9) The area of ​​continuous epithelial structures is 80% or more of the total area of ​​the apical surface of the neuroretinal layer. (10) Expression of neuroretinal cell-related genes is observed in the neuroretinal sheet for transplantation, and expression of non-neuroretinal cell-related genes is not observed, and the non-neuroretinal cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eye-related tissue marker genes. This is a neuroretinal sheet for transplantation characterized by the following features.

[0156] In one embodiment, the above-mentioned neuroretinal sheet for transplantation is preferably a sheet-like cell aggregate obtained by cutting out a portion containing the neuroretina from a sphere-like cell aggregate derived from pluripotent stem cells. A specific method of preparation is described, for example, in international application PCT / JP2020 / 011254.

[0157] In one embodiment, the neuroretina includes a neuroretinal layer having a plurality of layered structures, including (3) a photoreceptor cell layer and an inner layer. As described in (6) and (7), the photoreceptor cell layer is located on the outer (surface) side of the neuroretina, but an ectopic photoreceptor cell layer may also be present in the inner layer.

[0158] In one embodiment, the neuroretina (5) has an inner layer which contains one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells and bipolar cells, but which may also contain one or more cells selected from the group consisting of ectopic photoreceptor progenitor cells and photoreceptor cells. In one embodiment, there are also neuroretinas in which the content of ganglion cells, amacrine cells and horizontal cells is 30% or less of the total number of cells, neuroretinas in which the content of ganglion cells, amacrine cells, horizontal cells and bipolar cells is 30% or less of the total number of cells, and / or neuroretinas in which the content of bipolar cells is 10% or less of the total number of cells.

[0159] In one embodiment, the neuroretina is such that (8) the area of ​​the neuroretinal layer is 40% or more, preferably 50% or more, and more preferably 60% or more, of the total surface area of ​​the neuroretina. The neuroretina is such that (9) the area of ​​the continuous epithelial structure is 60% or more, preferably 70% or more, and more preferably 80% or more, of the total surface area of ​​the apical surface of the neuroretinal layer.

[0160] The genes related to neuroretinal cells and non-neuroretinal cells (brain and spinal cord tissue marker genes and eye-related tissue marker genes) are the genes mentioned above.

[0161] (10) Whether the neuroretinal cell-related genes are expressed and non-neuroretinal cell-related genes are not expressed in the neuroretinal sheet for transplantation can be determined by taking a portion of the neuroretinal sheet for transplantation and detecting gene expression. Furthermore, if the neuroretinal sheet for transplantation is isolated from a cell aggregate in which the expression of neuroretinal cell-related genes is substantially observed and the expression of non-neuroretinal cell-related genes is substantially not observed, then detection of gene expression in the neuroretinal sheet for transplantation itself is unnecessary. Whether the gene expression is substantially observed or not is determined by whether it is at a level that can be substantially detected by the detection method.

[0162] The neuroretinal cell-related genes in the neuroretinal sheet for transplantation may be one or more selected from the group consisting of, for example, Rx, Chx10, Pax6, and Crx. The proportion of cells expressing neuroretinal cell-related genes (positive cells) relative to the total number of cells varies depending on the differentiation stage of the neuroretina.

[0163] In one embodiment, the proportion of Rx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 30% or more, 40% or more, 50% or more, or 60% or more. In one embodiment, the proportion of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. In one embodiment, the proportion of Crx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0164] In one embodiment, the proportion of Rx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 30% to 80%, 40% to 70%, 45% to 60%, or 50% to 60%. In one embodiment, the proportion of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%. In one embodiment, the proportion of Crx-positive cells to the total number of cells in the neuroretinal sheet for transplantation is 10% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%.

[0165] In one embodiment, the proportion of cells in the neuroretinal sheet for transplantation may be: (1) Chx10-positive and Pax6-positive cells (neuroretinal progenitor cells) being 10% to 50% or 10% to 30%; (2) Chx10-positive and Pax6-negative cells (bipolar progenitor cells) being 10% to 25% or 15% to 25%; and (3) Chx10-negative and Pax6-positive cells (ganglion cells and amacrine cells) being 10% to 25% or 10% to 20%.

[0166] In another embodiment, the proportion of (1) Chx10-positive and Pax6-positive cells (neuroretinal progenitor cells) relative to the total number of cells in the neuroretinal sheet for transplantation may be 20% to 40%, (2) Chx10-positive and Pax6-negative cells (bipolar progenitor cells) may be 5% to 20%, and (3) Chx10-negative and Pax6-positive cells (ganglion cells and amacrine cells) may be 5% to 20% or 5% to 15%.

[0167] In one aspect, the neuroretinal sheet for transplantation according to the present invention is a neuroretinal sheet for transplantation that has been determined to be usable as a neuroretinal sheet for transplantation by the above-described quality evaluation method for neuroretinal sheet for transplantation, and may be an isolated sheet-like neuroretinal sheet for transplantation.

[0168] In one embodiment, the neuroretinal sheet for transplantation according to the present invention may be isolated from a cell aggregate containing neuroretina and may include a region near the center of the continuous epithelial tissue in the cell aggregate.

[0169] In one aspect, the neuroretina is a neuroretinal sheet for transplantation, (1) It is isolated from cell aggregates containing the neuroretinal layer, (2) The cell aggregate includes a region near the center of the continuous epithelial tissue, (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm. This is a neuroretinal sheet for transplantation.

[0170] In one embodiment, the neuroretinal sheet for transplantation according to the present invention is isolated from a cell aggregate including neuroretina, retinal pigment epithelial cells, and ciliary body peripheral structures, and may be a neuroretinal sheet for transplantation that has a continuity of tangent slopes on a surface different from the continuity of tangent slopes on the surface of the neuroretinal epithelial structure, and includes the region on the epithelial structure furthest from the portion containing retinal pigment epithelial cells.

[0171] In one embodiment, the neuroretinal sheet for transplantation according to the present invention is isolated from a cell aggregate including neuroretina and cerebrospinal tissue, and may be a neuroretinal sheet for transplantation that has a continuity of tangent slopes on a surface different from the continuity of tangent slopes on the surface of the neuroretinal epithelial structure, and includes the region on the epithelial structure furthest from the portion including cerebrospinal tissue.

[0172] In one embodiment, the neuroretinal sheet for transplantation according to the present invention is isolated from a cell aggregate containing two or more neuroretinal epithelium cells, and may be a neuroretinal sheet for transplantation that includes the central portion of the morphologically good and / or large neuroretinal epithelium cells that are suitable for isolation.

[0173] In one aspect, the neuroretina is a neuroretinal sheet for transplantation, (1) Isolated from a cell aggregate comprising at least a first epithelial tissue and a second epithelial tissue, wherein the first epithelial tissue comprises human neuroretina, and the second epithelial tissue has a continuity of tangent slopes on a surface different from that of the first epithelial tissue, and comprises non-neuroretinal cells, (2) Including the region on the first epithelial tissue furthest from the second epithelial tissue, (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm. The second epithelial tissue is selected from a group of tissues that are different from the neuroretinal tissue of the first epithelial tissue, including ocular tissues, brain and spinal cord tissues, and other tissues related to the eyeball. This is a neuroretinal sheet for transplantation.

[0174] In one embodiment, the major axis of the neuroretinal sheet for implantation according to the present invention may be, for example, 300 μm to 3300 μm, preferably 600 μm to 2500 μm, and more preferably 1100 μm to 1700 μm.

[0175] In one embodiment, the short diameter of the neuroretinal sheet for implantation according to the present invention may be, for example, 100 μm to 2000 μm, preferably 200 μm to 1500 μm, and more preferably 400 μm to 1100 μm.

[0176] In one embodiment, the height of the neuroretinal sheet for implantation according to the present invention may be, for example, 50 μm to 1500 μm, preferably 100 μm to 1000 μm, and more preferably 200 μm to 700 μm.

[0177] In one embodiment, the volume of the neuroretinal sheet for implantation according to the present invention is, for example, 0.001 mm 3 ~4.0mm 3 It may be, preferably 0.01 mm 3 ~1.5mm 3 More preferably 0.07 mm 3 ~0.57mm 3 That is the case.

[0178] The method for measuring the major axis, minor axis, and height of a neuroretinal sheet for transplantation is not particularly limited and can be measured, for example, from images taken under a microscope. For example, for a neuroretinal sheet for transplantation excised from a cell aggregate, a frontal image taken with the cut surface facing the objective lens and a lateral image taken with the cut surface tilted so that it is perpendicular to the objective lens can be taken with a stereomicroscope and measured from the images. Here, the major axis refers to the longest line segment and its length among the line segments connecting the two endpoints on the sheet cross-section in the frontal image. The minor axis refers to the longest line segment and its length among the line segments that connect the two endpoints on the sheet cross-section in the frontal image and are perpendicular to the major axis. The height refers to the longest line segment and its length among the line segments perpendicular to the sheet cross-section, with the intersection with the sheet cross-section and the vertex of the retinal sheet as endpoints. The volume of the sheet refers to the volume calculated according to the following formula, assuming that the graft is an ellipsoid obtained by approximating it as a cross-section obtained by halving it so that the cross-section passes through the major axis. Volume = 2 / 3 × Pi (π) × (major axis / 2) × (minor axis / 2) × height

[0179] (2) Matrix The matrix in this invention (hereinafter sometimes referred to as the matrix gel) can embed two or more cell aggregates containing neuroretina in a living state, and can be gel-like, preferably hydrogel-like. Examples include fibrin gel, gelatin, collagen, pectin, hyaluronic acid, and alginic acid, with fibrin gel or gelatin being preferred. In this specification, "hydrogel" refers to a material in which a liquid such as water is incorporated inside a polymer crosslinked into a three-dimensional network structure. These hydrogels are biodegradable and have a desired melting point, making them easy to handle and allowing for a high engraftment rate. Biodegradability means that they are broken down by enzymes in the body and absorbed or excreted.

[0180] The hydrogel in this invention may be a hydrogel with a melting point in the range of 20°C to 40°C. That is, it has the characteristic of melting at body temperature when implanted. Below, the physical properties will be described in detail using "gelatin" as an example. Hydrogels other than gelatin, such as fibrin gels, are preferably said to have the same physical properties as gelatin.

[0181] The matrix gel is preferably a fibrin gel. A fibrin gel is a gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution. In this specification, a substance that forms a gel upon reaction is called a matrix precursor, and for example, thrombin and fibrinogen, which form a fibrin gel upon reaction, are examples of matrix precursors. The fibrinogen solution can be prepared by dissolving fibrinogen powder or the like in a solvent that can dissolve fibrinogen and contains aprotinin. Its concentration is not particularly limited, but is, for example, 40 to 480 mg / ml, preferably 80 mg / mL to 320 mg / mL (e.g., 160 mg / mL). Also, when the blood coagulation factor VIII activity contained in 1 ml of normal human plasma is considered as 1 unit, it may be 37.5 units / mL to 225 units / mL (e.g., 75 units / mL). A thrombin solution can be prepared by dissolving thrombin powder or the like in a thrombin solution containing calcium chloride hydrate. Its concentration is not particularly limited, but is, for example, 125 units / mL to 750 units / mL (e.g., 75 units / mL). The fibrinogen solution and the thrombin solution can be reacted by contacting or mixing them. In this case, it is preferable to use the fibrinogen solution and the thrombin solution in an activity ratio of 1:1 to 1:9, preferably 1:3 to 1:4.

[0182] The matrix gel is preferably gelatin. Gelatin is obtained by solubilizing water-insoluble collagen, for example, by pretreatment with an acid or alkali and then thermal hydrolysis. In this specification, unless otherwise specified, it refers to gelatin in the form of a hydrogel, but it may also be explicitly stated as "hydrogel of gelatin." Heating breaks down the molecular structure of the three-chain helix of collagen, causing it to separate into three random molecules and thus become solubilized. The raw materials for gelatin mainly include bovine bones and hides, pigskin, pig bones, and fish scales. To extract gelatin from these collagen raw materials, the raw materials are pretreated using inorganic acids such as hydrochloric acid or sulfuric acid, or lime. Depending on the pretreatment conditions of the raw materials, the former may be called acid-treated gelatin (or type A gelatin), and the latter may be called alkali-treated (lime-treated) gelatin (type B gelatin). Acid-treated gelatin and alkali-treated gelatin have different properties. Furthermore, hydrolysis through heat treatment reduces the molecular weight of gelatin, improves its solubility, and decreases its gel strength under the same conditions. For example, heat treatment can be performed with warm water (50°C to 80°C) and may be repeated multiple times. Commercially available gelatin is also available, such as Gelatin LS-H (Nitta Gelatin Co., Ltd., porcine alkali-treated gelatin, unheat-treated gelatin, high gel strength) and Gelatin LS-W (Nitta Gelatin Co., Ltd., porcine alkali-treated gelatin, heat-treated gelatin, low gel strength). Alkali-treated (lime-treated) gelatin (Type B gelatin) is preferred, and heat-treated gelatin is preferred.

[0183] Collagen is composed of three polypeptide chains (α-chains) with a molecular weight of approximately 100,000. When collagen molecules are denatured by heat treatment, they separate into three α-chains (α-components). In addition, α-chain dimers (β-components: molecular weight of approximately 200,000) and trimers (γ-components: molecular weight of approximately 300,000) can also be produced. Furthermore, due to the processing steps of gelatin, some of the intermolecular or intramolecular bonds between collagen and gelatin are randomly cleaved, so gelatin is an aggregate of molecules with various molecular weights. Although it varies depending on whether or not heat treatment is performed, commercially available gelatin usually has a molecular weight distribution of tens of thousands to several million. In one embodiment, it is preferable that gelatin has a molecular weight distribution in which 50% or more (preferably 60% or more, 70% or more, 80% or more, or 90% or more) are in the range of approximately 100,000 to approximately 300,000. Furthermore, the preferred average molecular weight of gelatin is in the range of approximately 50,000 to 1,000,000, 100,000 to 800,000, 100,000 to 600,000, 200,000 to 500,000, and 300,000 to 500,000.

[0184] Furthermore, the molecular weight distribution and average molecular weight can be measured by methods well known to those skilled in the art. For example, the molecular weight distribution can be estimated by obtaining a chromatogram of an aqueous gelatin solution using a high-performance liquid chromatograph via gel filtration, and the average molecular weight can be further estimated by converting it to pullulan equivalent.

[0185] The term "isoionic point" refers to the pH of an aqueous solution of a protein or amphoteric electrolyte in the absence of other ions (excluding hydrogen ions and hydroxide ions produced by the ionization of water, and ions of the amphoteric electrolyte itself). The isoionic point can be measured by methods well known to those skilled in the art. For example, the hydrogen ion concentration can be measured using a pH meter on a test solution (gelatin aqueous solution) that has been desalted with an ion exchange resin. For example, if alkali treatment is performed during the gelatin manufacturing process, most of the gel is deamided, resulting in a low isoionic point of approximately pH 5. In contrast, acid-treated gelatin has a low deamide rate and exhibits an isoionic point of pH 7-9, similar to collagen. Aqueous gelatin solutions are positively charged at pH lower than the isoionic point and negatively charged at pH higher than the isoionic point. In this specification, hydrogels (such as gelatin) are preferably found to exhibit isoionic points of approximately pH 4-7, approximately pH 5-7, and approximately pH 6-7.

[0186] Gelatin-containing hydrogels undergo a phase change from gel to sol and back again upon heating or cooling. Hydrogels such as gelatin become gel (jelly) when cooled and lose fluidity, and become sol (aqueous solution) when heated and regain fluidity. Taking gelatin as an example, gelatin becomes gel because, upon cooling, some of the gelatin molecules adopt a helical structure similar to collagen, forming a network between molecules. Here, "melting point" refers to the temperature at which sol formation occurs under constant pressure, and "freezing point" refers to the temperature at which gel formation occurs under constant pressure. As described above, continued cooling forms a stronger gel. The hydrogels used herein have a melting point of 20°C to 40°C (e.g., 20°C to 35°C, 25°C to 35°C, 30°C to 40°C, 35°C to 40°C). Generally, the melting point of a gel is a measure of the strength of its network, and the melting point of a hydrogel (e.g., gelatin) increases with increasing concentration and molecular weight. Furthermore, increasing the solid content with sugars, for example, tends to increase both the melting and freezing points. Thus, it is possible to vary the melting and freezing points within a certain range.

[0187] The method for measuring the melting point of a hydrogel is not particularly limited and can be measured by, for example, the method specified in JIS K6503. Specifically, a sample solution (e.g., a 10 w / w% gelatin solution) is prepared in a 10 mm diameter glass tube to create a gel that rises 45 mm from a point 5 mm from the end. The tube is then placed in a water bath with the end 5 mm at the bottom, and the temperature is raised. The melting point is defined as the temperature at which the gel melts and the top of the bubble at the end rises 10 mm.

[0188] The method for measuring the freezing point of a hydrogel is not particularly limited and can be measured by, for example, the method specified in JIS K6503. Specifically, a test solution (e.g., a 10 w / w% gelatin solution) heated to 35°C is gradually cooled while being stirred in a 15°C water bath with a 35°C buffer bath. The freezing point is defined as the temperature at which the return phenomenon (the phenomenon in which bubbles, etc., generated when the solution is stirred are pulled back in the opposite direction rather than moving in the direction of stirring due to inertia when stirring stops) is observed. To make the return phenomenon easier to observe, it is advisable to add a piece of filter paper or similar to the test solution.

[0189] The "jelly strength" of a hydrogel refers to the mechanical strength of the object in which the gel is formed. It typically refers to the force required to deform a gel of a certain shape or to break the gel (unit: g, dyne(s) / cm). 2 or g / cm 2) is expressed as a unit and is primarily a measure of gel hardness. One dyne (dyne(s)) is defined as the force that, when acting on an object with a mass of 1 gram (g), imparts an acceleration of 1 centimeter per second squared (cm / s²) in that direction. For example, the gel strength of a hydrogel containing gelatin varies depending on its concentration, temperature, pH, and coexisting substances, but under the same conditions, it changes depending on its inherent properties such as molecular weight. The "gel strength" of gelatin is tested by methods specified in, for example, the Japanese Pharmacopoeia, Yakushokuhatsu 0531 No. 3 or JIS K6503. Specifically, the gel strength is defined as the load required to push down 4 mm on the surface of a gelatin solution prepared by cooling a gelatin solution at 10°C for 17 hours using a plunger with a diameter of 1 / 2 inch (12.7 mm). Generally, as the gelatin concentration increases, the gel strength also increases. Furthermore, the gelling strength of gelatin generally decreases significantly at acidic pH (e.g., pH 4 or lower) and alkaline pH (e.g., pH 8 or higher). Also, within a certain range (e.g., molecular weight 30,000 to 70,000), the gelling strength increases with increasing molecular weight, but beyond a certain point (e.g., molecular weight 100,000 or higher), the gelling strength becomes constant. In addition, gelation usually requires cooling to below 20°C, and within a cooling temperature range of, for example, 4°C to 20°C, the lower the cooling temperature, the higher the gelling strength. Since the reactions that orient molecules and form networks proceed at a relatively slow rate, intermolecular networks are formed within the gel even after gel formation, so the gelling strength increases over a period of 1 to 5 hours after the start of cooling. Moreover, rapid cooling increases the gelling strength because a fine network is formed between molecules without sufficient time for molecular orientation.

[0190] In this specification, the gel strength should be such that, for example, at 10°C to 20°C, it does not affect the embedded cells or tissues, and the hydrogel does not collapse during normal transplantation procedures of the complex. For hydrogel (gelatin) of about 5% to 30% by weight, the gel strength of the hydrogel (gelatin) may be 50g or more, 100g or more, 200g or more, 500g or more, 1000g or more, 1200g or more, 1300g or more, 1400g or more, or 1500g or more, according to the method specified in JIS K6503. Alternatively, the gel strength of the hydrogel (gelatin) may be 3000g or less, 2500g or less, or 2000g or less.

[0191] Viscosity is an indicator of the viscosity of a fluid. Those skilled in the art can measure viscosity using well-known methods. For example, it can be measured using the method specified in JIS K6503. That is, the time it takes for a certain amount of gelatin solution (60°C, 6.67%) to flow through a pipette-type viscometer is converted into a viscosity value (unit: mPa·s).

[0192] The viscosity of a gelatin-containing hydrogel in its sol state is influenced by the concentration of gelatin and other components, the system's temperature, pH, and coexisting salts. Generally, viscosity increases with increasing gelatin concentration and decreasing temperature. For example, with type B gelatin, viscosity is pH-dependent, with minimum viscosity occurring near the isoionic pH. On the other hand, with type A gelatin, no significant relationship is observed between viscosity and pH.

[0193] The viscosity of the gelatin-containing hydrogel is not particularly limited, as long as it has sufficient viscosity to form the complex described later. To embed living cells, and considering the risk of damaging the cells, it is necessary to embed the cells using a hydrogel at approximately 40°C (approximately 30°C to 50°C). Therefore, hydrogels exhibiting a viscosity sufficient for cell embedding at approximately 40°C (approximately 30°C to 50°C), such as 2 to 50 mPa·s (5 to 30 mPa·s), are recommended.

[0194] "Foaming rate" refers to the ratio (V1 / V0) of the total volume (V1) containing foam to the original volume (V0) of the sample. A lower foaming rate is preferable. Specifically, it should be 1.2 or less (preferably 1.15 or less, 1.1 or less, or 1.05 or less). The foaming rate can be measured, for example, by the Baggie method, 10th edition (2006 edition), which is a photographic gelatin test method. Specifically, 50 mL of the sample solution at 50°C placed in a graduated cylinder is vibrated for 1 minute at an amplitude of 300 mm and a frequency of 145 times / minute, and then the total volume including foam is read 3 minutes after the vibration stops.

[0195] Since the hydrogels described herein are administered into the body by transplantation, it is preferable that they meet the purity test standards specified in the Japanese Pharmacopoeia. For example, the Japanese Pharmacopoeia specifies quality standards and test methods for gelatin and purified gelatin, and specifically, the following standards must be met. (1) Unpleasant odor and insoluble matter: When 1.0 g of this product is added to 40 mL of water and heated to dissolve, the solution has no unpleasant odor. Furthermore, the solution is clear or only slightly cloudy, and its color is not darker than that of color comparison solution A. (2) Sulfites: 60 ppm or less (3) Heavy metals 50ppm or less (20ppm or less) (4) Arsenic 1 ppm or less (5) Mercury 0.1ppm or less (6) Loss on drying 15.0% or less (7) Ignition residue 2.0% or less

[0196] A higher concentration of the hydrogel is preferable from the viewpoint of preventing breakdown during various operations and dissolution during operations. The hydrogel concentration is the ratio of hydrogel to the medium (solvent) in which the hydrogel is dissolved. For example, when using gelatin, the hydrogel concentration may be 10% to 50% by weight, preferably 25% to 50% by weight, 30% to 50% by weight, or 20% to 40% by weight.

[0197] The hydrogel concentration can be adjusted by dissolving the measured hydrogel in an appropriate medium so that it falls within the aforementioned range. The medium can be any medium that does not affect cells, such as a buffer salt solution like HBSS.

[0198] The pH of the hydrogel is preferably near neutral to reduce damage to the embedded tissue and the living body after transplantation. For example, it may be 6 to 8 or 6.5 to 7.5, and preferably 7 to 7.5. The pH of the hydrogel can be measured as the pH in solution or sol state. As mentioned above, within this pH range, the gel strength will not decrease significantly. The method for measuring the pH of the hydrogel is not particularly limited and can be measured, for example, by a commercially available pH meter or pH test strip.

[0199] In one embodiment, the hydrogel is preferably a biodegradable hydrogel. After the complex is implanted in a human, the biodegradation of the biodegradable hydrogel causes the implanted neuroretina to gradually come into contact with the living retina, resulting in a high engraftment rate. Gelatin obtained by processing collagen, which is the main biological component, is a biodegradable hydrogel.

[0200] In one embodiment, the hydrogels used herein are preferably hydrogels having a melting point in the range of 20°C to 40°C and satisfying one or more of the following physical properties. (1) Preparation method: Alkali treatment and / or heat treatment (2) Average molecular weight: about 100,000 to about 500,000 (3) Concentration: 10% to 50% by weight (preferably 25% to 50% by weight, 30% to 50% by weight, or 20% to 40% by weight) (4) Jelly strength: 50g or more, 100g or more, 200g or more, 500g or more, 1000g or more, 1200g or more, 1300g or more, 1400g or more, or 1500g or more (3000g or less, 2500g or less, or 2000g or less) (5) pH: 6-8 (preferably 6.5-7.5, 7-7.5) (6) Isoionic points: Acidic region (approximately pH 4 to pH 7, approximately pH 5 to pH 7, approximately pH 6 to pH 7) (7) Foaming rate: 1.2 or less (preferably 1.15 or less, 1.1 or less, or 1.05 or less) (8) Viscosity: Approximately 5 to 30 mPa·s at approximately 40°C (30°C to 50°C).

[0201] (3) Complex <Structure of the complex> In one embodiment, the complex comprises two or more cell aggregates containing neuroretina and a matrix, wherein the two or more cell aggregates are arranged within the matrix. Here, "arranged within the matrix" means that the two or more cell aggregates are embedded in the matrix gel, i.e., they exist covered by the matrix gel. Furthermore, because the two or more cell aggregates are covered by the matrix gel, the cell aggregates do not come into direct contact with the external environment of the complex (such as preservation solution). Since the neuroretinal sheets (Caps) for transplantation, which are cut from cell aggregates, especially sphere-shaped cell aggregates, are small and difficult to handle as they are, arranging them in the matrix gel makes them easier to handle. In addition, it becomes possible to transplant multiple cell aggregates simultaneously.

[0202] In the complex, two or more cell aggregates may or may not be in contact with each other, but from the viewpoint of reducing cell damage due to mechanical contact, it is preferable that two or more cell aggregates are separated by the matrix gel and not in contact with each other. Not being in contact with each other means that the cell aggregates are not physically touching each other. The fact that the cell aggregates are not in contact with each other can be confirmed, for example, by visual inspection (e.g., under a microscope).

[0203] In one embodiment, it is preferable that cell aggregates containing two or more neuroretinas are arranged in rows within the matrix gel. Here, "row" means that the cell aggregates containing two or more neuroretinas are connected in a linear fashion, and the linear fashion may be a straight line or a curve extending in a generally consistent direction. It is preferable that 2 to 20 cell aggregates are arranged in rows, and more preferable that 4 to 16, 4 to 14, 4 to 10, or 6 to 8 cell aggregates are arranged in rows. Furthermore, the apical faces or bases of the two or more neuroretinas may be oriented in the same direction or in different directions, but it is preferable that their apical faces are oriented in the same direction, approximately perpendicular to the linear direction, and arranged in a horizontal row.

[0204] In one embodiment, the neuroretina may be a neuroretina for transplantation (also called a graft or cap) excised from a cell aggregate, or a neuroretinal sheet formed by dispersing a cell aggregate into single cells. By including two or more cell aggregates containing neuroretina, the composite of the present invention can treat a wider range of retinal tissue disorders.

[0205] [Method for producing the composite] The present invention provides a method for producing a complex in which two or more cell aggregates containing neuroretina are arranged in a matrix, comprising: (1) a first step of producing two or more neuroretinal cell aggregates from pluripotent stem cells; and (2) a second step of contacting the two or more cell aggregates with a matrix or a matrix precursor in a predetermined arrangement, and then gelling the matrix.

[0206] (1) In the first step, the cell aggregate containing two or more neuroretinas is preferably derived from human pluripotent stem cells and is preferably a tissue for transplantation.

[0207] A method for producing cell aggregates containing neural retina (spheroid cell aggregates, neural retina sheets, neural retina sheets for transplantation) from pluripotent stem cells is as described above. When cultured and produced on an extracellular matrix (e.g., collagen), detachment using an enzyme that decomposes the extracellular matrix (e.g., collagenase) may be performed to recover the cell aggregates containing neural retina. In such cases, it is preferable to wash multiple times with a medium or the like in order to prevent contamination with the enzyme.

[0208] (2) In one aspect of the second step, the method comprises contacting two or more cell aggregates with a matrix precursor in a predetermined arrangement, then allowing the matrix to gel. When the matrix is a fibrin gel, the matrix precursors are thrombin and fibrinogen. Although thrombin is generally a gelling agent and is not referred to as a precursor, in the present specification thrombin is also referred to as a matrix precursor. All of the two or more cell aggregates may be contacted with either one of a fibrinogen solution as a matrix precursor and a thrombin solution, then further contacted with the other of the fibrinogen solution and the thrombin solution, and reacted to cause gelation; or a part of the two or more cell aggregates may be contacted with either one of the fibrinogen solution and the thrombin solution, then the remaining cell aggregates among the two or more cell aggregates are contacted with the other of the fibrinogen solution and the thrombin solution to react and cause gelation. Here, contact with a solution refers to bringing a cell aggregate into contact so as to be immersed in the fibrinogen solution or thrombin solution.

[0209] The fibrinogen solution and the thrombin solution are preferably used in an activity ratio in the range of 1:1 to 1:9, more preferably 1:3 to 1:4.

[0210] (2) In one aspect of the second step, the method comprises contacting two or more cell aggregates with a matrix in a predetermined arrangement, then allowing the matrix to gel. As described above, examples of the matrix include gelatin, collagen, pectin, hyaluronic acid, and alginic acid, and gelatin is preferable. The appropriate concentration of the gelatin solution and the gelling method are as described above.

[0211] Contacting two or more cell aggregates with a matrix or a matrix precursor in a predetermined arrangement means arranging two or more cell aggregates in a desired arrangement (e.g., in rows) and then contacting them with the matrix or the matrix precursor, or arranging two or more cell aggregates in a desired arrangement (e.g., in rows) in the matrix or the matrix precursor. For example, the method can be carried out by preparing a container having elongated grooves and arranging two or more cell aggregates at predetermined intervals and orientations. The shape of the groove may be V-shaped, U-shaped, or U-shaped (open on one side), and is not limited to any particular shape. The groove diameter can correspond to the diameter of the complex; for example, the depth may be 100 µm to 2000 µm. The groove length can correspond to the length of the complex; for example, the length may be 300 µm to 50000 µm, but the length is not particularly limited, and it is preferable to adjust the length to an implantable size. It is also preferable to leave an interval of about 10 µm to 50 µm between two or more cell aggregates. It is preferable that the apical surfaces of the neural retina of each of the two or more cell aggregates face the same direction, and particularly that they are arranged in a single horizontal row oriented substantially perpendicular to the extending direction of the groove. In one embodiment, setting the width of the groove to be from about the same as the minor axis of the cell aggregates to about twice the minor axis facilitates arranging the cell aggregates in a single horizontal row.

[0212] In the case of a matrix precursor, either a fibrinogen solution or a thrombin solution, which are precursors to fibrin gel, may be added to the groove, two or more cell aggregates may be placed therein at predetermined intervals and orientations, and the other of the fibrinogen solution or thrombin solution may be added gently so as not to cause the two or more cell aggregates to flow out of their predetermined positions, thereby causing gelation. Alternatively, in the case of gelatin, a gelatin solution may be added to the groove, two or more cell aggregates may be placed therein at predetermined intervals and orientations, and then a gelatin solution may be added gently so as not to cause the two or more cell aggregates to flow out of their predetermined positions, and then the gelation may be caused by cooling. Before this operation, an operation may be performed to allow the cell aggregates to blend with the matrix precursor or matrix. Specifically, by gradually blending them with a matrix precursor or matrix at a concentration lower than the concentration required for gelation, it becomes easier to reach the desired final gelatin concentration.

[0213] In one embodiment, the composite obtained by the manufacturing method of the present invention can be aspirated and dispensed using a syringe without disrupting the matrix gel. This allows for the introduction of more cell aggregates containing neuroretina into the transplantation site in a single transplant. Furthermore, because the matrix gel is biodegradable, the transplanted neuroretina gradually comes into contact with the living retina and engrafts.

[0214] [Pharmaceutical compositions, therapeutic drugs, and therapeutic methods] One aspect of the present invention is a pharmaceutical composition containing the complex obtained in the present invention as an active ingredient. The pharmaceutical composition preferably further includes a pharmaceutically acceptable carrier in addition to the complex of the present invention.

[0215] The pharmaceutical composition may be used to treat diseases based on disorders of retinal cells or retinal tissue, or damage to retinal tissue. Examples of diseases based on disorders of retinal cells or retinal tissue include ophthalmic diseases such as retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone-rod dystrophy. Examples of retinal tissue damage include conditions in which photoreceptor cells or retinal pigment epithelial cells have degenerated and died.

[0216] Acceptable carriers for pharmaceutical use include physiological aqueous solvents (such as physiological saline, buffer solutions, and serum-free culture media). If necessary, the pharmaceutical composition may also contain preservatives, stabilizers, reducing agents, isotonic agents, etc., that are commonly used in pharmaceuticals containing transplanted tissue or cells in transplant medicine.

[0217] One aspect of the present invention is a therapeutic agent for diseases based on disorders of retinal cells or retinal tissue, or damage to retinal tissue, comprising the complex obtained in the present invention.

[0218] The therapeutic agent of the present invention can treat the disorder of retinal cells or retinal tissue or the state of retinal tissue damage in patients suffering from diseases based on retinal cell or retinal tissue damage, by transplanting the complex obtained in the present invention. Diseases based on retinal cell or retinal tissue damage or the state of retinal tissue damage include the diseases mentioned above.

[0219] One aspect of the present invention is an implantation composition containing the complex obtained in the present invention. The complex of the present invention can be used for implantation into the fundus of a patient's eye. Furthermore, the complex of the present invention can be used to ensure that, in the patient to whom the implanted complex is implanted, the neuroretina of the implanted complex is oriented toward the patient's neuroretinal layer. In addition to the complex of the present invention, the implantation composition preferably further comprises a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier being as described above.

[0220] One aspect of the present invention is a method for treating a disease based on damage to retinal cells or retinal tissue or damage to retinal tissue, comprising the following steps. (1) A step of implanting the composite obtained by the present invention into the fundus of the patient's eye, (2) The process by which the transplanted complex of the neuroretina is engrafted in the patient's body in a state facing the patient's neuroretinal layer.

[0221] One aspect of the present invention is a method for treating diseases based on retinal cell or retinal tissue damage or retinal tissue injury, which includes transplanting the composite obtained in the present invention to a target requiring transplantation (for example, subretinal tissue of an eye suffering from an ophthalmic disease). The composite of the present invention can be used as a therapeutic agent for diseases based on retinal cell or retinal tissue damage, or to replenish the damaged area in a state of retinal tissue damage. By transplanting the composite of the present invention to a patient with a disease based on retinal cell or retinal tissue damage requiring transplantation, or to a patient with a state of retinal tissue damage, the disease based on retinal tissue damage or the state of retinal tissue damage can be treated by replenishing the damaged retinal tissue. An example of a transplantation method is to transplant the composite of the present invention for transplantation subretinal tissue at the damaged site by making an incision in the eyeball. An example of a transplantation method is to inject using a thin tube or to transplant by grasping with forceps, and an example of a thin tube is an injection needle. [Examples]

[0222] <Example 1: Preparation of a composite gel consisting of fibrin gel and multiple neuroretinal implants> We investigated a complex in which cell aggregates (grafts) containing multiple neuroretinal cells are encapsulated in a fibrin gel. Neuroretinal tissue fragments (sometimes called caps) were used as grafts. The caps were prepared as follows: Genetically modified human ES cells (derived from the KhES-1 strain (Non-Patent Literature 6)) possessing the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports, 4,3594 (2014)". StemFit medium (trade name: AK03N, Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (trade name, Nippi Corporation) was used as a scaffold in place of feeder cells.

[0223] The specific maintenance culture procedure for human ES cells was performed as follows: First, the subconflated human ES cells (where approximately 60% of the culture area was covered by cells) were washed with PBS and then dispersed into single cells using TrypLE Select (trade name, Life Technologies). Subsequently, the dispersed human ES cells were seeded into plastic culture dishes coated with Laminin511-E8 and cultured in StemFit medium under feeder-free conditions in the presence of Y27632 (ROCK inhibitor, 10 μM). The plastic culture dishes used were 6-well plates (Iwaki Corporation, for cell culture, culture area 9.4 cm²). 2 When using the above method, the number of seeded human ES cells dispersed into the single cell is 1.2 × 10⁶ per well. 4 The cells were prepared. One day after seeding, the medium was changed to StemFit medium without Y27632. Thereafter, the medium was changed once every 1-2 days with StemFit medium without Y27632. Subsequently, the cells were cultured under feeder-free conditions until one day before subconfretion. The human ES cells one day before subconfretion were cultured for one day under feeder-free conditions in the presence of SB431542 (TGFβ signaling pathway inhibitor, 5 μM) and SAG (Shh signaling pathway activator, 300 nM) (preconditioning).

[0224] Human ES cells were washed with PBS, treated with a cell dispersion solution using TrypLE Select, and then dispersed into single cells by pipetting. The dispersed single-cell human ES cells were then placed in a non-cell-adherent 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) at a rate of 1.2 × 10⁶ cells per well. 4 To induce cell formation, the cells were suspended in 100 μL of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium used was a 1:1 mixture of F-12 medium and IMDM medium to which 10% KSR, 450 μM 1-monothioglycerol, and 1 × Chemically defined lipid concentrate were added.

[0225] At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (ROCK inhibitor, final concentration 10 μM) and SAG (Shh signaling pathway activator, 300 nM, 30 nM, or 0 nM) were added to the serum-free medium. On day 3 after the start of suspension culture, 50 μL of medium containing exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, R&D Co., Ltd.) at a final concentration of 1.5 nM was added using a medium that did not contain Y27632 or SAG. From day 6 after the start of suspension culture onward, half of the medium was replaced every three days with a medium that did not contain Y27632, SAG, or human recombinant BMP4.

[0226] Furthermore, the aggregates 14 days after the start of the suspension culture were transferred to a 90 mm low-adhesion culture dish (Sumitomo Bakelite Co., Ltd.) and cultured for 3 days at 37°C and 5% CO2 in serum-free medium (DMEM / F12 medium supplemented with 1% N2 Supplement) containing a Wnt signaling pathway activator (CHIR99021, 3 μM) and an FGF signaling pathway inhibitor (SU5402, 5 μM). Subsequently, culture was performed in a 90 mm low-adhesion culture dish (Sumitomo Bakelite Co., Ltd.) using DMEM / F12 medium containing serum but without the Wnt signaling pathway activator or FGF signaling pathway inhibitor (NucT0 medium). From 40 days after the start of the suspension culture, culture was performed using a mixed medium of NucT0 medium and NucT2 medium (NucT1 medium). From 60 days after the start of the suspension culture, culture was performed using Neurobasal medium containing the thyroid hormone signaling pathway activator T3 (NucT2 medium). Multiple caps were excised from aggregates 82 days after the start of suspension culture using microscissors. The formation of complexes was then investigated using the neuroretinal tissue fragments (caps) obtained in this way.

[0227] To create a container for arranging and resting the caps, a groove approximately 1 mm wide, 1 mm deep, and 20 mm long was cut into the silicone gel using a scalpel. 8 to 13 cut-out caps were then placed in the groove at roughly equal intervals using tweezers.

[0228] First, 50 μL of fibrinogen was added to the groove where the Caps were arranged, mixed in, and then removed. Another 50 μL of fibrinogen was added and distributed throughout. Next, 50 μL of thrombin was added and incubated at room temperature for 20 minutes to induce fibrinization. In this way, a complex (composite gel) was created in which the fibrin-gelled Caps were arranged vertically (Figure 1).

[0229] The ease of handling of the composite gel was examined. When recovered from the grooved composite gel that had undergone fibrin gelation using tweezers and a scalpel, the gel could be recovered while remaining solid. This fibrin gel was transferred into HBSS, and it was checked whether it could withstand suction and discharge operations using a 20G Surflo catheter; it was confirmed that the gel could be transferred in and out without disintegration, while maintaining the state of the composite without any problems (Figure 1). It was confirmed that this composite gel has sufficient strength to withstand handling during transplantation.

[0230] As described above, it was found that by adding thrombin and fibrinogen separately to neural retinal tissue pieces (Cap) and allowing fibrin gelation to occur, a plurality of neural retinal Caps can be solidified to prepare a composite gel with good handleability.

[0231] <Example 2: Preparation of Composite Gel of Gelatin and Multiple Grafts of Neural Retina for Transplantation> A study was conducted on composites in which cell aggregates (grafts) containing multiple neural retinas are encapsulated in gelatin. A neural retinal tissue piece (sometimes referred to as cap) was used as the graft. Caps were prepared as follows. Human ES cells (derived from the KhES-1 strain (Non-Patent Document 1)) genetically modified to carry the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used as a scaffold replacing feeder cells.

[0232] Multiple Caps were cut out using micro-scissors from aggregates on day 82 after the start of suspension culture, which were prepared in the same manner as in Example 1. Composite formation was examined using the thus-obtained neural retinal tissue pieces (Caps).

[0233] To create a container for arranging and resting the caps, a groove approximately 1 mm wide, 1 mm deep, and 20 mm long was cut into the silicone gel using a scalpel. Eight to ten cut-out caps were then placed in the groove at roughly equal intervals using tweezers.

[0234] First, 50 μL of 10% Gelatin (w / v) (Nitta Gelatin Co., Ltd. LS-W) was added to the groove where the Caps were arranged, mixed, and then removed. Next, 50 μL of 20% Gelatin (w / v) was added, mixed, and then removed. Then, 50 μL of 30% Gelatin was added and incubated at 4°C for 20 minutes. In this way, a composite (composite gel) was created in which the solidified Caps were arranged vertically (Figure 2).

[0235] The ease of handling the composite gel was investigated. When the gelatin-solidified composite gel was retrieved from the groove using tweezers and a scalpel, it could be retrieved while remaining solid. This gelatin was then moved into HBSS, and its aspiration and dispensing operation was checked using a 1 ml tip with a wide tip. It was confirmed that the gel could be inserted and removed without any problems in its composite state without collapsing (Figure 2). This confirmed that the composite gel has sufficient strength to withstand the procedures during implantation.

[0236] As described above, it was found that by adding gelatin to neuroretinal tissue fragments (Caps) and solidifying them, multiple neuroretinal Caps can be combined to create an easy-to-handle composite gel.

[0237] <Example 1: Manufacturing of a retinal sheet containing a neuroretina> Human iPS cells (DSP-SQ strain, established by Dainippon Sumitomo Pharma Co., Ltd.) were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, Ajinomoto Co., Ltd.) was used as the feeder-free medium, and Laminin511-E8 (Nippi Corporation) was used as the feeder-free scaffold.

[0238] For differentiation induction, human iPS cells (DSP-SQ strain) were cultured feeder-free in StemFit medium until two days before subconfretion (approximately 30% of the culture area was covered by cells). These human iPS cells, two days before subconfretion, were then cultured feeder-free for two days in the presence of SAG (300 nM) (preconditioning).

[0239] Preconditioned human iPS cells were treated with a cell dispersion using TrypLE Select (Life Technologies), and then dispersed into single cells by pipetting. These single-cell dispersed human iPS cells were then placed in a non-cell-adherent 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) at a rate of 1.3 × 10⁶ per well. 4 Cells were suspended in 100 μl of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium used was a 1:1 mixture of F-12 medium and IMDM medium to which 10% KSR, 450 μM 1-monothioglycerol, and 1 × Chemically defined lipid concentrate were added. At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (final concentration 20 μM) and SAG (final concentration 10 nM) were added to the serum-free medium. On day 2 after the start of suspension culture, 50 μl of the above serum-free medium was added to a medium containing human recombinant BMP4 (manufactured by R&D Co., Ltd.) but without Y27632 and SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0240] Four days later (six days after the start of suspension culture), the culture medium was changed with the serum-free medium described above, which does not contain Y27632, SAG, or human recombinant BMP4. The medium change procedure involved discarding 60 μl of the culture medium in the incubator and adding 90 μl of the new serum-free medium described above, so that the total volume of medium was 180 μl. Subsequently, every 2 to 4 days, a half volume of the culture medium was changed with the serum-free medium described above, which does not contain Y27632, SAG, or human recombinant BMP4. The half volume of the culture medium was discarded by half the volume, i.e., 90 μl, of the culture medium in the incubator and added 90 μl of the new serum-free medium described above, so that the total volume of medium was 180 μl.

[0241] The cell aggregates obtained in this manner, 13 days after the start of suspension culture, were cultured for 3 days, or until 16 days after the start of suspension culture, in serum-free medium containing CHIR99021 (3 μM) and SU5402 (5 μM) (DMEM / F12 medium supplemented with 1% N2 supplement).

[0242] The resulting cell aggregates, obtained 16 days after the start of suspension culture, were cultured under 5% CO2 conditions until 75 days after the start of suspension culture, using the serum media described in [1], [2], and [3] below for the culture medium described below. [1] From day 16 to day 40 after the start of suspension culture: DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine (hereinafter referred to as Medium A). [2] From day 40 to day 60 after the start of suspension culture: Medium A and medium B, which is Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, 60 nM T3 and 100 μM taurine, are mixed in a 1:3 ratio. [3] 60 days after the start of suspension culture: Medium B.

[0243] Cell aggregates 75 days after the start of suspension culture were observed using an inverted microscope to confirm their morphology. It was found that neuroepithelial structures had formed at this time.

[0244] Cell aggregates 75 days after the start of suspension culture were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor progenitor cell markers (Figure 3). Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers (Figure 4). Cell nuclei were stained with DAPI.

[0245] These stained sections were observed using a fluorescence microscope (Keyence), and immunohistochemical images were obtained. Photographs of the produced cells observed under a fluorescence microscope are shown in Figures 3 and 4. In Figures 3 and 4, the upper row shows images taken with a low-magnification lens, and the lower row shows images taken with a high-magnification lens.

[0246] From the DAPI staining images in Figures 3 and 4, it was found that densely packed nerve tissue was formed on the surface of the cell aggregate, and that this nerve tissue formed a continuous epithelial structure. Analysis of the image in Figure 3 revealed that this nerve tissue had a Crx-positive layer (photoreceptor layer) about 2-5 cells thick on the surface of the cell aggregate, a Chx10-positive layer about 5-20 cells thick inside the Crx-positive layer, and further inside that, a layer with sparsely distributed Crx-positive cells (Figure 3). Morphologically, the surface of this cell aggregate was found to be the apical surface. Furthermore, analysis of the image in Figure 4 revealed that this nerve tissue had a Recoverin-positive layer (photoreceptor layer) and an Rx-positive layer. From these results, it was found that this nerve tissue had a photoreceptor layer containing Crx-positive and Recoverin-positive cells on its surface, a retinal progenitor cell layer containing Chx10-positive cells inside the photoreceptor layer, and a cell layer also formed inside the retinal progenitor cells. In other words, this method allows for the creation of a neuroretina containing photoreceptor cells and retinal progenitor cells from human iPS cells, and it was found that this neuroretina possesses a continuous epithelial structure.

[0247] <Reference Example 2: Graft Preparation and Evaluation> The three-dimensional retina created from human iPS cells consists of a neuroretina with a neuroepithelial structure that exhibits continuity in the composition and distribution of cells. This neuroepithelial structure has a layered structure composed of a photoreceptor cell layer and an inner layer, and possesses a characteristic appearance and morphology (Figure 3).

[0248] Human three-dimensional retinas are approximately 1-2 mm in size, and each has a different shape. Furthermore, due to the characteristics of the manufacturing method using self-organizing culture, although the neuroretina used for transplantation is the main product, non-neuronal retinal tissues such as ophthalmoplastic tissue (RPE, ciliary body, etc.) and cerebrospinal tissue (telencephalon, spinal cord, etc.) are also produced as by-products. Therefore, the central part of the neuroretina that does not contain non-neuronal retina is excised to obtain retinal fragments (grafts, caps) (Figures 5 and 6). Specifically, the peripheral part of the retinal fragment (cap) is used as a quality evaluation sample (ring), and by analysis (preferably quantitative PCR), only the caps corresponding to rings that meet the criteria are used as neuroretina for transplantation.

[0249] Figures 5 and 6 are conceptual diagrams of a typical cell aggregate. The area where the neuroepithelial structure (preferably a continuous epithelial structure) unique to the neuroretina, in which the photoreceptor layer and inner layer appear to be separated into two layers, is observed is designated as the graft (cap). The area around the cap where a similar neuroepithelial structure (preferably a continuous epithelial structure) is observed is designated as the quality evaluation sample (ring). The area other than the cap and ring is called the root.

[0250] The following is a method for isolating caps and rings from neuroepithelial structures contained within a single cell aggregate.

[0251] <Example 3: Shape of the graft (cap)> Grafts (caps) were prepared using the following method (Figure 7). First, bright-field images (phase-contrast images) of cell aggregates prepared from human iPS cells (DSP-SQ strain) 99 days after the start of suspension culture were taken using an inverted microscope (Olympus Corporation) according to the method described in Reference Example 1. After confirming the presence of neuroretina on the cell aggregates, the cell aggregates were transferred to a stereomicroscope, and neuroretina of various sizes were excised as grafts using the following method. The influence of graft size on the transplantation procedure using the transplantation device was also investigated.

[0252] (Cutting out the retinal sheet of the neuroretina) Cell aggregates were observed using an inverted microscope (Nikon ECLIPSE Ti) to obtain bright-field images (phase-contrast images). Particular attention was paid to the morphology of each individual cell and the characteristics of cell adhesion. In the cell aggregates, areas with a continuous epithelial structure, where the outer neuroblastic layer (including the photoreceptor cell layer and the neuroretinal progenitor cell layer) and the inner neuroblastic layer appeared as two distinct layers, were identified as the neuroretina. Tissues lacking a continuous epithelial structure, or areas where a continuous epithelial structure was observed but the outer and inner neuroblastic layers could not be distinguished and appeared as a single layer, were considered byproducts. Subsequently, while observing with a stereomicroscope, the neuroretina was dissected from the cell aggregates under stereomicroscope conditions using tapered tweezers and scissors to prepare tissue samples.

[0253] (Effects on the transplantation procedure of the excised retinal sheet) For the excised graft, a frontal image was taken with the cut surface facing the objective lens, and a lateral image was taken with the cut surface tilted so that it was perpendicular to the objective lens, using a stereomicroscope. Subsequently, the major axis, minor axis, and height of the graft were measured from the acquired images. For measurement, the major axis was defined as the longest line segment connecting two endpoints on the retinal sheet cross-section in the frontal image and its length. The minor axis was defined as the longest line segment perpendicular to the major axis connecting two endpoints on the retinal sheet cross-section in the frontal image and its length. The height was defined as the longest line segment perpendicular to the retinal sheet cross-section in the lateral image, with the intersection with the retinal sheet cross-section and the surface of the retinal sheet as endpoints, and its length. Furthermore, the volume of the graft was approximated as an ellipsoid obtained by halving the graft so that its cross-section passes through the major axis, and was calculated according to the following formula. Volume = 2 / 3 × Pi (π) × (major axis / 2) × (minor axis / 2) × height

[0254] The results showed that the placement of grafts in the implantation device, the stability of grafts within the device, and the ejection of grafts from the device were all affected by the size of the graft. Furthermore, the short diameter was suggested to be a particularly useful parameter. For 11 grafts that underwent successful implantation using the implantation device, the long diameter, short diameter, height, and volume were calculated. The mean, maximum, and minimum values ​​for each parameter were calculated and summarized in Table 11. From these results, it was found that grafts (caps) should have at least a long diameter of 0.8–1.7 mm, a short diameter of 0.4–1.1 mm, a height of 0.2–0.7 mm, and an apparent volume of 0.07–0.57 mm. 3 It was found to be to that extent. [Table 11]

[0255] <Reference Example 4: Cell composition of the graft (cap)> The grafts (caps) were prepared by the following method (number: 18001MF, d89, H5). First, the grafts (caps) were isolated from cell aggregates 89 days after the start of suspension culture, which were prepared from human iPS cells (DSP-SQ strain) according to the method described in Reference Example 1, using the methods described in Reference Examples 2 and 3.

[0256] The grafts were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor progenitor cell markers (Figure 8). Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers (Figure 8). Cell nuclei were stained with DAPI. These stained sections were observed using a confocal laser microscope (Olympus), and immunostained images were obtained.

[0257] Staining revealed that densely packed nerve tissue was formed on the surface of the graft (cap) (left side in the figure), and that this nerve tissue formed a neuroepithelial structure (particularly a continuous epithelial structure) (Figure 8). Furthermore, it was found that a Crx-positive layer (photoreceptor layer, Figure 8) with a thickness of about 2 to 10 cells was formed on the surface of the cell mass in this nerve tissue, a Chx10-positive layer with a thickness of about 5 to 20 cells was formed inside the Crx-positive layer, and further inside that, a layer containing Crx-positive cells was formed (Figure 8). Morphologically, the surface of this graft (cap) was found to be the apical surface. In addition, it was found that a Recoverin-positive layer (photoreceptor layer, arrow in Figure 8) and an Rx-positive layer were formed in this nerve tissue. From these results, it was found that this nerve tissue had a photoreceptor layer containing Crx-positive and Recoverin-positive cells on its surface, a retinal progenitor cell layer containing Chx10-positive cells was formed inside the photoreceptor layer, and a cell layer was also formed inside the retinal progenitor cells. In other words, it was found that the graft (cap) could be used to create a neuroretina containing a photoreceptor cell layer and a retinal progenitor cell layer, and that this neuroretina had a continuous epithelial structure.

[0258] <Example 5: Verification of the equivalence of the cap and ring> Gene expression in the caps and rings was compared using the following method. First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) 99 days after the start of suspension culture according to the method described in Reference Example 1, and designated as Lot 1. Furthermore, cell aggregates were prepared from human iPS cells (DSP-SQ strain) 82 days after the start of suspension culture according to the method described in Reference Example 1, and designated as Lot 2. For these two lots, the main product, neuroretina, and by-products were identified using a microscope according to the method described in Reference Example 3, and the caps of the neuroretina and by-products were isolated, respectively. The rings were isolated by cutting them out under a stereomicroscope using tapered tweezers and scissors, similar to the grafts. Total RNA was extracted from the caps and rings isolated from the neuroretina and by-products using a spin column (QIAGEN, RNeasy Micro kit) according to the method described in the manual included with the kit.

[0259] The total RNA concentration was measured using a meter (Nanodrop, Thermo Scientific), and then reverse-transcribed to cDNA using reverse transcriptase and primer (Reverse Transcription Master Mix Kit, Fluidigm). The cDNA was subjected to multiplex-PCR (Pre-Run) using a PCR instrument (Veriti 96-well thermal cycler, Applied Biosystems) with all the probes used for validation. Subsequently, the pre-run reaction mixture was injected into a multi-well with a flow channel (96.96 Dynamic Array IFC, Fluidigm) using an IFC controller HX (Fluidigm), and the expression levels of marker genes for neuroretina and non-neurotinic byproducts were measured by real-time PCR using a multi-sample real-time PCR system (Biomark HD, Fluidigm). The PCR probes used for validation are shown in Table 12.

[0260] [Table 12]

[0261] The results are shown in heatmaps in Figures 9A and 9B. Gene expression levels were evaluated using the ΔCt value, which is calculated from the difference between the Ct value of the target gene and the Ct value of GAPDH, a gene used as an internal standard. A lower ΔCt value indicates higher gene expression, and a higher ΔCt value indicates lower gene expression. Gray indicates high gene expression, and black indicates low gene expression (lighter colors indicate higher gene expression). When gene expression was examined in each cap and ring, neuroretinal marker genes were expressed in the caps and rings isolated from the neuroretina in both Lot 1 and Lot 2. On the other hand, when gene expression was examined in the caps and rings isolated from the byproduct, in both lots, contrary to the neuroretina, the expression levels of the neuroretinal marker genes were low, and the expression levels of the byproduct marker genes were high. Furthermore, when gene expression was compared in caps and rings isolated from the same cell aggregate, it was found that the expression levels of the neuroretinal marker genes and the byproduct marker genes were equivalent in all caps and rings isolated from the neuroretina and byproducts.

[0262] These results demonstrate that if the ring is a neuroretina, then the cap is also a neuroretina. Furthermore, it was demonstrated that gene expression is equivalent in the cap and the ring.

[0263] <Reference Example 6: Results of graft transplantation> After analyzing the gene expression of the ring using the method described in Reference Example 5, the corresponding cap (neuroretinal sheet) was used as the retinal sheet. The retinal sheet was immersed in a 4-fold dilution of "Viscote (Viscote:Opeguard = 1:3)" as the transplantation medium to prepare the transplantation composition. This was transplanted into retinal degenerated nude rats, and the engraftment after transplantation was evaluated.

[0264] First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) according to the method described in Reference Example 1. Then, caps and rings were isolated from the cell aggregates 75 days or more after the start of suspension culture, according to the method described in Reference Example 5. The isolated caps were stored in a commercially available preservation solution while genetic analysis of the rings was performed. Gene expression analysis of the isolated rings was performed using real-time PCR with Biomark HD (Fluidigm) according to the method described in Reference Example 5. From the results of the gene expression analysis, rings that expressed the neuroretinal marker gene and did not express the byproduct marker gene were selected, and the caps corresponding to these rings were selected as grafts (retinal sheets to be transplanted). The grafts were washed with buffer (Thermo Fisher Scientific), and then immersed in a transplantation medium to prepare the transplantation composition. This was implanted subretinally into retinal degenerative nude rats (a photoreceptor degeneration model, SD-Foxn1 Tg(S334ter)3LavRrrc nude rat) using an injector described in a publicly available document (Shirai et al. PNAS 113, E81-E90).

[0265] Eye tissue from mice aged 230-240 days after the start of suspension culture was fixed with paraformaldehyde (PFA) and sucrose. Frozen sections were prepared from the fixed eye tissue using a cryostat. These frozen sections were immunostained for human nuclei (anti-HuNu antibody, Millipore, mouse or anti-HNA antibody), Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers, and PKCα (anti-PKCα antibody, R&D Systems, goat), one of the bipolar cell markers.

[0266] Table 13 summarizes the results of graft quality evaluation based on gene expression analysis of the ring and the transplantation results. The method for calculating the ΔCt value was the same as the method described in Reference Example 5. For the gene expression analysis of the ring, the quality evaluation test (ring-PCR test) was considered successful if the ΔCt value of Recoverin, one of the neuroretinal marker genes, was 10 or less, and the ΔCt values ​​of the by-product marker genes FOXG1, HOXB2, ZIC1, and OCT3 / 4 were each 5 or more. For the transplantation results, the engraftment was evaluated as good if human nucleus-positive and Recoverin-positive photoreceptor cells could be detected under the retina. Furthermore, if the transplantation site did not become significantly thicker than the appropriate engraftment size, it was determined that no hypertrophy was detected.

[0267] A representative engraftment image is shown in Figure 10. The post-transplant engraftment image was evaluated for 14 eyes that passed the pre-transplant quality assessment test. In all 14 eyes, Recoverin-positive photoreceptor cells were detected, indicating good engraftment. Since these cells were HuNu-positive, it was determined that the Recoverin-positive photoreceptor cells originated from the transplanted cap. Furthermore, no hypertrophy was detected in any of the 14 eyes.

[0268] Based on these results, we were able to select grafts that would successfully engraft beneath the retina, meaning that photoreceptor cells would engraft and hypertrophy would not occur, by examining the expression levels of neuroretinal and byproduct marker genes in the ring before transplantation.

[0269] Furthermore, we were able to demonstrate the safety of subretinal administration of "Viscote 4x dilution (Viscote:Opegard = 1:3)," which is used as a transplantation medium. Specifically, although Viscote 4x dilution contains hyaluronic acid and chondroitin sulfate, we were able to demonstrate the safety of subretinal administration of hyaluronic acid and chondroitin sulfate at the concentrations used in this study.

[0270] [Table 13]

[0271] <Reference Example 7: Marker expression on the cap and ring>. In Reference Example 5, the equivalent cell composition of the cap and ring was demonstrated using the PCR method. Next, immunohistochemical staining was used to investigate whether the cell composition and tissue structure of the cap and ring were equivalent.

[0272] First, human iPS cells (DSP-SQ strain) were differentiated into retinal cells using the method described in Reference Example 1. Subsequently, the neuroretinal caps and rings were isolated from cell aggregates 120 days after the start of suspension culture using the method described in Reference Example 5. After washing the caps and rings, they were fixed with 4% paraformaldehyde (PFA fixation) and sucrose replacement. Frozen sections were prepared from the fixed caps and rings using a cryostat. These frozen sections were immunostained for DAPI, which stains the nucleus; Crx (anti-Crx antibody, Takara, rabbit), one of the markers for photoreceptor progenitor cells; Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the markers for neuroretinal cells; NRL (anti-NRL antibody, Bio-Techne, goat), one of the markers for rod photoreceptor progenitor cells; FOXG1 (anti-FOXG1 antibody, Takara Bio, rabbit), one of the telencephalon markers; PAX2 (anti-PAX2 antibody, Thermo Fisher Scientific, rabbit), one of the Optic Stalk markers; and NANOG (anti-NANOG antibody, Merck, mouse), one of the markers for undifferentiated pluripotent stem cells.

[0273] The results of immunohistochemical staining are shown in Figure 11. For caps and rings excised from the same cell aggregate, the immunohistochemical staining results for the rings are shown in the upper panel, and the results for the caps are shown in the lower panel. From these results, it was found that in both the caps and rings, Crx-positive photoreceptor progenitor cells, Chx10-positive neuroretina, and NRL-positive rod photoreceptor progenitor cells were expressed in a continuous, layered manner. Furthermore, in neither the caps nor the rings were telencephalon (FOXG1-positive), optic stalk (PAX2-positive), or pluripotent stem cells (NANOG-positive) detected. In addition, a comparison of the staining patterns for Crx, Chx10, and NRL confirmed that these neuroretinal markers showed almost identical distributions in the caps and rings.

[0274] <Reference Example 8: Proportion of photoreceptor progenitor cells and neuroretinal progenitor cells constituting a neuroretinal sheet for transplantation> The proportion of photoreceptor progenitor cells and neuroretinal progenitor cells among the constituent cells of a neuroretinal sheet prepared from cell aggregates differentiated from pluripotent stem cells was analyzed and quantified using immunohistochemistry (IHC), a type of immunostaining method.

[0275] Human iPS cells (DSP-SQ strain) were differentiated into retinal cells using the method described in Reference Example 1. Subsequently, caps and rings were isolated from cell aggregates at 84, 92, and 93 days after the start of suspension culture using the method described in Reference Example 3. Gene expression analysis of the isolated rings was performed using the method described in Reference Example 6. Using the method described in Reference Example 6, rings expressing neuroretinal marker genes and not expressing by-product marker genes were selected, and the caps corresponding to these rings were used as neuroretinal sheets for transplantation. In this way, one neuroretinal sheet for transplantation was prepared from the cell aggregate at 84 days after the start of suspension culture, two neuroretinal sheets for transplantation were prepared from the cell aggregate at 92 days after the start of suspension culture, and one neuroretinal sheet for transplantation was prepared from the cell aggregate at 93 days after the start of suspension culture. In total, four neuroretinal sheets for transplantation were prepared.

[0276] The obtained neuroretinal sheets for transplantation were cultured in medium B for 7 days for analysis. The cultured neuroretinal sheets were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal progenitor cell markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor cell progenitor cell markers. Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers. Cell nuclei were stained with DAPI. These stained sections were observed using a fluorescence microscope (Keyence), and immunostained images were obtained. One example (D3) is shown in Figure 12.

[0277] Immunostaining images were analyzed using ImageJ (version 1.52a, NIH) to determine the number of DAPI-positive cells, DAPI-positive and Chx10-positive cells, and DAPI-positive and Crx-positive cells for each of the four neuroretinal sheets for transplantation. Similarly, immunostaining images were analyzed to determine the number of DAPI-positive cells and DAPI-positive and Rx-positive cells. From these values, the percentage of Chx10-positive cells, Crx-positive cells, and Rx-positive cells were calculated. The results are shown in Table 14. [Table 14]

[0278] From these results, it was found that the proportion of Chx10-positive cells in the neuroretinal sheets for transplantation, excised from the cell aggregates, was approximately 23-45%, the proportion of Crx-positive cells was approximately 30-56%, and the proportion of Rx-positive cells was approximately 40-54%.

[0279] In other words, it was suggested that the neuroretinal sheets for transplantation contained approximately 34% (23-45%) Chx10-positive neuroretinal progenitor cells, approximately 40% (30-56%) Crx-positive photoreceptor progenitor cells, and approximately 47% (40-54%) Rx-positive cells.

[0280] <Reference Example 9: Proportion of photoreceptor progenitor cells and neuroretinal progenitor cells constituting a neuroretinal sheet for transplantation> The composition of the constituent cells of a neuroretinal sheet for transplantation, prepared from cell aggregates differentiated from various pluripotent stem cells, was investigated using flow cytometry (also known as FACS), a type of immunohistochemical staining method.

[0281] Human iPS cells (QHJI01s04 strain) were differentiated into retinal cells using the method described in Reference Example 1. Subsequently, caps and rings were isolated from cell aggregates 88 days after the start of suspension culture using the method described in Reference Example 3. The caps were then used as the neuroretinal sheets for transplantation. The neuroretinal sheets for transplantation were stored at 17°C for 2 days. Five of the obtained neuroretinal sheets were bundled together to form one sample, washed with PBS, and enzymatically treated with a neuronal cell dispersion (WAKO, containing papain) at 37°C for approximately 30 minutes. The cells were then dispersed into single cells by pipetting to obtain a single-cell suspension. The obtained single-cell suspension was fixed using a fixative (BD, CytoFix) to obtain a sample for FACS. The FACS sample was blocked and permeabilized (cell membrane puncture) using a serum-containing Perm / Wash solution (BD). Immunostaining was then performed using the following fluorescently labeled antibodies: anti-Chx10 antibody (Santa Cruz), anti-Pax6 antibody (BD), and anti-Crx antibody (Santa Cruz). The samples were then analyzed by flow cytometry using an analyzer (BD).

[0282] The results showed that the Chx10-positive and Pax6-positive fraction (neuroretinal progenitor cell fraction) accounted for 11.5%, the Chx10-positive and Pax6-negative fraction (progenitor cell fraction biased towards bipolar cells) accounted for 23.4%, the Chx10-negative and Pax6-positive fraction (ganglion cells and amacrine cells) accounted for 10.7%, and the Crx-positive cell fraction (photoreceptor cell progenitor cell fraction) accounted for 17.4%.

[0283] Furthermore, human iPS cells (DSP-SQ strain) were differentiated into retinal cells using the method described in Reference Example 1. Subsequently, 11 cell aggregates were prepared 88 days after the start of suspension culture, and 11 caps and rings were isolated from each cell aggregate using the method described in Reference Example 3. The 11 caps were combined to form one cap sample. Similarly, the 11 rings were combined to form one ring sample. The cap samples and ring samples were washed with PBS and enzymatically treated with a nerve cell dispersion (WAKO, containing papain) at 37°C for approximately 30 minutes to obtain single-cell suspensions of the caps and rings, respectively. The obtained single-cell suspensions of the caps and rings were fixed using a fixative (BD, CytoFix) to obtain samples for FACS. FACS samples were blocked and drilled using serum-containing Perm / Wash solution (BD), and immunostained with the following fluorescently labeled antibodies: anti-Chx10 antibody (Santa Cruz), anti-Crx antibody (Santa Cruz), and anti-SSEA-4 antibody. An isotype control was used as a negative control for immunostaining. The samples were then analyzed by flow cytometry using an analyzer (BD). The percentages of Chx10-positive cells, Crx-positive cells, and SSEA-4-positive cells were calculated as the difference compared to the respective isotype controls. The results are shown in Table 15. [Table 15]

[0284] In the cap samples, 29.4% of cells were positive for Chx10, a neuroretinal progenitor cell marker; 15.8% were positive for Crx, a photoreceptor progenitor cell marker; and less than 1% were positive for SSEA-4, a pluripotent stem cell marker (untargeted cells). In the ring samples, 28.1% were positive for Chx10, a neuroretinal progenitor cell marker; 21.7% were positive for Crx, a photoreceptor progenitor cell marker; and less than 1% were positive for SSEA-4, a pluripotent stem cell marker (untargeted cells).

[0285] These results first revealed that the cap and ring samples were neuroretina containing Chx10-positive and Crx-positive cells, and substantially free of undifferentiated iPS cells. Furthermore, it was demonstrated that the ratio of Chx10-positive to Crx-positive cells in the cap sample was equivalent to that in the ring sample. Finally, if the ring sample is neuroretina, then the cap sample is also neuroretina.

[0286] Furthermore, when these caps or rings (preferably caps) are used as a neuroretinal sheet for transplantation, it was found that the Chx10-positive cell fraction (neuroretinal progenitor cell fraction) contained in this neuroretinal sheet is approximately 30% (20-40%), and the Crx-positive cell fraction (photoreceptor progenitor cell fraction) is approximately 17% (10-30%).

Claims

1. A complex comprising cell aggregates and a matrix, wherein two or more of the cell aggregates are arranged in the matrix, The cell aggregate comprises two or more neuroretinae, The matrix is ​​fibrin gel or gelatin, Two or more of the aforementioned cell aggregates are arranged in rows within the matrix, The aforementioned neuroretina is a composite neuroretinal sheet for transplantation having the following characteristics (1) to (10): (1) Derived from pluripotent stem cells, (2) Having a three-dimensional structure, (3) A neuroretinal layer having a multilayer structure including a photoreceptor cell layer and an inner layer, (4) The photoreceptor layer comprises one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, (5) The inner layer comprises one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells and bipolar cells, (6) The surface of the neuroretinal layer has an apical surface, (7) The inner layer is located inside the photoreceptor layer that is located along the apical surface, (8) The area of ​​the neuroretinal layer is 50% or more of the total surface area of ​​the neuroretinal sheet for transplantation, (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neuroretinal layer. (10) Expression of neuroretinal cell-related genes is observed in the neuroretinal sheet for transplantation, and expression of non-neuroretinal cell-related genes is not observed, and the non-neuroretinal cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eyeball-related tissue marker genes.

2. The composite according to claim 1, wherein the cell aggregates have a major axis of 600 μm to 2500 μm.

3. The composite according to claim 1 or 2, wherein the spacing between two or more cell aggregates in the matrix is ​​10 μm to 50 μm.

4. A method for producing a complex in which cell aggregates are arranged in a matrix, (1) The first step involves creating two or more neuroretinal cell aggregates from pluripotent stem cells, (2) A second step of bringing two or more of the cell aggregates into contact with a matrix or matrix precursor in a predetermined arrangement, and then gelling the matrix, Includes, The cell aggregate comprises two or more neuroretinae, The matrix is ​​fibrin gel or gelatin, A method for producing cells, wherein two or more of the aforementioned cell aggregates are arranged in rows within the matrix.

5. The manufacturing method according to claim 4, further comprising arranging 2 to 20 cell aggregates in a row in the second step.

6. The manufacturing method according to claim 4 or 5, wherein the matrix is ​​a fibrin gel, and in the second step, two or more of the cell aggregates are arranged in a row, the two or more arranged cell aggregates are brought into contact with either a fibrinogen solution or a thrombin solution, and then brought into contact with the other of the fibrinogen solution or thrombin solution, thereby causing the fibrinogen and thrombin to react and gel.

7. The manufacturing method according to claim 4 or 5, wherein the matrix is ​​gelatin.

8. The manufacturing method according to any one of claims 4 to 7, wherein the distance between two or more cell aggregates in the matrix is ​​10 μm to 50 μm.

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