Methods for preserving nerve tissue

Abstract

The present invention aims to provide a method for preserving neural tissue for several hours to several weeks without freezing, a preservation solution therefor, a method for transporting neural tissue using the method and preservation solution, and a composition for transplantation. [Solution] The method for preserving neural tissue of the present invention comprises preserving the neural tissue in a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 8°C to 30°C. The preservation solution of the present invention is a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM, for preserving neural tissue derived from pluripotent stem cells at a storage temperature of 8°C to 30°C.

Description

[Technical field]

[0001] The present invention relates to a method for preserving neural tissue, a preservation solution for neural tissue, and a method for transporting neural tissue, and in particular to a method for preserving neural tissue for several hours to several weeks without freezing, a preservation solution therefor, and a method for transporting neural tissue while preserving it. The present invention also relates to a composition for transplantation. [Background technology]

[0002] In the nervous tissue of a living organism, one or more types of nervous system cells form a layer structure. Retinal tissue, which is one type of nervous tissue, is mainly composed of five types of nerve cells, namely photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and glial cells, forming a three-dimensional layer structure. It has been suggested that transplantation therapy using nervous tissue is effective as a treatment for nerve diseases, such as retinal degenerative diseases, but it has been difficult to obtain tissue that maintains the layer structure and function that reflects the nervous tissue of a human living organism, making it difficult to generalize as a treatment. In recent years, it has become possible to stably produce nervous tissue (e.g., retinal tissue) by inducing differentiation from pluripotent stem cells (Non-Patent Documents 1 and 2). Therefore, as the next challenge to realize transplantation therapy using nervous tissue, it was necessary to develop a technology to stably preserve the produced nervous tissue during the period required for quality testing or transportation to a transplant facility.

[0003] Cryopreservation methods have been developed as a method for preserving neural tissue (e.g., Patent Document 1), but freezing can sometimes cause deterioration in the quality of the tissue, and cryopreservation can also require skilled techniques. On the other hand, it has been reported that liver tissue can be preserved by perfusing it using a huge device and preserving it at 22°C (Non-Patent Document 3). However, for neural tissues such as retinal tissues that are composed of one or more types of cells in a layered structure, there are no appropriate preservation methods other than freezing, and the challenge was to establish a method suitable for preservation for a few hours to a few weeks. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent number 6012164 [Non-patent literature]

[0005] [Non-Patent Document 1] Cell Stem Cell,3,519-32(2008) [Non-Patent Document 2] Nature Communications,6,6286(2015) [Non-Patent Document 3] Scientific Reports,5,9563(2015) [Non-Patent Document 4] Transplantation,74,1414-9(2002) Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a method for preserving neural tissue for several hours to several weeks without freezing, a preservation solution therefor, a method for transporting neural tissue using said method and preservation solution, and a composition for transplantation. [Means for solving the problem]

[0007] The inventors conducted extensive research to solve the above problems and discovered that neural tissue can be preserved for several days while maintaining its function in a preservation solution having a potassium ion concentration of less than 115 mM at a storage temperature of 8°C to 30°C, thereby arriving at the present invention.

[0008] That is, the present invention relates to the following. [1] A method for preserving neural tissue, comprising preserving the neural tissue in a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 8°C to 30°C. [2] The storage temperature should be between 12℃ and 22℃. [3] The preservation method according to [1] or [2], wherein the nervous tissue is of human origin. [4] Any of the preservation methods [1] to [3], wherein the neural tissue is tissue derived from pluripotent stem cells. [5] A preservation method according to any of [1] to [4], in which the neural tissue is to be used for transplantation. [6] Any of the preservation methods [1] to [5], wherein the nervous tissue is a cellular aggregate having a layered structure. [7] The method of [6], wherein the neural tissue is a cellular aggregate having a neuroepithelial structure. [8] The preservation method of [7], wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of a cell aggregate and / or a rosette structure present on the surface or inside of a cell aggregate. [9] The preservation method according to any one of [1] to [8], wherein the neural tissue is one or more neural tissues selected from the group consisting of forebrain tissue, midbrain tissue, and retinal tissue.

[10] Any of the storage methods [1] to [9] for storage for a period of 14 days or less.

[11] Any of the storage methods [1] to [9] for storage for a period of 5 days or less.

[12] The preservation method according to any one of [1] to

[11] , wherein the preservation solution is an aqueous solution that contains a buffer substance and has the effect of buffering the pH to a range of 6.0 to 8.6.

[13] The preservation method according to any one of [1] to

[12] , wherein the preservation solution is an aqueous solution further containing glycosaminoglycans.

[14] The storage method according to

[13] , wherein the concentration of glycosaminoglycans is 0.1% (w / v) or more and 10% (w / v) or less.

[15] The method for preservation described in

[13] or

[14] , wherein the glycosaminoglycans are one or more glycosaminoglycans selected from the group consisting of chondroitin sulfate and hyaluronic acid.

[16] A storage solution for storing neural tissue derived from pluripotent stem cells at a storage temperature of 8°C to 30°C, with a potassium ion concentration of greater than 0 mM and less than 115 mM.

[17] The preservation solution of

[16] , the storage temperature of which is 12°C to 22°C.

[18] A preservation solution according to

[16] or

[17] , in which the neural tissue is a cellular aggregate having a layered structure.

[19] A preservation solution of

[18] in which neural tissue is a cellular aggregate with neuroepithelial structures.

[20] A preservation solution according to

[19] , wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of the cell aggregates and / or a rosette structure present on the surface or inside of the cell aggregates.

[21] A method for transporting neural tissue, comprising transporting the neural tissue in a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 8°C to 30°C.

[22] A transportation method according to

[21] , wherein the storage temperature is between 12°C and 22°C.

[23] The method of

[21] or

[22] , wherein the neural tissue is tissue derived from human pluripotent stem cells.

[24] Any of the transportation methods

[21] to

[23] , in which neural tissue is to be used for transplantation.

[25] Any of the transportation methods

[21] to

[24] , wherein the nervous tissue is a cellular aggregate having a layered structure.

[26] The transportation method of

[25] , in which nervous tissue is a cellular aggregate having a neuroepithelial structure.

[27] The transportation method of

[26] , wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of a cell aggregate and / or a rosette structure present on the surface or inside of a cell aggregate.

[28] The transportation method according to any one of

[21] to

[27] , wherein the neural tissue is one or more neural tissues selected from the group consisting of forebrain tissue, midbrain tissue, and retinal tissue.

[29] A composition for transplantation, comprising: an aqueous solution having a potassium ion concentration of more than 0 mM and less than 115 mM, and a pH adjusted to the range of 6.0 to 8.6 using a buffering substance; and human neural tissue derived from pluripotent stem cells, maintained at 12°C to 22°C.

[30] The composition described in

[29] , wherein the neural tissue is a cellular aggregate having a neuroepithelial layer structure.

[31] The composition described in

[30] , wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of the cell aggregate and / or a rosette structure present on the surface or inside the cell aggregate.

[32] Any of the compositions

[29] to

[31] , wherein the neural tissue is one or more neural tissues selected from the group consisting of forebrain tissue, midbrain tissue, and retinal tissue.

[33] Any of the compositions

[29] to

[32] , wherein the aqueous solution further contains 0.1% (w / v) or more and 10% (w / v) or less of glycosaminoglycans. Effect of the Invention

[0009] According to the present invention, for example, during the period from when nervous tissue is removed from a living body or when differentiation is induced from pluripotent stem cells until when it is provided for transplantation, for example during the period required for quality control and transportation until when it is provided for transplantation, it is possible to store the tissue for up to several weeks without freezing, making it more feasible to treat diseases based on nervous tissue disorders or damaged states of nervous tissue through transplantation therapy. [Brief description of the drawings]

[0010] [Figure 1] The results of bright field observation of cell aggregates containing retinal tissue prepared from human iPS cells were shown, which were stored under various storage conditions and then further cultured for recovery at 37°C for 7 days. [Diagram 2] The results of immunohistochemical staining of cell aggregates containing retinal tissue prepared from human iPS cells were compared for Chx10 expression in the cell aggregates obtained after storage under various conditions and then recovery culture at 37°C for 7 days. [Diagram 3] The results of immunohistochemical staining of Crx expression in cell aggregates containing retinal tissue prepared from human iPS cells were compared after storage under various conditions and subsequent recovery culture at 37°C for 7 days. [Figure 4] The results of bright field observation of cell aggregates containing retinal tissue prepared from human ES cells that were stored under various storage conditions and then further recovered and cultured at 37°C for 7 days are shown. [Diagram 5]The results of immunohistochemical staining of cell aggregates containing retinal tissue prepared from human ES cells were compared for Chx10 expression in the cell aggregates obtained after storage under various conditions and then recovery culture at 37°C for 7 days. [Figure 6] The results of immunohistochemical staining of Crx expression in cell aggregates containing retinal tissue prepared from human ES cells were compared after storage under various conditions and subsequent recovery culture at 37°C for 7 days. [Figure 7] Cell aggregates containing retinal tissues prepared from human iPS cells were stored under various conditions, and then cultured for recovery at 37°C for 7 days. The results of immunohistochemical staining of the expression of various proteins in the cell aggregates were compared: (Top) Chx10 and Crx, (middle) Chx10, (bottom) Crx. [Figure 8] Cell aggregates containing retinal tissues prepared from human iPS cells were stored under various storage conditions, and then cultured for recovery at 37°C for 7 days. The results of immunohistochemical staining of the expression of various proteins in the cell aggregates were compared. (Top) Recoverin, (middle) Rx, (bottom) Ki67. [Figure 9] The results of bright field observation of cell aggregates containing retinal tissue prepared from human iPS cells were shown immediately after storage under various storage conditions (storage conditions A and B), and after recovery culture at 37°C for 7 days. [Figure 10] 1 shows the results of measuring the viability of cell aggregates containing retinal tissue prepared from human iPS cells before and after storage. [Figure 11] Regarding cell aggregates containing retinal tissue prepared from human iPS cells, the following results are shown: bright field observation of the cell aggregates immediately after storage under various storage conditions (storage conditions B to F) (top row), bright field observation of the cell aggregates after recovery culture at 37°C for 7 days (middle row), and comparison of Crx expression in cell aggregates after recovery culture by immunohistochemical staining (bottom row). [Figure 12] The number of Crx-positive cells was quantified from the results shown in FIG. 11 and the results are shown in graph form. [Figure 13] Regarding cell aggregates containing retinal tissue prepared from human iPS cells, the following results are shown: bright field observation of the cell aggregates immediately after storage under various storage conditions (storage conditions B to F) (top row), bright field observation of the cell aggregates after recovery culture at 37°C for 7 days (middle row), and comparison of Crx expression in cell aggregates after recovery culture by immunohistochemical staining (bottom row). [Figure 14] The number of Crx-positive cells was quantified based on the results of immunohistochemical staining shown in FIG. 11 and the results are shown in graph form. [Figure 15] Regarding cell aggregates containing retinal tissue prepared from human iPS cells, the following results are shown: bright field observation of the cell aggregates immediately after storage under various storage conditions (storage conditions A and B) (top row); bright field observation of the cell aggregates after recovery culture at 37°C for 7 days (middle row); and comparison of Crx expression in the cell aggregates after recovery culture by immunohistochemical staining (bottom row). [Figure 16] Regarding cell aggregates containing retinal tissue prepared from human iPS cells, the following results are shown: bright field observation of the cell aggregates immediately after storage under various storage conditions (storage conditions A and B) (top row); bright field observation of the cell aggregates after recovery culture at 37°C for 7 days (middle row); and comparison of Crx expression in the cell aggregates after recovery culture by immunohistochemical staining (bottom row). [Figure 17] The following shows the results of bright field observation of cell aggregates containing forebrain and / or midbrain tissue prepared from human iPS cells immediately after storage under various storage conditions (storage conditions A to D) (top row), the results of bright field observation of cell aggregates after recovery culture at 37°C for 7 days (middle row), and the results of immunohistochemical staining to compare the expression of Rx and Otx2 in cell aggregates after recovery culture (bottom row). [Figure 18] Regarding the results of immunohistochemical staining shown in Figure 17, the ratio of the number of cell aggregates in which luminal structures composed of Otx2-positive cells were observed to the total number of cell aggregates under each storage condition was calculated, and the results are shown in graph form. [Figure 19]Cell aggregates containing retinal tissue prepared from human ES cells were left for 48 hours under conditions without (A, C) or with (B, D) preservation, and then subretinal transplantation was performed on nude rats with retinal degeneration. Immunohistochemical staining of frozen sections of nude rat retinas is shown. [Figure 20] Regarding cell aggregates containing retinal tissue prepared from human iPS cells, the results of bright field observation of the cell aggregates immediately after storage under various storage conditions (storage conditions A to D) (top row), the results of bright field observation of the cell aggregates after recovery culture at 37°C for 7 days (second row from the top), and the results of immunohistochemical comparison of the expression of the photoreceptor precursor cell marker Crx (second row from the bottom) and the retinal precursor cell marker Chx10 (bottom row) in the cell aggregates after recovery culture are shown. [Figure 21] Cell aggregates containing retinal tissue prepared from human iPS cells were incubated in Optisol solution at 17°C for 96 hours and then transplanted under the retina of a nude rat with retinal degeneration. The immunohistochemical staining of frozen sections of the nude rat retina is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The method for preserving neural tissue of the present invention comprises preserving the neural tissue in a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 8°C to 30°C.

[0012] As used herein, the term "tissue" refers to a structure of a cell population (also called a "cell aggregate") in which one type of cell with uniform morphology and properties, or multiple types of cells with different morphologies and properties, are arranged three-dimensionally in a certain pattern. Tissues are either harvested from a living body or artificially produced using stem cells, and are maintained in an isolated state outside the body.

[0013] In the present invention, cells include epithelial cells and mesenchymal cells based on the morphological classification of cells in developmental biology. Epithelial cells have apical-basal polarity. The apical side is often the interstitial side, while the basal side has a basement membrane and contacts the extracellular matrix. Epithelial cells can form epithelial tissue by forming strong bonds between epithelial cells through adherens junctions or tight junctions on the apical side. Epithelial tissue is also called epithelial sheet, and includes simple squamous epithelium, simple columnar epithelium, and stratified squamous epithelium. Mesenchymal cells are cells with weak apical-basal polarity, and are less likely to form sheets due to weak contribution of adherens junctions and / or tight junctions. In vivo, mesenchymal cells are often present scattered in star-like configurations within the extracellular matrix.

[0014] As used herein, the term "neural tissue" refers to a tissue composed of nervous system cells, in which one or more types of nervous system cells are arranged three-dimensionally in layers. Representative examples of neural tissue include forebrain tissue, midbrain tissue, and retinal tissue, as well as tissues such as the cerebrum, cerebellum, diencephalon, hindbrain, telencephalon, and spinal cord, and also include structures in which at least multiple types of cells and / or progenitor cells constituting these are arranged three-dimensionally in layers, as well as multiple combinations of these tissues. Neural tissue can be confirmed by microscopic observation using an optical microscope (e.g., bright-field microscope, phase-contrast microscope, differential interference microscope, Hoffman interference microscope, stereomicroscope), or by using the expression of neural tissue markers such as PSA-NCAM and N-cadherin as indicators.

[0015] Nervous tissue may form a structure called a neuroepithelial structure. A neuroepithelial structure is a structure composed of nervous system cells, in which one or more types of nervous system cells are arranged three-dimensionally in layers. In one embodiment, the neuroepithelial structure has apical-basal polarity.

[0016] In some embodiments, neuroepithelial structures may be present over the surface of the cell aggregates, while in other embodiments, neuroepithelial structures may also form within the cell aggregates.

[0017] In some embodiments, the neuroepithelial structure may be a continuous epithelial structure on the surface of the cell aggregate, as described below, or a rosette structure (also called a neural rosette, a luminal structure) on the surface of the cell aggregate or within the cell aggregate, as described below.

[0018] The abundance of neuroepithelial structures in the cell aggregates can be assessed by bright field observation using an optical microscope.

[0019] In the present invention, the term "neural cells" refers to cells of ectoderm-derived tissues other than epidermal cells (e.g., epidermis, hair, nails, sebaceous glands, etc.). In other words, the term includes cells such as neural progenitor cells, neurons (nerve cells), glia, neural stem cells, neuronal progenitor cells, and glial progenitor cells. Neural cells also include cells constituting retinal tissue (retinal cells), retinal progenitor cells, retinal layer-specific nerve cells, neural retinal cells, and retinal pigment epithelial cells, which will be described below.

[0020] Neurons (or neuronal cells) are functional cells that form neural circuits and contribute to signal transmission, and can be identified using the expression of immature neuronal markers such as TuJ1, Dcx, and HuC / D, and / or mature neuronal markers such as Map2 and NeuN.

[0021] Examples of glia include astrocytes, oligodendrocytes, Muller glia, etc. Examples of astrocyte markers include GFAP, oligodendrocyte markers include O4, and Muller glia markers include CRALBP.

[0022] Neural stem cells are cells that have the ability to differentiate into neurons and glial cells (multipotency) and the ability to proliferate while maintaining multipotency (also called self-renewal). Neural stem cell markers include Nestin, Sox2, Musashi, Hes family, CD133, etc., but these markers are markers for progenitor cells in general and are not considered to be specific to neural stem cells. The number of neural stem cells can be evaluated by neurosphere assay, clonal assay, etc.

[0023] Neuronal progenitor cells are cells that have the ability to proliferate, produce nerve cells, and do not produce glial cells. Examples of markers for neuronal progenitor cells include Tbr2 and Tα1. Alternatively, cells that are positive for immature neuronal markers (TuJ1, Dcx, HuC / D) and proliferation markers (Ki67, pH3, MCM) can be identified as neuronal progenitor cells.

[0024] Glial precursor cells are cells that have the ability to proliferate and differentiate into glial cells but do not differentiate into neural cells.

[0025] Neural precursor cells are a collection of precursor cells, including neural stem cells, neuronal precursor cells, and glial precursor cells, and have the ability to proliferate and differentiate into neurons and glia. Neural precursor cells can be identified using markers such as Nestin, GLAST, Sox2, Sox1, Musashi, and Pax6. Alternatively, cells that are positive for neural cell markers and proliferation markers (Ki67, pH3, MCM) can be identified as neural precursor cells.

[0026] In this specification, "brain tissue" refers to the anterior region of neural tissue in the anterior-posterior axis. The brain of a living organism develops from fetal brain tissue. Brain tissue is divided into the forebrain, midbrain, and hindbrain in the anterior-posterior axis.

[0027] As used herein, "forebrain tissue" refers to the anterior region of brain tissue in the anterior-posterior axis. Forebrain tissue includes telencephalon tissue, diencephalon tissue, cerebral tissue, diencephalon tissue, cerebral cortical tissue, hypothalamic tissue, thalamus, and the like, which are at advanced developmental stages. As used herein, "forebrain tissue" also includes tissues that develop from the above-mentioned tissues. Specifically, since retinal tissue develops from diencephalon tissue, retinal tissue is included in forebrain tissue. In some embodiments, forebrain tissue on a culture dish may form a neuroepithelial structure. In some embodiments, forebrain tissue on a culture dish may have a continuous epithelial structure and / or a rosette structure.

[0028] As used herein, "midbrain tissue" refers to a region of brain tissue that is posterior to the forebrain and anterior to the hindbrain in the anterior-posterior axis. Midbrain tissue includes the tegmentum, superior colliculus, inferior colliculus, substantia nigra, red nucleus, cerebral peduncle, etc. In some embodiments, midbrain tissue on a culture dish may form a neuroepithelial structure. In some embodiments, midbrain tissue on a culture dish may have a continuous epithelial structure and / or a rosette structure.

[0029] To identify which region of the brain tissue is involved, the expression of marker genes described in a publicly known publication (Shiraishi et al. Development 2017) can be examined.

[0030] Marker genes include, but are not limited to, Six3 (anterior forebrain), FoxG1 (telencephalon, the anterior part of the forebrain), Rx (part of the forebrain posterior to the telencephalon), Otx2 (part of the forebrain and midbrain), Otx2 positive and Rx negative (areas of the forebrain or midbrain, not retinal tissue), and Irx3 (midbrain or hindbrain).

[0031] In the present specification, the term "retinal tissue" refers to a tissue in which one or at least a plurality of types of cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, their precursor cells, or retinal progenitor cells, are arranged three-dimensionally in layers. The layer to which each cell belongs can be determined by a known method, for example, the presence or absence or the degree of expression of cell markers known to those skilled in the art (Chx10 (retinal progenitor cells or bipolar cells), L7 (bipolar cells), TuJ1 (retinal ganglion cells), Brn3 (retinal ganglion cells), Calretinin (amacrine cells), Calbindin (horizontal cells), Recoverin (photoreceptors), Rhodopsin (photoreceptors), RPE65 (retinal pigment epithelial cells), Mitf (retinal pigment epithelial cells), etc.).

[0032] As used herein, the term "retinal layer" refers to each layer constituting the retina, and specifically includes the retinal pigment epithelium layer and the neural retinal layer. The neural retinal layer includes the outer limiting membrane, photoreceptor layer (outer nuclear layer), outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.

[0033] As used herein, the term "retinal progenitor cells" refers to progenitor cells that can differentiate into any of the mature retinal cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells, etc. Markers of retinal progenitor cells include Rx (also called Rax), PAX6, and Chx10.

[0034] As used herein, the term "stem cell" refers to an undifferentiated cell having differentiation and proliferation capabilities (particularly self-renewal capabilities). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation capabilities. Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues. Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues or cells, but not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into a specific tissue or cell.

[0035] "Pluripotent stem cells" can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ 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 produced from germ cells (e.g., testes) are also included in pluripotent stem cells.

[0036] Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from a designated institution, 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 embryonic stem cells Crx::Venus strain (derived from KhES-1) are available from the National Institute of Physical and Chemical Research.

[0037] As used herein, the term "induced pluripotent stem cells" refers to cells in which pluripotency has been induced by reprogramming somatic cells using known methods.

[0038] In 2006, Yamanaka et al. established induced pluripotent stem cells in mouse cells (Cell, 2006, 126(4), pp.663-676). In 2007, induced pluripotent stem cells were also established in human fibroblast cells, and they have the same pluripotency and self-renewal ability as 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).

[0039] Specifically, induced pluripotent stem cells include cells in which pluripotency is induced by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., by expressing any combination of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. Preferred combinations of reprogramming factors 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).

[0040] In addition to producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp. 651-654).

[0041] It is also possible to obtain established induced pluripotent stem cells, and 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 established induced pluripotent stem cells, for example, Ff-I01 cells and Ff-I14 cells established at Kyoto University are available from Kyoto University.

[0042] In this specification, the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0043] In this specification, pluripotent stem cells refer to mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, more preferably human pluripotent stem cells, and even more preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0044] 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.

[0045] In the present specification, the nervous tissue is preferably derived from a human, and is preferably derived from a pluripotent stem cell. Specific examples of the nervous tissue derived from a pluripotent stem cell include nervous tissue derived from an embryonic stem cell or an induced pluripotent stem cell. In other words, the nervous tissue is preferably produced by using pluripotent stem cells, specifically embryonic stem cells or induced pluripotent stem cells, as a raw material, through a process of inducing differentiation.

[0046] In the present specification, the retinal tissue is preferably derived from pluripotent stem cells. Specific examples of retinal tissue derived from pluripotent stem cells include retinal tissue derived from embryonic stem cells or artificial pluripotent stem cells. In other words, the retinal tissue is preferably a retinal tissue produced by using pluripotent stem cells, specifically embryonic stem cells or artificial pluripotent stem cells, as a raw material and inducing differentiation.

[0047] Pluripotent stem cells can be used to produce neural tissues such as retinal tissues by methods well known to those skilled in the art, such as those described in WO2011 / 055855, WO2011 / 028524, WO2013 / 077425, WO2015 / 025967, WO2016 / 063985, and WO2016 / 063986. Examples of such a method include those described in non-patent literature: Nature 472, pp51-56 (2011), Proc Natl Acad Sci US A. 111 (23): 8518-8523 (2014), Nat Commun. 5: 4047 (2014), Nature Communications, 6, 6286 (2015), Stem Cells. (2017); doi: 10.1002 / stem.2586, etc.

[0048] Forebrain and midbrain tissues can be produced using pluripotent stem cells by methods well known to those skilled in the art, such as those described in Cell Stem Cell, 3, 519-32 (2008), WO2009 / 148170, and WO2016 / 063985.

[0049] In addition to the method of producing them from pluripotent stem cells, cells such as neural progenitor cells and neural retinal progenitor cells derived from the body can be separated and then reaggregated to produce multilayered neural tissue and retinal tissue.

[0050] The neural or retinal tissue produced by the above-described method forms cellular aggregates, and in one aspect, the aggregates exhibit characteristics such as (1) being round, (2) having a smooth surface, (3) not losing their shape, and (4) having a dense interior.

[0051] As used herein, the term "cell aggregate" refers to a mass formed by adhesion of multiple cells to each other, and is not particularly limited. Embryoid bodies, spheres, spheroids, and organoids are also included in cell aggregates. Cell aggregates include proliferating cells, non-proliferating cells (cells whose proliferation has stopped), or both.

[0052] In one embodiment, the "cell aggregate" comprises a neuroepithelial structure as described above. In one embodiment, the cell aggregate comprises one or more neuroepithelial structures.

[0053] In one embodiment, the retinal tissue produced by the above method has a structure in which one or at least multiple types of retinal cells are three-dimensionally arranged in layers (vesicular layered morphology).

[0054] Whether or not a retinal tissue has a vesicular lamellar morphology can be determined by a person skilled in the art by observing the retinal tissue under a bright-field microscope, specifically by confirming that the periphery of the retinal tissue aggregate is bright and that the retinal tissue has a vesicular or cup-shaped structure.

[0055] In the present specification, when the nervous tissue has a neuroepithelial structure, in one embodiment, the nervous tissue has a continuous epithelial structure. Here, the continuous epithelial structure refers to a state in which the epithelial tissue is continuous. The continuous epithelial tissue refers to a state in which, for example, 10 cells to 10,000,000 cells are lined up in the tangential direction of the epithelial tissue, preferably 30 cells to 10,000,000 cells, more preferably 100 cells to 10,000,000 cells are lined up in the tangential direction of the epithelial tissue.

[0056] For example, the continuous epithelial structure formed in the retinal tissue has an apical surface characteristic of epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to and continuous with at least the photoreceptor layer (outer nuclear layer) among the layers forming the neural retina. For example, in the case of a cell aggregate containing retinal tissue produced from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and 10 or more, preferably 30 or more, more preferably 100 or more, and even more preferably 400 or more photoreceptor cells or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface.

[0057] As used herein, the tangential direction to the epithelial tissue refers to the direction in which cells are arranged when each cell in the epithelial tissue is arranged in a fixed direction, and refers to the parallel or lateral direction to the epithelial tissue (or epithelial sheet).

[0058] In one embodiment, the retinal tissue produced by the above-mentioned method has a rosette-like structure. In this specification, the "rosette-like structure" in the retinal tissue refers to a structure in which cells are arranged radially or spirally so as to surround a lumen in the center. In the retinal tissue in which a rosette-like structure is formed, the apical surface and photoreceptor cells or photoreceptor precursor cells are present along the center (lumen), and the apical surface is divided into each rosette-like structure.

[0059] In this specification, the term "apical surface" refers to the surface (superficial surface) of epithelial tissue formed on the opposite side to the basement membrane side where the basal layer (basement membrane) of 50-100 nm produced by epithelial cells is rich in mucopolysaccharides (positive for PAS staining) and contains a lot of laminin and type IV collagen. In one aspect, in retinal tissue whose developmental stage has progressed to the extent that photoreceptor cells or photoreceptor precursor cells can be found, the term refers to the surface where the outer limiting membrane is formed and contacts the photoreceptor layer (external nuclear layer) where photoreceptors and photoreceptor precursor cells are present. In addition, such an apical surface can be identified by immunostaining 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).

[0060] Whether or not the nervous tissue has a continuous epithelial structure can be confirmed by the continuity of the apical surface of the nervous tissue (i.e., an uninterrupted morphology). The continuity of the apical surface can be determined, for example, by immunostaining for apical markers aPKC, E-cadherin, and N-cadherin, or by staining of cell nuclei (DAPI staining, PI staining, Hoechst staining, or staining of marker proteins localized in cell nuclei (Rx, Chx10, Ki67, Crx, etc.)).

[0061] In the case of retinal tissue, specifically, it can be determined by immunostaining apical surface markers (e.g., aPKC, E-cadherin, N-cadherin) and markers of photoreceptors or photoreceptor precursor cells located on the apical surface side (e.g., Crx or recoverin), and analyzing the positional relationship between the apical surface, the photoreceptor layer, and each retinal layer for the acquired images, etc. Retinal layers other than the apical surface and the photoreceptor layer (outer nuclear layer) can be identified by DAPI staining, PI staining, Hoechst staining, which stains cell nuclei, or immunostaining with marker proteins localized in cell nuclei (Rx, Chx10, Ki67, Crx, etc.).

[0062] Whether or not neural tissue has rosette-like structures can be determined by fixing the cell aggregates with 4% paraformaldehyde, preparing frozen sections, and observing the malformation of rosette-like structures (e.g., a disrupted apical surface or the infiltration of the apical surface into the cell aggregate) by immunostaining, typically performed with antibodies against the apical surface markers aPKC, E-cadherin, and N-cadherin, or DAPI, which specifically stains nuclei.

[0063] The nervous tissue produced by the above-mentioned method contains the above-mentioned nervous system cells, and the proportion of specific nervous system cells varies depending on the differentiation stage.

[0064] Some of the forebrain tissue produced by the above-mentioned method exists as aggregates with Otx2-positive lumens.

[0065] Some of the midbrain tissue prepared by the above-mentioned method exists as aggregates with Otx2-positive lumens.

[0066] The retinal tissue produced by the above-mentioned method contains Chx10-positive neural retinal progenitor cells, Crx-positive photoreceptor progenitor cells, etc. The ratio of these cells varies depending on the differentiation stage. In one embodiment, in the retinal tissue at a certain differentiation stage, Crx-positive photoreceptor progenitor cells are present in a layer on the surface (apical side) of the retinal tissue as a photoreceptor layer, and Chx10-positive neural retinal progenitor cells are present inside the layer as a progenitor cell layer (also called Neuroblastic layer).

[0067] The "preservation solution" used in the present specification for preserving or transporting neural tissue has a potassium ion concentration of more than 0 mM and less than 115 mM. The preservation solution is not particularly limited as long as it is an aqueous solution suitable for the survival of animal cells or animal tissues, but preferably contains a buffer substance.

[0068] The buffering action means the action of keeping the hydrogen ion concentration constant when a weak acid and its conjugate base or a weak base and its conjugate acid are mixed. In other words, the buffering action means the action of keeping the pH within a certain range even if external factors in the aqueous solution change. Examples of situations in which changes in external factors can occur include the addition of a small amount of an acidic or basic substance to an aqueous solution, dilution of a liquid, changes in the CO2 concentration in the atmosphere, production of metabolic products from cells or tissues, and changes in temperature.

[0069] The term "buffering substance" refers to a substance that maintains the pH within a certain range by interacting with other components in an aqueous solution, and is a substance that provides a buffering effect to the aqueous solution. That is, the preservation solution in this specification is an aqueous solution that has a buffering effect to keep the pH within the range of 6.0 to 8.6. The pH of the preservation solution is preferably within the range of 6.2 to 8.4, more preferably within the range of 6.7 to 7.9, more preferably within the range of 7.2 to 7.8, and even more preferably within the range of 7.2 to 7.4.

[0070] In this specification, the buffering substance contained in the preservative solution is not particularly limited as long as it can maintain the aqueous solution within the above-mentioned pH range, and may be a combination of a weak acid and its conjugate base (e.g., an alkali metal salt such as sodium or potassium, an alkaline earth metal salt such as calcium or magnesium, or an ammonium salt), or a combination of a weak base and its conjugate acid (e.g., an amine compound and a hydrochloride). One or more substances well known to those skilled in the art may be appropriately combined and used. For example, carbonate buffer (sodium bicarbonate, sodium carbonate), phosphate buffer (phosphoric acid, potassium phosphate, sodium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, trisodium phosphate, tripotassium phosphate), Good's buffer (HEPES, MES, PIPES, etc.), Tris, citrate buffer (trisodium citrate), acetate buffer (sodium acetate, potassium acetate), borate buffer (sodium borate, sodium tetraborate), tartaric acid (sodium tartrate), amino acid buffer (histidine, taurine, aspartic acid), etc. may be used, but are not limited thereto. In one embodiment, the preservation solution may contain a combination of one or more buffering substances appropriately selected from a plurality of buffering substances. Preferable examples of the buffering substances include sodium bicarbonate, phosphate buffer (sodium dihydrogen phosphate, disodium hydrogen phosphate), HEPES, etc. In one embodiment, the buffering substances contained in the preservation solution may include, in addition to sodium bicarbonate, one or more buffering substances appropriately selected from other buffering substances.

[0071] In this specification, an aqueous solution refers to a solution that has water (H2O) as the basic solvent, and may contain a small amount of water-soluble liquid that does not affect cell viability.

[0072] The preservation solution used herein for preserving neural tissue may contain an inorganic salt in addition to a buffering substance.

[0073] Inorganic salts contained in the preservation solution include inorganic salts that are known as components contained in mammalian living bodies or components useful for cell survival, specifically, metal salts such as nitric acid, sulfuric acid, and hydrochloric acid. In this specification, inorganic salts that provide a buffering effect to an aqueous solution are treated as buffering substances. The metal salts are not particularly limited as long as they form stable inorganic salts and are useful for cell survival, and include alkali metal (sodium, potassium) salts, alkaline earth metal (calcium, magnesium) salts, copper, zinc, iron, and other salts. Specific examples include potassium salts, sodium salts, calcium salts, magnesium salts, and iron salts, and specific examples include potassium chloride, sodium chloride, calcium chloride, iron nitrate, and magnesium chloride.

[0074] The preservation solution in this specification may contain a potassium salt as a buffer substance or other inorganic salt, and the potassium ion concentration contained in the preservation solution is not particularly limited as long as it is more than 0 mM and lower than 115 mM. It is preferably a concentration of 60 mM, more preferably 30 mM, and even more preferably lower than 10 mM. The potassium ion concentration is preferably 0 mM to 60 mM, more preferably 0 mM to 30 mM, even more preferably 0.01 mM to 20 mM, even more preferably 0.1 mM to 10 mM, and even more preferably 1 mM to 8 mM.

[0075] In addition, in this specification, the ratio of the potassium ion concentration to the sodium ion concentration contained in the preservation solution is preferably lower than 0.8 times, more preferably 0.5 times, more preferably 0.4 times, 0.3 times, 0.2 times, 0.1 times, and even more preferably 0.05 times.

[0076] In this specification, the "potassium ion concentration" refers to the concentration of the inorganic ions when all salts present in the preservation solution are dissociated into ions, and is a value that does not depend on whether potassium salts are dissociated in the preservation solution. For example, when potassium phosphate (K3PO4) and potassium chloride (KCl) are both contained in the preservation solution, the potassium ions (K+) and phosphate ions (PO4 3-) are completely dissociated, and furthermore, potassium ions (K+) and chloride ions (Cl-) are also completely dissociated. The combined potassium ion concentration from both salts is defined as the potassium ion concentration contained in this preservative solution.

[0077] In this specification, osmotic pressure can be mentioned as one of the important characteristics that the preservation solution should satisfy. The concentration of the buffering substance and other inorganic salts can be mentioned as one of the factors that affect the osmotic pressure of the preservation solution. The osmotic pressure of the preservation solution is preferably adjusted to such an extent that the nerve tissue to be preserved does not expand or contract during the preservation period. In one aspect, the preservation solution is an aqueous solution having an osmotic pressure such that the nerve tissue to be preserved does not expand or contract. The preservation solution is preferably an aqueous solution having a buffering effect that is prepared to maintain a pH within the range of 6.0 to 8.6, and having an osmotic pressure such that the nerve tissue to be preserved does not expand or contract.

[0078] The osmotic pressure at which the nerve tissue does not expand or shrink during storage is specifically 200 to 500 mOsm / kg, for example 200 to 400 mOsm / kg, or 250 to 360 mOsm / kg. The osmotic pressure of the preservation solution is determined by the concentration of all substances, including ions, contained in the preservation solution. The osmotic pressure of the preservation solution can be appropriately adjusted by changing the content of ions, etc., contained in the preservation solution, or an osmotic pressure regulator (e.g., hydroxyethyl starch, etc.) may be added. The osmotic pressure can be measured, for example, using a commercially available osmotic pressure measuring device.

[0079] In the present specification, the preservation solution may contain, in addition to the above-mentioned buffering substances and other inorganic salts, one or more additives appropriately selected from nutrients such as amino acids, vitamins, sugars, nucleic acid bases, pyruvates, etc., antibiotics (e.g., penicillin, streptomycin, gentamicin, amphotericin), steroids such as cholesterol, 2-mercaptoethanol, serum, serum substitutes, etc., within the scope in which cells contained in the nerve tissue are viable and the nerve tissue can be preserved, but the additives are not limited to these. Specifically, the components contained in the culture medium listed in Tables 1 to 9 of the present specification can be exemplified.

[0080] As the preservation solution, commercially available preservation solutions, culture media, etc. for cells or biological tissues that satisfy the above conditions can be appropriately used. The preservation solution in this specification is preferable if it has a high ability to maintain the morphology and properties of neural tissue without changing them, and in that sense, it is preferable if it has a low ability to proliferate or differentiate cells. Whether or not cells proliferate in the preservation solution can be confirmed by methods well known to those skilled in the art, such as immunostaining of Ki67, a cell proliferation marker, or the incorporation rate of nucleic acid analogs (tritium-labeled products, BrdU, CldU, IdU, EdU, etc.).

[0081] In one embodiment, the preservation solution may contain glycosaminoglycans. Examples of glycosaminoglycans include linear polysaccharides having a basic skeleton containing amino sugar and uronic acid. The glycosaminoglycans used in the preservation solution in this specification are not particularly limited as long as they are soluble in water and do not adversely affect the survival of nerve tissue. In this specification, the glycosaminoglycans are preferably chondroitin sulfate or hyaluronic acid. One of the advantages of containing glycosaminoglycans is that the viscosity of the preservation solution is added, thereby reducing vibration caused by transportation.

[0082] Here, the concentration of glycosaminoglycans in the preservation solution is, for example, 10% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, or 2.5% (w / v) or less in terms of the concentration of total glycosaminoglycans. The concentration of total glycosaminoglycans is, for example, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.5% (w / v) or more, or 0.7% (w / v) or more. The concentration of total glycosaminoglycans can be, for example, 0.1% (w / v) or more and 10% (w / v) or less, 0.2% (w / v) or more and 4% (w / v) or less, 0.5% (w / v) or more and 3% (w / v) or less, and 0.7% (w / v) or more and 2.5% (w / v) or less.

[0083] When chondroitin sulfate is contained in the preservation solution as the glycosaminoglycan, the concentration of chondroitin sulfate, expressed as sodium chondroitin sulfate, is, for example, 10% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, or 2.5% (w / v) or less. The concentration of chondroitin sulfate, expressed as sodium chondroitin sulfate, is, for example, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.4% (w / v) or more, or 0.5% (w / v) or more. Furthermore, the concentration of chondroitin sulfate, in terms of sodium chondroitin sulfate ester, can be, for example, 0.1% (w / v) to 10% (w / v), 0.2% (w / v) to 4% (w / v), 0.4% (w / v) to 3% (w / v), and 0.5% (w / v) to 2.5% (w / v).

[0084] Although one type of glycosaminoglycan can be used alone, multiple types of glycosaminoglycans can also be used in combination. For example, chondroitin sulfate and hyaluronic acid can be used in a weight ratio of 1:1 to 3:1 or 2:1 to 3:1.

[0085] In one embodiment, the preservation solution may have low activity in promoting cell metabolism. The metabolic state of the cell can be evaluated by measuring the amount of metabolic pathway substances (e.g., starting materials, intermediates, end metabolites, etc.). The amount of metabolic pathway substances can be measured by analytical instruments such as liquid chromatography, gas chromatography, gel filtration, NMR, mass spectrometry, UV spectrometer, IR spectrometer, etc., or a combination of such analytical instruments.

[0086] In one embodiment, the preservation solution may contain serum and / or a serum substitute, but the concentration may be low. When the concentration of serum and / or a serum substitute contained in the preservation solution is low, the effect of reducing cell proliferation and / or cell metabolism can be expected.

[0087] As the preservation solution, a medium used for the maintenance culture of cells or tissues can also be used. A medium for cell or tissue culture, preferably an aqueous solution commercially available as a medium for the culture of animal cells or animal tissues, can be used as the preservation solution of the present invention as long as cell proliferation or differentiation that would substantially change the morphology of neural tissue does not occur at the preservation temperature of the present invention. Examples of the medium include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Neurobasal medium medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof, which can be used for the culture of animal cells. Preferably, DMEM (Gibco), DMEM / F-12, Neurobasal medium (Thermo Fisher), or a mixture thereof can be used. For the maintenance culture of cells or tissues, serum, serum substitutes, antibiotics, etc. may be added as appropriate. On the other hand, since serum, serum substitutes and / or antibiotics are not essential components of a preservation solution, when used as a preservation solution in the present invention, it is preferable not to add these substances, or to add them at a lower concentration than that for normal cell culture, from the viewpoint of reducing the number of quality control items in the preservation solution. In one embodiment, D-PBS or the like containing a buffer substance and inorganic salts but not containing amino acids, vitamins, or sugars may be used as the preservation solution.

[0088] In one embodiment, the preservation solution preferably contains a buffering agent and an inorganic salt, and may contain amino acids and / or vitamins. More preferably, the preservation solution includes an aqueous solution containing a buffering agent, amino acids, and vitamins. Specifically, the preservation solution includes HBSS (available from Thermo Fisher), DMEM (available from Thermo Fisher), DMEM-no-glucose (available from Thermo Fisher), Neurobasal medium (available from Thermo Fisher), a corneal preservation solution having the composition shown in Table 1 below (see U.S. Patents 5,104,787; 5,407,669), or Optisol (available from Bausch and Lomb), and more preferably, Optisol. [Table 1]

[0089] In one aspect, the present invention provides a transplantation composition comprising a preservation solution and human neural tissue, the transplantation composition being kept at 12°C to 22°C, preferably 15°C to 20°C, i.e., a transplantation composition whose temperature is adjusted to 12°C to 22°C or 15°C to 20°C.

[0090] Here, the preservation solution may be an aqueous solution having a potassium ion concentration of more than 0 mM and less than 115 mM, and having a pH adjusted to a range of 6.0 to 8.6 by a buffering substance. The aqueous solution may further contain amino acids and vitamins. The aqueous solution may be an aqueous solution for maintaining or culturing cells or tissues, having a potassium ion concentration of more than 0 mM and less than 115 mM, and having a pH adjusted to a range of 6.0 to 8.6 by a buffering substance. Specific examples of the aqueous solution include Optisol, HBSS, DMEM, DMEM-no-glucose, Neurobasal medium, and a corneal preservation solution having the composition shown in Table 1.

[0091] The aqueous solution preferably further contains glycosaminoglycans, specifically chondroitin sulfate and / or hyaluronic acid, the concentrations of which are as described above.

[0092] Human neural tissue includes human neural tissue derived from pluripotent stem cells, for example, human neural tissue that is a cell aggregate having a neuroepithelial structure.

[0093] Specific examples of the cell aggregate include retinal tissue having a continuous epithelial structure present on the surface of the cell aggregate, and forebrain / midbrain tissue having a continuous epithelial structure and / or rosette structure present on the surface or inside of the cell aggregate. Examples of the retinal tissue include the above-mentioned retinal tissue, for example, neural retinal tissue having a continuous epithelial structure containing CRX and / or N-cadherin positive cells. Examples of the forebrain / midbrain tissue include forebrain / midbrain tissue having a continuous epithelial structure containing Otx2 positive cells and / or rosette (lumen) structure.

[0094] The composition of the preservation solution used in the preservation method or transportation method of the present invention may be changed during the preservation period to the extent that preservation of the nerve tissue is possible. Also, the preservation solution may be replaced with a preservation solution of the same composition during the preservation period. Examples of replacement include, but are not limited to, half replacement, 80% replacement, and full replacement.

[0095] The container used in the preservation method or transportation method of the present invention is not particularly limited, and can be appropriately determined by a person skilled in the art. Examples of such containers include flasks, tissue culture flasks, culture dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, tubes, screw-cap tubes, vials, trays, and culture bags. These containers are preferably non-adhesive to cells in order to enable floating preservation of neural tissue. As the non-adhesive container, a container whose inner surface has not been artificially treated (for example, coating with extracellular matrices such as basement membrane preparations, laminin, entactin, collagen, and gelatin, or with polymers such as polylysine and polyornithine, or surface treatment such as positive charge treatment) for the purpose of improving adhesion to cells can be used. As the non-cell-adhesive container, a container whose surface has been artificially treated (for example, superhydrophilic treatment with MPC polymer, low protein adsorption treatment, etc.) for the purpose of reducing adhesiveness to cells can be used. Materials for the container include, but are not limited to, polystyrene, polypropylene, etc. The bottom of the container may be flat or may have an uneven surface.

[0096] In one embodiment, the container is preferably sterile. A method for sterilizing the container to make it sterile can be a method known to those skilled in the art. Alternatively, a sterile container may be purchased and used.

[0097] In one embodiment, the container may be a cell storage tube, for example, a cryotube, a conical tube, a centrifuge tube, a plastic tube, etc., but is not limited thereto.

[0098] In the preservation method or transportation method of the present invention, the nervous tissue is preserved at a storage temperature of 8° C. or higher or 12° C. or higher and 30° C. or lower or 22° C. or lower. The storage temperature is 8° C. to 30° C., preferably 12° C. to 22° C., and more preferably about 17° C. (15° C. to 19° C. or 16° C. to 18° C.). For the purpose of controlling the storage temperature, a temperature maintenance device described below may be used.

[0099] In one embodiment, the storage temperature may be the temperature of the storage solution actually measured with a thermometer or the like, the temperature inside a temperature maintaining device actually measured, or the temperature set in the temperature maintaining device. When the temperature is actually measured, for example, a thermometer can be used.

[0100] The temperature maintaining device is not particularly limited as long as it has a function of maintaining the temperature of the sealed gas phase or liquid phase within the above range. Specifically, a CO2 incubator for cell culture, a refrigerator, a centrifuge, a thermostatic water bath, a thermostatic bath, a block incubator, a heat retaining gel, etc. can be used. Those skilled in the art can appropriately set other settings (oxygen concentration, carbon dioxide concentration, etc.) depending on the tissue to be preserved, etc.

[0101] Nervous tissue may be transported between facilities (e.g., from a nervous tissue manufacturing facility to a transplantation facility), and it is preferable that the tissue is appropriately preserved during the transport period, and the preservation method of the present specification can be applied. The transport method is not particularly limited, and may be any method such as a vehicle, ship, or aircraft. Therefore, it is preferable that the transport means is equipped with an appropriate temperature maintenance device, or that the temperature maintenance device can be included in the transport. In addition, it is preferable that the temperature maintenance device has a structure in which vibrations caused by transport are not transmitted to the internal preservation solution. Therefore, a method of transporting nervous tissue in a preservation solution having a potassium ion concentration of more than 0 mM and less than 115 mM at a preservation temperature of 8°C to 30°C, preferably 12°C to 22°C, and more preferably about 17°C (15°C to 19°C or 16°C to 18°C) is also within the scope of the present invention.

[0102] As used herein, preservation of neural tissue is initiated by placing the neural tissue in a temperature-maintaining device set at a temperature within the ranges described above and is completed by removing the neural tissue from the temperature-maintaining device.

[0103] By using the preservation solution of the present invention, the nerve tissue can be preserved for a period not exceeding 14 days, not exceeding 5 days, or not exceeding 3 days. The period is usually 2 hours or more because of the need for quality control or transportation before transplantation. As used herein, "a period not exceeding X days" refers to the period from the day the preservation started to the day X, which is designated as day 0. In one embodiment, the nerve tissue may be preserved by the method for a period not exceeding, for example, one month, although the quality of the nerve tissue may be deteriorated.

[0104] In the preservation method or transportation method of the present invention, preservation of neural tissue is distinguished from the culture step. In the case of a culture step (e.g., maintenance culture or differentiation-inducing culture) of neural tissue, cell metabolism, proliferation, fate determination, differentiation, maturation, migration, etc. may occur intermittently. In the above culture step (e.g., maintenance culture or differentiation-inducing culture), the neural cells are cultured under conditions suitable for survival and proliferation or differentiation induction. Such conditions include medium composition (including nutrients such as sugars, amino acids, vitamins, etc.), temperature (usually 36 to 37°C), humidity (usually 95%), carbon dioxide concentration (usually 5 to 10%), oxygen concentration (usually 2 to 60%), etc.

[0105] On the other hand, the storage temperature in the present invention is 30° C. or lower, which is different from the optimal temperature in the culture process (e.g., maintenance culture or differentiation-inducing culture of neural tissue). During storage in the present invention, it is not essential to maintain or adjust the nutritional components, humidity, and carbon dioxide concentration in the medium.

[0106] In the present specification, the preservation of neural tissue preferably does not substantially involve cell proliferation and differentiation, and the state of the neural tissue before and after preservation is substantially equivalent. For example, when the neural tissue is used for transplantation, the produced neural tissue may be subjected to a quality test, which may take several hours to several days depending on the content of the quality test. The equivalent of the neural tissue before and after preservation can be demonstrated by performing a quality test that can be set by a person skilled in the art. In one embodiment, the equivalent of the neural tissue before and after preservation can be demonstrated by comparing the effect when transplanted into an animal model.

[0107] In addition, when neural tissue is produced at a manufacturing facility such as a CPC cell culture center, it needs to be transported to the medical institution of the patient (recipient) requiring the transplant. However, in remote locations, transportation may take several hours to several days depending on the distance.

[0108] Therefore, it is important that the condition of the nervous tissue remains substantially the same during the period required for the quality inspection or transportation.

[0109] In one aspect, "preservation" of neural tissue is distinguished from "culture" accompanied by cell proliferation. During maintenance culture or differentiation induction, cell proliferation or differentiation is intermittently performed, and conditions such as medium composition (including nutritional components such as sugars, amino acids, vitamins, etc.) and external environment such as temperature (usually 37°C), humidity (usually 95%), and carbon dioxide concentration (usually 5%) suitable for survival and proliferation or differentiation induction of neural cells are required. In this specification, "preservation" means that the properties of the cell aggregates are maintained without change without substantial cell proliferation and differentiation, i.e., substantial equivalence is maintained.

[0110] In one aspect, "preservation" of neural tissue is distinguished from "culture" which involves morphological changes in tissue. When "culture" tissue, morphological changes in tissue (e.g., elongation, invagination, inversion) may occur, and conditions such as medium composition (including nutrients such as sugars, amino acids, vitamins, etc.) and external environment such as temperature (usually 37°C), humidity (usually 95%), and carbon dioxide concentration (usually 5%) suitable for "culture" of tissue are required.

[0111] The storage temperature in the storage method of the present invention is 30° C. or lower, which is different from the optimal temperature for cell proliferation or differentiation.

[0112] In one embodiment, it is preferable that the viability of nervous cells contained in the nervous tissue is equivalent before and after preservation by the method of the present specification. Here, equivalent viability means that the ratio of the viability of cells after preservation to the viability of cells before contact with the preservation solution of the present invention does not change substantially, specifically, it is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

[0113] Here, for example, if the viability of nervous system cells contained in nervous tissue used for transplantation is 95% or more before contact with the preservation solution of the present invention, the viability of nervous system cells in the tissue preserved by the method of the present invention, i.e., after storage in the preservation solution for several hours to 14 days, will be maintained at 80% or more, 90% or more, or 95% or more, depending on the storage period and storage conditions.

[0114] In one embodiment, by preserving a cell aggregate containing a retinal tissue by the method of the present invention, the number of CRX-positive cells in the cell aggregate is equivalent before and after preservation. Here, the equivalent number of CRX-positive cells means that the ratio of the number of CRX-positive cells after preservation in the preservation solution of the present invention for several hours to 14 days to the number of CRX-positive cells before contact with the preservation solution is maintained at 70% or more, 80% or more, or 95% or more, depending on the preservation period and conditions.

[0115] In one embodiment, by preserving cell aggregates containing neural tissues using the method of the present invention, the number of aggregates having Otx2-positive rosette structures (lumens) is equivalent before and after preservation. Here, the equivalent number of aggregates having Otx2-positive rosette structures means that the ratio of the number of aggregates having Otx2-positive lumens after preservation in the preservation solution of the present invention for several hours to 14 days to the number of aggregates having Otx2-positive rosette structures before contact with the preservation solution is maintained at 50% or more, 80% or more, or 90% or more, depending on the preservation period and conditions.

[0116] In one embodiment, by preserving by the method of the present invention, it is possible to maintain a layer structure or a three-dimensional cell arrangement specific to a substantially equivalent aggregate of neural tissue before and after preservation. For example, in the case of retinal tissue including a neural retinal layer, specific examples include a continuous epithelial structure, a vesicular layered morphology, and a cell arrangement in which Crx-positive cells are present in a layer on the surface and Chx10-positive cells are present in a layer on the inside. "Maintaining a substantially equivalent layer structure or a three-dimensional cell arrangement" means that the ratio of the number of aggregates having a layer structure or a three-dimensional cell arrangement exhibiting a specific property after preservation in the preservation solution of the present invention for several hours to 14 days to the number of aggregates having the layer structure or the three-dimensional cell arrangement before contacting the preservation solution is 80% or more, preferably 90% or more, more preferably 95% or more. In another aspect, a layered structure or three-dimensional cell arrangement exhibiting a specific property being equivalent means that the proportion of the area exhibiting the layered structure or three-dimensional cell arrangement contained within the outer periphery of the aggregate is maintained at 70% or more, 80% or more, or 95% or more, depending on the storage period and storage conditions, compared to before storage and before contact according to the present invention.

[0117] The neural tissue preserved by this preservation method can be immediately used for the intended purpose, specifically, for quality testing and transplantation into a patient (recipient). After the preservation period, the neural tissue may be subjected to recovery culture in a medium for a short period (e.g., 3 hours to 7 days) or a long period (e.g., 8 days to several months). The recovery culture may be performed by a method well known to those skilled in the art, for example, in a CO2 incubator set at 37°C. In one embodiment, the recovery culture may be performed under exactly the same conditions as normal differentiation culture or maturation culture.

[0118] Neural tissues preserved by the preservation or transportation method of the present invention, specifically, forebrain tissue, midbrain tissue, retinal tissue, etc., can be used, for example, for transplantation into a living body (recipient).

[0119] In one embodiment, the preserved neural tissue, specifically, forebrain tissue, midbrain tissue, retina tissue, etc., may be directly transplanted after being removed from the preservation solution when transplanted into a living body. In one embodiment, the preserved neural tissue may be washed with a preservative, stabilizer, reducing agent, isotonicity agent, etc., which are commonly used in pharmaceuticals containing tissues or cells, and may be transplanted using these as a carrier.

[0120] The nervous tissue (e.g., retinal tissue, forebrain tissue, midbrain tissue) preserved by the preservation or transportation method of the present invention is useful for transplantation therapy of diseases caused by damage to the nervous tissue. Thus, the present invention provides a therapeutic agent for diseases caused by damage to the nervous tissue, comprising the nervous tissue preserved by the preservation method of the present invention. The nervous tissue preserved by the preservation method of the present invention can be used as a therapeutic agent for diseases caused by damage to the nervous tissue, or to replenish the corresponding damaged site in a damaged state of the nervous tissue. By transplanting the nervous tissue preserved by the preservation method of the present invention into a patient who is in need of transplantation and has a disease caused by damage to the nervous tissue or a damaged state of the nervous tissue, and replenishing the damaged or damaged nervous tissue itself, the disease caused by damage to the nervous tissue or the damaged state of the nervous tissue can be treated.For example, diseases based on nervous tissue damage include, for example, neurodegenerative diseases (e.g., cerebral ischemic disorder, cerebral infarction, Parkinson's disease, spinal cord injury, cerebrovascular disorder, brain / spinal cord traumatic disorder (e.g., cerebral infarction, head trauma / cerebral contusion (TBI), spinal cord injury multiple system atrophy), typical neurodegenerative diseases (amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease). , multiple system atrophy (MSA), spinocerebellar degeneration (SCD)), demyelinating diseases, neuromuscular diseases (multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), inflammatory diffuse sclerosis (Schilder's disease), subacute sclerotic panencephalitis, progressive multifocal leukoencephalopathy, hypoxic encephalopathy, central pontine myelinopathy, Binswanger's disease, Guillain-Barré syndrome, Fisher's syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, syringomyelia, spinocerebellar degeneration, nigrostriatal degeneration (SND), olivopontocerebellar atrophy (OPCA), Shy ·Drager syndrome (Shy-Drager syndrome), eye diseases (macular degeneration, age-related macular degeneration, retinitis pigmentosa, cataracts, glaucoma, corneal diseases, retinopathy), intractable epilepsy, progressive supranuclear palsy, syringomyelia, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), primary lateral sclerosis (PLS), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Huntington's disease (HD), acanthocytic chorea, syringomyelia, frontotemporal lobar degeneration, Charcot-Marie-Tooth Examples of diseases caused by damage to cerebral tissue or cerebral-related cells include neurodegenerative diseases (e.g., cerebral ischemic disorder, cerebral infarction, motor neuron disease, ALS, Alzheimer's disease, polyglutamine disease, corticobasal degeneration). Examples of diseases caused by damage to retinal tissue or retinal-related cells include retinal degeneration, retinitis pigmentosa, age-related macular degeneration, organic mercury poisoning, chloroquine retinopathy, glaucoma, diabetic retinopathy, neonatal retinopathy, etc.Further, conditions of nerve tissue damage include patients after nerve tissue removal, patients after radiation exposure to intraneural tumors, and trauma. EXAMPLES

[0121] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way. Example 1: Preservation of cell aggregates containing retinal tissues prepared from human iPS cells under various preservation conditions (screening experiment of preservation temperature and preservation solution)

[0122] Human iPS cells (1231A3 line, obtained from Kyoto University) were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (manufactured by Nippi Co., Ltd.) was used as the feeder-free scaffold.

[0123] As a specific maintenance culture procedure, first, subconfluent human iPS cells (1231A3 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded onto a plastic culture dish coated with Laminin511-E8 and cultured feeder-free in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 μM). The plastic culture dish used was a 6-well plate (Iwaki, for cell culture, culture area 9.4 cm). 2 ), the number of human iPS cells dispersed into the single cells was 1.0 × 10 4 One day after seeding, the medium was replaced with StemFit medium not containing Y27632. Thereafter, the medium was replaced with StemFit medium not containing Y27632 once every 1 to 2 days. Six days after seeding, the cells were cultured until they became subconfluent (about 60% of the culture area was covered with cells).

[0124] For differentiation induction, human iPS cells (1231A3 strain) were cultured in a feeder-free manner using StemFit medium until 1 day before subconfluency (approximately 50% of the culture area was covered with cells). The human iPS cells 1 day before subconfluency were cultured in a feeder-free manner for 1 day in the presence of SB431542 (5 μM) and SAG (300 nM) (Preconditioning treatment).

[0125] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) and further dispersed into single cells by pipetting. The dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) at 1.2 × 10 cells per well. 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× chemically defined lipid concentrate. At the start of suspension culture (day 0 after the start of suspension culture), IWR-1e (final concentration 3 μM), Y27632 (final concentration 20 μM), and SAG (final concentration 30 nM) were added to the serum-free medium. On the third day after the initiation of suspension culture, 50 μl of the above-mentioned serum-free medium was added to the medium containing IWR-1e and human recombinant BMP4 (manufactured by R&D) but not containing Y27632 or SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) and the final concentration of IWR-1e was 3 μM.

[0126] Three days later (six days after the start of suspension culture), half of the medium was replaced with the above serum-free medium containing IWR-1e but not containing Y27632 or human recombinant BMP4. Four days later (ten days after the start of suspension culture), 80% medium replacement was performed twice using the above serum-free medium containing IWR-1e, Y27632, and human recombinant BMP4 so that the concentration of exogenous IWR-1e was 3% or less compared to before the medium replacement. Thereafter, half of the medium was replaced with the above serum-free medium containing IWR-1e, Y27632, and human recombinant BMP4 once every 2 to 4 days. For the half-medium replacement, half of the medium in the culture vessel, i.e., 75 μl, was discarded, and 75 μl of the above serum-free medium was added, making the total medium volume 150 μl.

[0127] The cell aggregates thus obtained, 17 days after the start of suspension culture, were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM), a substance that acts on the Wnt signaling pathway, and SU5402 (5 μM), an FGF signaling pathway inhibitor, for 3 days, i.e., until the 20th day after the start of suspension culture.

[0128] Furthermore, a medium (hereinafter referred to as "Retina medium") was prepared by adding 10% fetal bovine serum, 1% N2 supplement, 0.5 μM retinoic acid, and 100 μM taurine to DMEM / F12 medium as a serum medium free of Wnt signaling pathway active substances and FGF signaling pathway inhibitors, and the cell aggregates on the 20th day after the start of the suspension culture were suspension cultured in the Retina medium for 65 days, i.e., up to the 85th day after the start of the suspension culture. From the 20th day to the 85th day after the start of the suspension culture, about half of the medium was replaced with Retina medium once every 2 to 4 days.

[0129] When bright-field images (phase contrast images) of the cell aggregates obtained in this manner on the 85th day after the start of suspension culture were observed using an inverted microscope (Nikon, ECLIPSE Ti), it was confirmed that the cell aggregates contained three-dimensional neural tissue, some of which contained continuous epithelial structures.

[0130] The following three types of preservation solutions were examined (see Tables 2 to 4). Retina medium: DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, 0.5 μM retinoic acid, and 100 μM taurine. Hank's balanced salt solution (Gibco, hereafter referred to as "HBSS") Optisol-GS (registered trademark) (manufactured by Bausch and Lomb, hereinafter referred to as "Optisol" or "Optisol")

[0131] [Table 2]

[0132] [Table 3]

[0133] [Table 4]

[0134] The following three storage temperatures were considered: 37℃ (incubator, 5% CO2, Panasonic) 17℃ (incubator, 5% CO2, Astec) 4℃ (refrigerator, sealed, Panasonic)

[0135] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0136] Specifically, the following storage conditions A to I were examined. Storage condition A (normal culture conditions). Storage solution is Retina medium, storage temperature is 37℃, storage container is low-attachment plate (5% CO2) Storage condition B: Storage solution is Retina medium, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage condition C: Storage solution is Retina medium, storage temperature is 4℃, storage container is low-adhesion plate (sealed) Storage condition D: Storage solution is HBSS, storage temperature is 37℃, storage container is low-adhesion plate (5% CO2) Storage conditions E. Storage solution is HBSS, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage conditions F. Storage solution is HBSS, storage temperature is 4℃, storage container is low-adhesion plate (sealed) Storage conditions G. Storage solution is Optisol, storage temperature is 37℃, storage container is low adhesion plate (5% CO2) Storage conditions H. Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2) Storage conditions I. Storage solution is Optisol, storage temperature is 4℃, storage container is low adhesion plate (sealed)

[0137] The cell aggregates on day 85 after the start of suspension culture were stored for 48 hours under the above storage conditions A to I. Immediately after storage (day 87 after the start of suspension culture), bright field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon, ECLIPSE Ti). As a result, the morphology of three-dimensional tissue was maintained in most cell aggregates immediately after storage under storage conditions A, B, E, F, G, and H. Under storage conditions D and I, the morphology of three-dimensional tissue was maintained in some cell aggregates, but many aggregates lost their morphology. On the other hand, under storage condition C, the morphology of three-dimensional tissue was significantly destroyed in almost all cell aggregates.

[0138] Next, recovery culture was performed to verify whether the cells contained in the preserved cell aggregates could be cultured normally and whether any abnormalities such as cell death occurred in differentiated cells. Specifically, the cell aggregates preserved for 48 hours under storage conditions A to I were subjected to suspension culture in Retina medium using low-adhesion plates in an incubator at 37°C and 5% CO2 (recovery culture).

[0139] The resulting cell aggregates after recovery culture were observed as bright-field images (phase contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 1).

[0140] As a result, in the cell aggregates after recovery culture under storage conditions A, B, E, F, G, and H, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates. Under storage conditions D and I, although there were some cell aggregates in which neural tissue with a continuous epithelial structure with a layered structure was observed, the morphology of the cell aggregate (three-dimensional retina) was distorted in approximately 70% of the cell aggregates, as shown in Figure 1. On average, the morphologies shown in D and I in Figure 1 were observed. On the other hand, under storage condition C, as shown in C in Figure 1, there was a tendency for the morphology of the three-dimensional tissue to be significantly distorted in almost all cell aggregates.

[0141] The resulting cell aggregates after recovery culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a retinal tissue marker, and Crx (anti-Crx antibody, Takara, rabbit), a photoreceptor precursor cell marker. Cell nuclei were stained with DAPI. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (Figures 2 and 3).

[0142] We then morphologically analyzed the immunostained images to determine whether neural retinal tissue was present, characterized by a layer of Crx-positive photoreceptor precursor cells on the surface and a layer of Chx10-positive neural retinal precursor cells on the inside. We further examined whether the neural retinal tissue formed a continuous epithelial structure that continued tangentially for 100 μm or more around the periphery of the aggregate (hereafter referred to as the "continuous epithelial structure of the neural retinal tissue").

[0143] As a result, it was found that the continuous epithelial structure of neural retinal tissue was present in most cell aggregates after recovery culture under storage conditions A, B, E, F, G, and H (Figures 2 and 3). Although there were some cell aggregates in which the continuous epithelial structure of neural retinal tissue was observed under storage conditions D and I, the three-dimensional retinal morphology was disrupted in about 70% of the cell aggregates, as shown in the figures. On the other hand, under storage condition C, the continuous epithelial structure of neural retinal tissue was hardly detected in most cell aggregates.

[0144] These results show that, compared to storage condition A, which is the same as the normal culture condition, cell aggregates can be preserved for 48 hours while maintaining the continuous epithelial structure of the neural retinal tissue under storage conditions B, E, F, G, and H. It was also found that the continuous epithelial structure of the neural retinal tissue can be partially maintained under storage conditions D and I.

[0145] It was also found that under storage conditions B, E, and H, i.e., a storage temperature of 17°C, the protein could be stably stored in various storage solutions.

[0146] Example 2: Preservation of cell aggregates containing retinal tissue prepared from human ES cells under various preservation conditions (screening experiment of preservation temperature and preservation solution) Crx::Venus knock-in human ES cells (derived from KhES-1; Nakano, T. et al. Cell Stem Cell 2012,10(6),771-785; obtained from Kyoto University, established at RIKEN CDB and used) were cultured under feeder-free conditions in StemFit medium until 1 day before subconfluency, as described in Example 1. The human ES cells 1 day before subconfluency were cultured feeder-free for 1 day in the presence of SB431542 (5 μM) and SAG (300 nM) (Precondition treatment).

[0147] The preconditioned human ES cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution, and then dispersed into single cells by pipetting. The dispersed human iPS cells were then cultured in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) at 1.2 × 10 cells per well. 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2 (day 0 after the start of suspension culture). The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× chemically defined lipid concentrate.

[0148] At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (final concentration 20 μM) and SAG (30 nM) were added to the serum-free medium. On the third day after the start of suspension culture, 50 μL of a medium containing human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Co.) but not Y27632 or SAG was added so that the final concentration of human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0149] On the sixth day after the start of suspension culture, 60 μL of the medium was removed and 90 μL of new medium was added. On the ninth, twelfth, and fifteenth days after the start of suspension culture, 85 μL of the medium was removed and 90 μL of new medium was added, and the cells were cultured until the 18th day after the start of suspension culture.

[0150] The aggregates on the 18th day after the start of suspension culture were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM) and SU5402 (5 μM) for 3 days, i.e., until the 21st day after the start of suspension culture.

[0151] The resulting cell aggregates on the 21st day after the start of suspension culture were cultured under 5% CO2 conditions using the serum medium shown in [1], [2], and [3] below, until the 80th day after the start of suspension culture. [1] From the 21st to the 40th day 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: A medium and Neurobasal medium (see Table 5) supplemented with 10% fetal bovine serum, 2% B27 supplement, 200 mM glutamine, and 100 μM taurine (hereinafter referred to as "B medium") mixed in a 1:3 ratio. [3] After 60 days from the start of suspension culture: Medium B.

[0152] The cell aggregates obtained 80 days after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific) and were found to contain a continuous epithelial structure of neural tissue. Storage conditions were examined using the cell aggregates 80 days after the start of suspension culture.

[0153] The following four types of preservation solutions were examined: -B medium mentioned above HBSS (described in Example 1) Optisol (see Example 1) University of Wisconsin solution (product name Viaspan, sold by Astellas Pharma, hereafter referred to as "UW solution"; see Table 6) [Table 5] [Table 6]

[0154] The following three storage temperatures were considered: 37℃ (incubator, 5% CO2, Panasonic) 17℃ (incubator, 5% CO2, Astec) 4℃ (refrigerator, sealed, Panasonic)

[0155] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0156] Specifically, the following storage conditions A to H were examined. Storage condition A (normal culture conditions). Storage solution is medium B, storage temperature is 37℃, storage container is low-attachment plate (5% CO2) Storage condition B: Storage solution is B medium, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage condition C: Storage solution is HBSS, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage condition D: Storage solution is HBSS, storage temperature is 4℃, storage container is low-adhesion plate (sealed) Storage conditions E. Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2) Storage conditions F. Storage solution is Optisol, storage temperature is 4℃, storage container is low adhesion plate (sealed) Storage conditions G. Storage solution is UW solution, storage temperature is 17℃, storage container is low adhesion plate (5% CO2) Storage conditions H: Storage solution is UW solution, storage temperature is 4°C, storage container is low-adhesion plate (sealed)

[0157] The cell aggregates on day 80 after the start of the suspension culture were stored for 48 hours under the above storage conditions A to H. Immediately after storage, bright field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti).

[0158] As a result, the morphology of three-dimensional tissue was maintained in most cell aggregates immediately after storage under storage conditions A (normal culture conditions) and storage conditions B, C, and E. Under storage conditions D and F, although there were some cell aggregates in which the morphology of three-dimensional tissue was maintained, many cell aggregates lost their morphology. On the other hand, under storage conditions G and H, which used UW solution, the morphology of three-dimensional tissue was significantly destroyed in almost all cell aggregates.

[0159] Next, the cell aggregates preserved under preservation conditions A to H were subjected to suspension culture in medium B using low-adhesion plates in an incubator at 37° C. and 5% CO 2 (recovery culture).

[0160] The resulting cell aggregates after recovery culture were observed as bright-field images (phase contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 4).

[0161] As a result, neural tissue with a continuous epithelial structure with a layered structure was frequently observed in cell aggregates after recovery culture under storage condition A (normal culture conditions) and storage conditions B, C, and E. Under storage conditions D and F, neural tissue with a continuous epithelial structure with a layered structure was observed in some cell aggregates. On the other hand, under storage conditions G and H, which used UW solution, the three-dimensional tissue morphology tended to be significantly distorted in most cell aggregates.

[0162] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde, and frozen sections were prepared. These frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), which is a retinal tissue marker, and Crx (anti-Crx antibody, Takara, rabbit), which is a marker for photoreceptor precursor cells. The cell nuclei were stained with DAPI. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (Figures 5 and 6). Furthermore, whether a continuous epithelial structure of the neural retina tissue was formed was examined using the method described in Example 1.

[0163] As a result, in the cell aggregates after recovery culture under storage condition A (normal culture condition) and storage conditions B, C, and E, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates. Under storage conditions D and F, although there were some cell aggregates in which neural tissue with a continuous epithelial structure with a layered structure was observed, in about 70% of the cell aggregates, many rosette structures were formed as shown in Figures 5 and 6, and the morphology of the cell aggregate (three-dimensional retina) was distorted. On average, the morphology was as shown in D and I in Figures 5 and 6. On the other hand, under storage conditions G and H using UW solution, the morphology of the three-dimensional retina tended to be significantly distorted in almost all cell aggregates, as shown in G and H in Figures 5 and 6.

[0164] These results showed that, compared with storage condition A, which is the same as the normal culture conditions, storage conditions B, C, and E, i.e., storage temperatures of 17°C, allowed cell aggregates to be preserved for 48 hours using various preservation solutions while maintaining the continuous epithelial structure of the neural retinal tissue.

[0165] On the other hand, under storage conditions G and H, in which UW solution was used as the storage solution, it was found that the continuous epithelial structure of the neural retinal tissue was hardly maintained.

[0166] Example 3: An example of preserving cell aggregates containing retinal tissues produced from human iPS cells at 17°C using Optisol as a preservation solution Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner and induced to differentiate by the method described in Example 1, to produce cell aggregates 20 days after the start of suspension culture.

[0167] The resulting cell aggregates on the 20th day after the start of the suspension culture were cultured up to the 97th day after the start of the suspension culture using serum media [1], [2], and [3] as described in Example 2. When the resulting cell aggregates on the 97th day after the start of the suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0168] The cell aggregates on day 97 after the start of suspension culture were stored under the following storage conditions A to C. Storage conditions A: Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2), storage period is 48 hours Storage condition B: Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2), storage period is 72 hours Storage condition C: Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2), storage period is 120 hours

[0169] The cell aggregates preserved under preservation conditions A to C were subjected to recovery culture for 7 days using medium B according to the method described in Example 2.

[0170] The resulting cell aggregates after recovery culture were fixed with 4% paraformaldehyde and frozen sections were prepared. These frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a retinal tissue marker, Crx (anti-Crx antibody, Takara, rabbit), a photoreceptor precursor cell marker, Recoverin (anti-Recoverin antibody, Proteintech, rabbit), Rx (anti-Rx antibody, Takara, guinea pig), a retinal tissue marker, and Ki67 (anti-Ki67 antibody, BD, mouse), a marker for proliferating cells. The cell nuclei were stained with DAPI. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (Figures 7 and 8). The presence of a continuous epithelial structure of neural tissue was then examined.

[0171] As a result, it was found that the continuous epithelial structure of neural tissue was frequently present in cell aggregates preserved under any of the preservation conditions A to C. Furthermore, it was found that in the continuous epithelial structure of neural tissue in any of the preservation conditions A to C, Crx-positive photoreceptor precursor cells were present on the surface (apical side), and Chx10-positive neural retinal precursor cells were present inside. Analysis of serial sections revealed that the Chx10-positive cell layer and the Ki67-positive cell layer overlapped. Furthermore, it was found that in the continuous epithelial structure of neural tissue in any of the conditions A to C, Recoverin-positive photoreceptor cells were present on the surface (apical side).

[0172] These results demonstrated that, regardless of the storage temperature of 17°C and the storage period of 48, 72, or 120 hours, a continuous epithelial structure of neural tissue was maintained, characterized by the presence of a photoreceptor layer containing both Crx-positive photoreceptor precursor cells and Recoverin-positive photoreceptors on the surface (apical side) of the aggregate, a neural retinal progenitor cell layer containing both Chx10-positive neural retinal progenitor cells and Ki67-positive proliferating cells on the inner side, and Crx-positive photoreceptors on the basal side on the inner side of that.

[0173] Example 4: Cell aggregates containing retinal tissues prepared from human iPS cells were incubated in a 5% CO 2 Examples of storage in the presence or absence of Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner by the method described in Example 1. StemFit medium (AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (manufactured by Nippi Co., Ltd.) was used as the feeder-free scaffold.

[0174] For differentiation induction, human iPS cells (1231A3 strain) were cultured in a feeder-free manner using StemFit medium until 1 day before subconfluency (approximately 50% of the culture area was covered with cells). The human iPS cells 1 day before subconfluency were cultured in a feeder-free manner for 1 day in the presence of SB431542 (5 μM) and SAG (300 nM) (Preconditioning treatment).

[0175] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) as a cell dispersion solution and further dispersed into single cells by pipetting. The dispersed human iPS cells were then placed in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) at 1.3 × 10 cells per well. 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× chemically defined lipid concentrate. At the start of suspension culture (day 0 after the start of suspension culture), IWR-1e (final concentration 3 μM), Y27632 (final concentration 20 μM), and SAG (final concentration 30 nM) were added to the serum-free medium. On the third day after the initiation of suspension culture, 50 μl of the above-mentioned serum-free medium was added to the medium containing IWR-1e and human recombinant BMP4 (R&D) but not containing Y27632 or SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml) and the final concentration of IWR-1e was 3 μM.

[0176] After 3 days (6 days after the start of suspension culture), 60 μl of medium in the culture vessel was discarded, and 90 μl of the above serum-free medium containing IWR-1e (final concentration 3 μM) but not Y27632 or human recombinant BMP4 was added (total medium volume was 180 μl).

[0177] After 4 days (10 days after the start of suspension culture), 67% medium exchange was performed twice using the above serum-free medium that did not contain IWR-1e, Y27632, or human recombinant BMP4 so that the concentration of exogenous IWR-1e was 10% or less compared to before the medium exchange. After that, half of the medium was exchanged once every 2 to 4 days with the above serum-free medium that did not contain Y27632, human recombinant BMP4, or IWR-1e. For the half-medium exchange, half the volume of the medium in the culture vessel, i.e., 90 μl, was discarded, and 90 μl of the above serum-free medium was added (total medium volume was 180 μl).

[0178] The cell aggregates thus obtained, 22 days after the start of suspension culture, were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM) and SU5402 (5 μM) for 3 days, until the 25th day after the start of suspension culture.

[0179] The resulting cell aggregates on the 25th day after the start of suspension culture were cultured until the 66th day after the start of suspension culture using serum media [1], [2], and [3] as described in Example 2. When the cell aggregates on the 66th day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0180] The storage conditions were examined using the obtained cell aggregates 66 days after the start of suspension culture.

[0181] The preservative solutions examined were all Optisol (described in Example 1).

[0182] The storage temperature and surrounding conditions that were examined were as follows: 17℃: Incubator (5% CO2, Astec) 17℃: Centrifuge (atmospheric pressure, sealed conditions, Hitachi)

[0183] The storage containers considered were the following two: 60mm low adhesion plate (low adhesion plate, manufactured by Sumitomo Bakelite Co., Ltd.) 2.0mL cryovial (Thermo Fisher Scientific)

[0184] Specifically, the following storage conditions A and B were examined. Storage conditions A. Storage solution is Optisol, storage temperature is 17℃ (incubator), storage container is low-adhesion plate (5% CO2) Storage condition B: The storage solution was Optisol, the storage temperature was 17°C (centrifuge, Hitachi), and the storage container was a 2.0 mL cryovial (sealed at atmospheric pressure). Under these conditions, the centrifuge was used without centrifugation for the purpose of temperature control. It was confirmed by measuring the temperature inside the centrifuge with a temperature logger that the temperature was maintained at 16.2-17.8°C during the storage period.

[0185] The cell aggregates on day 66 after the start of the suspension culture were stored for 72 hours under the above storage conditions A and B. Immediately after storage, bright-field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) ( FIG. 9 , top).

[0186] As a result, the cell aggregates immediately after storage under storage conditions A and B tended to retain the three-dimensional tissue morphology with equal frequency.

[0187] Next, the cell aggregates preserved under storage conditions A and B were subjected to suspension culture in medium B described in Example 2 using a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0188] Seven days after the start of recovery culture, bright-field images (phase-contrast images) of the cell aggregates were observed using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 9, lower panel).

[0189] As a result, continuous epithelium with a layered structure was frequently observed in the cell aggregates after recovery culture under storage conditions A and B. Furthermore, microscopic examination using Hoffman interference images with an inverted microscope (Nikon, ECLIPSE Ti) confirmed that neural retinal tissue with continuous epithelium with a layered structure was frequently present under both storage conditions A and B.

[0190] These results demonstrated that storage condition B, in which the tissue was tightly sealed and stored in air, enabled the preservation of three-dimensional neural tissue for 72 hours while maintaining a continuous epithelial structure, with a level that was essentially insignificant compared to storage condition A, in which the tissue was stored at 5% CO2.

[0191] Example 5: An example in which cell aggregates containing retinal tissues prepared from human iPS cells were stored at 17°C using Optisol as a storage solution, and then the viability of the cells was measured. Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner and induced to differentiate by the method described in Example 1, to produce cell aggregates 20 days after the start of suspension culture.

[0192] The obtained cell aggregates on the 20th day after the start of suspension culture were cultured until the 91st day after the start of suspension culture using serum media [1], [2], and [3] by the method described in Example 2. After that, they were prepared under the following condition A (without storage) and condition B (with storage). Condition A (without preservation): Two cell aggregates 91 days after the start of suspension culture were cultured in medium B at 37°C and 5% CO2 for 72 hours. Condition B (with storage): Two cell aggregates 91 days after the start of suspension culture were stored for 72 hours at 17°C and atmospheric pressure (sealed) using Optisol as the storage solution and a 17°C incubator (manufactured by Wakebeetech Co., Ltd.) as the storage device.

[0193] The cell aggregates thus prepared under condition A (without preservation) and condition B (with preservation) were dispersed using a nerve cell dispersion solution (papain-based, manufactured by Wako Pure Chemical Industries, Ltd.) to prepare cell suspensions. Furthermore, the viability of the obtained cell suspensions was measured using a nucleocounter (manufactured by ChemoMetec) (Figure 10).

[0194] As a result, the viability of two cell aggregates under condition A (without preservation) was 99.8% and 99.1%, while the viability of two cell aggregates under condition B (with preservation) was 99.3% and 98.2%. In other words, even after preservation, the viability of the aggregates was very high at 98% or more, and it was found that there was no substantial difference in the viability of cells with or without preservation.

[0195] These results demonstrated that conditions with neural tissue preservation did not substantially differ in cell viability compared to conditions without neural tissue preservation.

[0196] Example 6: Examination of storage temperature using cell aggregates containing retinal tissue prepared from human iPS cells (1) Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner, preconditioned, and induced to differentiate, as described in Example 4, and treated with CHIR99021 and SU5402 to produce "cell aggregates 25 days after the start of suspension culture."

[0197] The resulting cell aggregates on the 25th day after the start of suspension culture were cultured under 5% CO2 conditions using serum media [1], [2], and [3] described in Example 2 until the 73rd day after the start of suspension culture. When the cell aggregates on the 73rd day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0198] The storage conditions were examined using the cell aggregates obtained 73 days after the start of suspension culture.

[0199] The preservative solutions examined were all Optisol (described in Example 1).

[0200] The following five storage temperatures were examined: 4℃: Refrigerator (sealed, Panasonic) 8℃: Centrifuge (sealed, Hitachi) 17℃: Centrifuge (sealed, Hitachi) 30℃: Thermostatic chamber (sealed, AS ONE) 37℃: Incubator (sealed or 5% CO2, Panasonic)

[0201] The storage containers considered were the following two: 60mm low adhesion plate (low adhesion plate, manufactured by Sumitomo Bakelite Co., Ltd.) 2.0mL cryovial (Thermo Fisher Scientific)

[0202] Specifically, the following storage conditions A to F were examined. Storage condition A (normal culture conditions). Storage solution is medium B, storage temperature is 37℃, storage container is low-attachment plate (5% CO2) Storage condition B: Storage solution is Optisol, storage temperature is 4℃, storage container is 2.0mL cryovial (sealed) Storage condition C: Storage solution is Optisol, storage temperature is 8℃, storage container is 2.0mL cryovial (sealed) Storage condition D: Storage solution is Optisol, storage temperature is 17℃, storage container is 2.0mL cryovial (sealed) Storage conditions E. Storage solution is Optisol, storage temperature is 30℃, storage container is 2.0mL cryovial (sealed) Storage conditions F. Storage solution is Optisol, storage temperature is 37℃, storage container is 2.0mL cryovial (sealed)

[0203] The cell aggregates on day 73 after the start of the suspension culture were stored for 72 hours under the above storage conditions A to F. Immediately after storage, bright-field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) ( FIG. 11 , top).

[0204] As a result, the 3D tissue morphology was maintained in most cell aggregates immediately after storage under storage condition A (normal culture conditions) and storage condition D. Although there were some cell aggregates in which the 3D tissue morphology was maintained under storage conditions B, C, and E, many aggregates lost their morphology. On the other hand, under storage condition F, the 3D tissue morphology was significantly destroyed in most cell aggregates.

[0205] Furthermore, as a recovery culture treatment, the cell aggregates after storage under storage conditions A to F were subjected to suspension culture in medium B described in Example 2 using a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0206] The resulting cell aggregates after recovery culture were observed as bright-field images (phase contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 11, middle).

[0207] As a result, in the cell aggregates after recovery culture under storage condition A (normal culture condition) and storage condition D, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates. In the cell aggregates after recovery culture under storage conditions B, C, and E, neural tissue with a continuous epithelial structure with a layered structure was observed in some cell aggregates, but in approximately 50% of the cell aggregates, the morphology of the cell aggregate (three-dimensional retina) was distorted as shown in the figure. On average, the morphologies shown in B, C, and E in Figure 11 were observed. On the other hand, under storage condition F, as shown in F in Figure 11, the morphology of the three-dimensional tissue tended to be significantly distorted in almost all cell aggregates.

[0208] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde to prepare frozen sections. These frozen sections were immunostained for Crx (anti-Crx antibody, Takara, rabbit), which is one of the markers for photoreceptor precursor cells. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (FIG. 11, lower). Then, using the method described in Example 1, it was examined whether Crx-positive photoreceptor precursor cells existed in layers on the surface and whether a continuous epithelial structure of neural retinal tissue existed.

[0209] As a result, it was found that the continuous epithelial structure of neural retinal tissue was present in most cell aggregates after recovery culture under storage condition A (normal culture condition) and storage condition D. On the other hand, although there were some cell aggregates under storage conditions B, C, and E in which the continuous epithelial structure of neural retinal tissue was observed, the three-dimensional retinal morphology was disrupted in about 50% of the cell aggregates, as shown in the figure. Furthermore, under storage condition F, most cell aggregates did not have Crx-positive photoreceptor precursor cells present in the surface layer, did not have a continuous epithelial structure, and had formed rosettes.

[0210] Furthermore, the number of Crx-positive cells was quantified under storage conditions A to E. To quantify the number of Crx-positive cells, an image of the area where Crx-positive cells were observed for each aggregate was taken with a 20x lens of a Keyence inverted microscope (BZ-X710) and cut out at a size of 400 x 400 pixels, and the number of positive cells in the entire image was compared (Figure 12). Although many Crx-positive photoreceptor precursor cells were observed in storage condition F, these cells were not present in the surface layer, there was no continuous epithelial structure, and the morphology of the cell aggregate was greatly disrupted, so it was not examined. As a result, the number of Crx-positive photoreceptor precursor cells in the cell aggregates stored under storage condition D was higher than that in storage condition A. On the other hand, the number of Crx-positive photoreceptor precursor cells in the cell aggregates stored under storage conditions B, C, and E was lower than that in storage condition A.

[0211] These results show that in cell aggregates stored under storage condition D, i.e. at a storage temperature of 17°C, the morphology of three-dimensional tissue was maintained in most cell aggregates. On the other hand, in cell aggregates stored under storage conditions B, C, and E, i.e. at storage temperatures of 4, 8, and 30°C, although some cell aggregates maintained the morphology of three-dimensional tissue, many aggregates lost their morphology. In addition, under storage condition F, i.e. at a storage temperature of 37°C, the morphology of three-dimensional tissue was significantly destroyed in almost all cell aggregates.

[0212] Example 7: Examination of storage temperature using cell aggregates containing retinal tissue prepared from human iPS cells (2) Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner, preconditioned, and induced to differentiate, as described in Example 4, and treated with CHIR99021 and SU5402 to produce "cell aggregates 25 days after the start of suspension culture."

[0213] The resulting cell aggregates on the 25th day after the start of suspension culture were cultured under 5% CO2 conditions using serum media [1], [2], and [3] described in Example 2 until the 80th day after the start of suspension culture. When the cell aggregates on the 80th day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0214] The storage conditions were examined using the obtained cell aggregates 80 days after the start of suspension culture.

[0215] The preservative solutions examined were all Optisol (described in Example 1).

[0216] The following five storage temperatures were examined: 4℃: Refrigerator (sealed, Panasonic) 12℃: Centrifuge (sealed, Hitachi) 17℃: Centrifuge (sealed, Hitachi) 22℃: Centrifuge (sealed, Hitachi) 37℃: Incubator (sealed or 5% CO2, Panasonic)

[0217] The storage containers considered were the following two: 60mm low adhesion plate (low adhesion plate, manufactured by Sumitomo Bakelite Co., Ltd.) 2.0mL cryovial (Thermo Fisher Scientific)

[0218] Specifically, the following storage conditions A to F were examined. Storage condition A (normal culture conditions). Storage solution is medium B, storage temperature is 37℃, storage container is low-attachment plate (5% CO2) Storage condition B: Storage solution is Optisol, storage temperature is 4℃, storage container is 2.0mL cryovial (sealed) Storage condition C: Storage solution is Optisol, storage temperature is 12℃, storage container is 2.0mL cryovial (sealed) Storage condition D: Storage solution is Optisol, storage temperature is 17℃, storage container is 2.0mL cryovial (sealed) Storage conditions E. Storage solution is Optisol, storage temperature is 22℃, storage container is 2.0mL cryovial (sealed) Storage conditions F. Storage solution is Optisol, storage temperature is 37℃, storage container is 2.0mL cryovial (sealed)

[0219] The cell aggregates on day 80 after the start of the suspension culture were stored for 72 hours under the above storage conditions A to F. Immediately after storage, bright-field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) ( FIG. 13 , top).

[0220] As a result, the morphology of three-dimensional tissue was maintained in most cell aggregates immediately after storage under storage condition A (normal culture conditions) and storage conditions C, D, and E. Under storage condition B, although some cell aggregates maintained the morphology of three-dimensional tissue, many aggregates lost their morphology. On the other hand, under storage condition F, the morphology of three-dimensional tissue was significantly destroyed in almost all cell aggregates.

[0221] Next, the cell aggregates preserved under storage conditions A to F were subjected to suspension culture in medium B described in Example 2 using a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0222] The resulting cell aggregates after recovery culture were observed as bright-field images (phase-contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 13, middle). As a result, in the cell aggregates after recovery culture under storage condition A (normal culture condition) and storage conditions C, D, and E, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates. In the cell aggregates after recovery culture under storage condition B, although there were some cell aggregates in which neural tissue with a continuous epithelial structure with a layered structure was observed, the morphology of the cell aggregate (three-dimensional retina) was distorted in about 50% of the cell aggregates, as shown in Figure 13. On the other hand, in the cell aggregates after recovery culture under storage condition F, as shown in F in Figure 13, the morphology of the three-dimensional tissue tended to be significantly distorted in almost all of the cell aggregates.

[0223] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde to prepare frozen sections. These frozen sections were immunostained for Crx (anti-Crx antibody, Takara, rabbit), which is one of the markers for photoreceptor precursor cells. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (FIG. 12, lower). Then, using the method described in Example 1, it was examined whether Crx-positive photoreceptor precursor cells existed in layers on the surface and formed a continuous epithelial structure of neural retinal tissue.

[0224] As a result, it was found that the continuous epithelial structure of neural retinal tissue was present in most cell aggregates after recovery culture under storage condition A (normal culture condition) and storage conditions C, D, and E (Figure 13, bottom). On the other hand, even under storage condition B, there were some cell aggregates in which the continuous epithelial structure of neural retinal tissue was observed, but in about 50% of the cell aggregates, the three-dimensional retinal morphology was distorted, as shown in the figure. Furthermore, under storage condition F, in most cell aggregates, Crx-positive photoreceptor precursor cells were not present in the surface layer, and rosettes were formed.

[0225] Furthermore, the number of Crx-positive photoreceptor precursor cells under storage conditions A to E was quantified. To quantify the number of Crx-positive cells, an image of the area where Crx-positive cells were observed for each aggregate was taken with a 20x lens of a Keyence inverted microscope (BZ-X710) and cut out at a size of 400 x 400 pixels, and the number of positive cells in the entire image was compared (Figure 14). Although many Crx-positive photoreceptor precursor cells were observed for storage condition F, these cells were not present on the surface and the morphology of the cell aggregate was also greatly distorted, so it was not examined. As a result, the number of Crx-positive photoreceptor precursor cells in the cell aggregates stored under storage conditions D and E was higher than that of storage condition A. On the other hand, the number of Crx-positive photoreceptor precursor cells in the cell aggregates stored under storage condition C was almost the same as that of storage condition A. In addition, the number of Crx-positive photoreceptor precursor cells in the cell aggregates stored under storage condition B was lower than that of storage condition A.

[0226] From these results, in the cell aggregates stored under storage conditions D and E, i.e., at storage temperatures of 17 and 22°C, nerve tissues having a continuous epithelial structure with a layered structure were observed in most of the cell aggregates. In addition, in the cell aggregates stored under storage condition C, i.e., at a storage temperature of 12°C, the continuous epithelial structure was observed at almost the same frequency as in the cell aggregates stored under storage condition C, i.e., at a storage temperature of 12°C, but the number of photoreceptor precursor cells was found to be smaller than in the cell aggregates stored under storage condition B, i.e., at a storage temperature of 4°C. It was found that in storage condition F, i.e., at a storage temperature of 37°C, not only the continuous epithelial structure but also the cell aggregate itself tends to collapse. In other words, it was found that the most preferable storage temperature for nerve tissue is 17 to 22°C, and a storage temperature of 12°C is also preferable.

[0227] Example 8: Examination of potassium ion concentration using cell aggregates containing retinal tissue prepared from human iPS cells The above-mentioned UW solution is a preservation solution that is actually used in clinical practice for organ transplantation of endodermal tissues such as pancreatic islets, and is considered the gold standard in the transplantation therapy industry. On the other hand, the results of Example 2 suggest that UW solution is unsuitable for preserving neural tissue. UW solution is known to have a potassium chloride (KCl) concentration of 120 mM, which is higher than other preservation solutions (Table 6). Therefore, in order to investigate what conditions are preferable for a preservation solution, the effect of potassium chloride on preservation was investigated.

[0228] Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner, preconditioned, and induced to differentiate, as described in Example 4, and treated with CHIR99021 and SU5402 to produce "cell aggregates 25 days after the start of suspension culture."

[0229] The resulting cell aggregates on the 25th day after the start of suspension culture were cultured under 5% CO2 conditions using serum media [1], [2], and [3] described in Example 2 until the 99th day after the start of suspension culture. When the cell aggregates on the 99th day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0230] The storage conditions were examined using the cell aggregates obtained 99 days after the start of suspension culture.

[0231] The preservation solutions examined were the following two: Optisol (see Example 1) Optisol containing high concentration of potassium ions (Optisoll to which potassium chloride has been added so that the final concentration of potassium ions becomes 120mM)

[0232] The storage temperature examined was 17°C in all cases (incubator, 5% CO2, manufactured by Astec Co., Ltd.).

[0233] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0234] Specifically, the following storage conditions A and B were examined. Storage conditions A. Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2) Storage condition B: The storage solution is Optisol containing high concentration potassium ions, the storage temperature is 17℃, and the storage container is a low adhesion plate (5% CO2).

[0235] The cell aggregates on day 99 after the start of the suspension culture were stored for 120 hours under the above storage conditions A and B. Immediately after storage, bright-field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) ( FIG. 15 , upper row).

[0236] As a result, the morphology of three-dimensional tissue was maintained in most cell aggregates immediately after storage under storage condition A. On the other hand, under storage condition B, although some cell aggregates maintained the morphology of three-dimensional tissue, the morphology of the surface layer was lost in many aggregates.

[0237] Next, the cell aggregates preserved under storage conditions A and B were subjected to suspension culture in medium B described in Example 2 using a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0238] The resulting cell aggregates after recovery culture were observed as bright-field images (phase-contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 15, middle).

[0239] As a result, continuous epithelium with a layered structure was frequently observed in cell aggregates after recovery culture under storage condition A. On the other hand, under storage condition B, the three-dimensional retinal structure tended to collapse in some cell aggregates.

[0240] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde, and frozen sections were prepared. These frozen sections were immunostained for Crx (anti-Crx antibody, Takara, rabbit), which is one of the markers for photoreceptor precursor cells. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (FIG. 15, lower). Then, using the method described in Example 6, it was examined whether the cell aggregates formed a continuous epithelial structure of neural retinal tissue.

[0241] As a result, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates after recovery culture under storage condition A. On the other hand, in the cell aggregates after recovery culture under storage condition B, Crx-positive photoreceptor precursor cells were hardly observed, and they did not appear to be present in a layered structure.

[0242] These results indicate that the three-dimensional retinal morphology is lost and photoreceptor precursor cells are killed in a preservation solution with a potassium ion concentration of 120 mM, which is the same as that of UW solution. In other words, it was demonstrated that it is important for the potassium ion concentration to be lower than 120 mM for preservation solutions of neural tissue.

[0243] Example 9: Preservation of cell aggregates containing retinal tissue produced from human iPS cells Human iPS cells (1231A3 strain, obtained from Kyoto University) were cultured in a feeder-free manner, preconditioned, and induced to differentiate, as described in Example 4, and treated with CHIR99021 and SU5402 to produce "cell aggregates 25 days after the start of suspension culture."

[0244] The resulting cell aggregates on the 25th day after the start of suspension culture were cultured under 5% CO2 conditions using serum media [1], [2], and [3] described in Example 2 until the 99th day after the start of suspension culture. When the cell aggregates on the 99th day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained a continuous epithelial structure of neural tissue.

[0245] The storage conditions were examined using the cell aggregates obtained 99 days after the start of suspension culture.

[0246] The following two preservation solutions were examined (the composition of each preservation solution is shown in Table 7): DMEM, no glucose (Thermo Fisher Scientific) DMEM, no glucose (Thermo Fisher Scientific) with sodium pyruvate solution (Thermo Fisher Scientific) added to a final concentration of 1 mM [Table 7]

[0247] The storage temperature examined was 17°C in all cases (incubator, 5% CO2, manufactured by Astec Co., Ltd.).

[0248] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0249] Specifically, the following storage conditions A and B were examined. Storage condition A: Storage solution is DMEM, no glucose, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage condition B: Storage solution is DMEM, no glucose, with sodium pyruvate solution added to a final concentration of 1 mM, storage temperature is 17°C, storage container is a low-adhesion plate (5% CO2)

[0250] The cell aggregates on day 99 after the start of the suspension culture were stored for 120 hours under the above storage conditions A and B. Immediately after storage, bright-field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) ( FIG. 16 , top).

[0251] As a result, immediately after storage under both storage conditions A and B, most of the cell aggregates maintained the morphology of three-dimensional tissue.

[0252] Next, the cell aggregates preserved under storage conditions A and B were subjected to suspension culture in medium B described in Example 2 using a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0253] The resulting cell aggregates after recovery culture were observed as bright-field images (phase contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 16, middle).

[0254] As a result, in both the cell aggregates after recovery culture under storage conditions A and B, continuous epithelium with a stratified structure was frequently observed.

[0255] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde, and frozen sections were prepared. These frozen sections were immunostained for Crx (anti-Crx antibody, Takara, rabbit), which is one of the markers for photoreceptor precursor cells. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (FIG. 16, lower). Then, using the method described in Example 6, it was examined whether the cell aggregates formed a continuous epithelial structure of neural retinal tissue.

[0256] As a result, in both cell aggregates after recovery culture under storage conditions A and B, neural tissue with a continuous epithelial structure with a layered structure was observed in most of the cell aggregates.

[0257] These results indicate that retinal tissue can be preserved without losing its morphology or the continuous epithelial structure of the surface layer if it is stored at 17°C in DMEM, a cell culture medium.

[0258] Example 10: Preservation example using cell aggregates containing anterior midbrain produced from human iPS cells Human iPS cells (1231A3 line) were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014) as described in Example 1. Stem Fit medium (AK03; Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (Nippi Co., Ltd.) was used as the feeder-free scaffold.

[0259] For differentiation induction, human iPS cells (1231A3 strain) were cultured in a feeder-free manner using StemFit medium until 1 day before subconfluency (approximately 50% of the culture area was covered with cells). The human iPS cells 1 day before subconfluency were cultured in a feeder-free manner for 1 day in the presence of SB431542 (5 μM) and SAG (300 nM) (Preconditioning treatment).

[0260] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) as a cell dispersion solution and further dispersed into single cells by pipetting. The dispersed human iPS cells were then placed in a non-cell-adhesive 96-well culture plate (Sumilon Spheroid V-bottom plate, PrimeSurface 96V-bottom plate, Sumitomo Bakelite) at 1.0 × 10 cells per well. 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37° C. and 5% CO. The serum-free medium (GMEM+KSR) used was a serum-free medium prepared by adding 20% ​​KSR (Life Technologies), 0.1 mM 2-mercaptoethanol, 1x non-essential amino acids (Life Technologies), and 1 mM pyruvic acid (Life Technologies) to GMEM medium (Life Technologies).

[0261] At the start of suspension culture (day 0 after the start of suspension culture), a Wnt signaling pathway inhibitor (IWR-1-endo, 3 μM), a TGFβR inhibitor (SB431542, 5 μM), and Y27632 (20 μM) were added to the above serum-free medium and cultured.

[0262] On the fourth day after the start of suspension culture, 80 μl of serum-free medium containing a Wnt signaling pathway inhibitor (IWR-1-endo, 3 μM) and a TGFβR inhibitor (SB431542, 5 μM) without Y27632 was added to make the total medium volume 180 μl. After that, half of the medium was replaced with the above serum-free medium containing a Wnt signaling pathway inhibitor (IWR-1-endo, 3 μM) and a TGFβR inhibitor (SB431542, 5 μM) without Y27632 every 2 to 4 days until the 17th day after the start of suspension culture.

[0263] The cell aggregates obtained in this way on the 17th day after the start of suspension culture were cultured in serum-free medium (DMEM / F12 medium) containing 1% N2 supplement and 1% chemically defined lipid concentrate (Thermo Fisher Scientific) under 5% CO2 conditions until the 45th day after the start of suspension culture. When the cell aggregates on the 45th day after the start of suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific), they contained neuroepithelial tissue.

[0264] The storage conditions were examined using the obtained cell aggregates 45 days after the start of suspension culture.

[0265] The following four preservation solutions were examined (the composition of each preservation solution is as described above): Serum-free medium (DMEM / F12 medium) containing 1% N2 supplement and 1% chemically defined lipid concentrate Optisol (see Example 1) HBSS (described in Example 1) UW solution (described in Example 2)

[0266] The storage temperatures considered were the following two: 37℃: Incubator, 5% CO2, Panasonic 17℃: Incubator, 5% CO2, Astec

[0267] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0268] Specifically, the following storage conditions A to D were examined. Storage condition A (normal culture condition). The storage solution is a serum-free medium (DMEM / F12 medium) containing 1% N2 supplement and 1% chemically defined lipid concentrate, the storage temperature is 37°C, and the storage container is a low-attachment plate (5% CO2). Storage condition B: Storage solution is Optisol, storage temperature is 17℃, storage container is low adhesion plate (5% CO2) Storage condition C: Storage solution is HBSS, storage temperature is 17℃, storage container is low-adhesion plate (5% CO2) Storage condition D: Storage solution is UW solution, storage temperature is 17℃, storage container is low adhesion plate (5% CO2)

[0269] The cell aggregates on day 45 after the start of the suspension culture were stored for 72 hours under the above storage conditions A to D. Immediately after storage, bright field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Nikon Corporation, ECLIPSE Ti) (Figure 17, top). Immediately after storage, neuroepithelial tissue was observed in the cell aggregates under all storage conditions.

[0270] Next, the cell aggregates preserved under storage conditions A to D were subjected to suspension culture in a serum-free medium (DMEM / F12 medium) containing 1% N2 supplement and 1% chemically defined lipid concentrate in a non-cell-adhesive 96-well culture plate (PrimeSurface 96U bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) at 37°C in an incubator with 5% CO2 (recovery culture).

[0271] The resulting cell aggregates after recovery culture were observed as bright-field images (phase-contrast images) using an inverted microscope (Nikon, ECLIPSE Ti) (FIG. 17, middle).

[0272] As a result, neuroepithelial tissue was observed in some of the cell aggregates after recovery culture under storage condition A (normal culture condition), and storage conditions B and C. On the other hand, no cell aggregates containing neuroepithelial tissue were observed in the cell aggregates after recovery culture under storage condition D.

[0273] The resulting cell aggregates after recovery culture were fixed with 4% paraformaldehyde to prepare frozen sections. These frozen sections were immunostained for Rx (anti-Rx antibody, Takara, guinea pig), a retinal tissue marker, and Otx2 (anti-Otx2 antibody, Abcam, rabbit), a forebrain and / or midbrain marker. These stained sections were observed using a fluorescent microscope (Keyence) to obtain immunostained images (FIG. 17, lower). The presence or absence of luminal structures (neural rosettes) that were negative for Rx and positive for Otx2 and did not contain retinal tissue but were composed of forebrain and / or midbrain tissue was examined. As a result, the above-mentioned tubular structure was observed in some of the cell aggregates after recovery culture under storage condition A (normal culture condition), and storage conditions B and C. On the other hand, no cell aggregates having the above-mentioned tubular structure were observed at all in the cell aggregates after recovery culture under storage condition D.

[0274] The number of cell aggregates having a luminal structure that does not contain retinal tissue but consists of forebrain and / or midbrain tissue was examined (FIG. 18). As a result, the above luminal structure was observed in four out of six cell aggregates after recovery culture under storage condition A (normal culture condition). The above luminal structure was observed in two out of six cell aggregates after recovery culture under storage condition B, and the above luminal structure was observed in three out of six cell aggregates after recovery culture under storage condition C. On the other hand, the above luminal structure was observed in none of the six cell aggregates after recovery culture under storage condition D.

[0275] These results demonstrated that, compared to storage condition A, which is the same as the normal culture conditions, storage conditions B and C, which have a storage temperature of 17°C and use Optisol or HBSS as the storage solution, enable storage for 72 hours while maintaining the morphology of cell aggregates with luminal structures consisting of forebrain and / or midbrain tissue.

[0276] On the other hand, it was found that under storage condition D using UW solution, the above-mentioned luminal structure was hardly maintained.

[0277] The results were almost the same as those of the neural retinal tissues preserved in Examples 2 and 3.

[0278] From the above results, it was found that neural tissue can be preserved while maintaining the neuroepithelial structure, whether it is neural retinal tissue with a continuous epithelial structure as the neuroepithelium, or anterior midbrain tissue with a luminal structure as the neuroepithelium, as long as the preservation temperature is around 17°C. As for the composition of the preservation solution, it was found that UW solution, with a potassium ion concentration of 120 mM, is not suitable even for preserving anterior midbrain tissue.

[0279] Example 11: Cell aggregates containing retinal tissue prepared from human ES cells were preserved and then transplanted into a rat with retinal degeneration Human ES cells (KhES1 strain, obtained from Kyoto University and used at RIKEN CDB) were cultured feeder-free, preconditioned, and induced to differentiate as described in Example 2, and treated with CHIR99021 and SU5402 to produce cell aggregates 21 days after the start of suspension culture.

[0280] The resulting cell aggregates 21 days after the start of suspension culture were cultured under 5% CO2 conditions using serum media [1], [2] and [3] as described in Example 2 until the 80th day after the start of suspension culture.

[0281] Using the obtained cell aggregates 80 days after the start of suspension culture, cell aggregates were prepared under the following condition A (without storage) and condition B (with storage). Condition A (no storage): Cell aggregates 80 days after the start of suspension culture were cultured in medium B at 37°C for 48 hours in 5% CO2. Condition B (with storage): Cell aggregates 80 days after the start of suspension culture were stored at 17°C for 48 hours in a 17°C incubator (5% CO2 condition, manufactured by Astec Co., Ltd.) using Optisol as a storage solution and 5% CO2 as a storage device.

[0282] The storage containers examined were all 60 mm low adhesion plates (low adhesion plates, manufactured by Sumitomo Bakelite Co., Ltd.).

[0283] The cell aggregates under condition A (without preservation) and condition B (with preservation) were transplanted subretinaly into nude rats with retinal degeneration (SD-Foxn1 Tg(S334ter)3LavRrrc nude rat), a photoreceptor degeneration model, using a syringe according to the method described in a published literature (Shirai et al. PNAS 113, E81-E90).

[0284] Ocular tissues aged 240 days after the start of suspension culture were fixed with paraformaldehyde (PFA) and sucrose was substituted. The fixed ocular tissues were frozen into sections using a cryostat. These frozen sections were immunostained for Rhodopsin (anti-RetP1 antibody, Sigma, mouse), a marker for rod photoreceptors, and S+M+L opsin (anti-S+M+L opsin antibody, Millipore, rabbit), a marker for cone photoreceptor precursors. Crx expression was observed using the fluorescent image of Crx::Venus. The immunostained frozen sections were observed for fluorescence using a confocal laser microscope (TCS SP8, Leica) (Figure 19).

[0285] First, in the rats, almost no photoreceptors were observed in the area where the transplanted cell aggregate-derived human cells were not present. In the retinas of rats transplanted with cell aggregates under condition A (without preservation), Rhodopsin-positive rod photoreceptors and S+M+L opsin-positive cone photoreceptors were observed to have formed rosette structures, engrafted, and matured. In the retinas of rats transplanted with cell aggregates under condition B (with preservation), Rhodopsin-positive rod photoreceptors and S+M+L opsin-positive cone photoreceptors were observed to have formed rosette structures, engrafted, and matured. Furthermore, when a wider area was examined under a microscope, it was found that photoreceptor rosettes had engrafted and matured at almost the same frequency under condition A (without preservation) as under condition B (with preservation).

[0286] These results demonstrated that preserved retinal tissue survived and matured to a level that was virtually indistinguishable from non-preserved retinal tissue when transplanted, demonstrating that the method of the present invention can preserve neural tissue suitable for transplantation.

[0287] Example 12: Examination of chondroitin sulfate concentration in cell masses containing retinal tissues produced from human iPS cells Human iPS cells (TFH-HA line, established by Sumitomo Dainippon Pharma) were established using commercially available Sendai virus vectors (four factors: Oct3 / 4, Sox2, KLF4, and c-Myc; CytoTune Kit, IDPharma) according to the protocol published by Thermo Fisher Scientific (iPS 2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and the protocol published by Kyoto University (Establishment and maintenance of human iPS cells in a feeder-free environment; CiRA_Ff-iPSC_protocol_JP_v140310; http: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html) using StemFit medium (AK03; Ajinomoto Co.) and Laminin511-E8 (Nippi Co.).

[0288] The human iPS cells (TFH-HA line) were cultured under feeder-free conditions in StemFit medium until 1 day before subconfluency, by the method described in Example 1. The human iPS cells (TFH-HA line) 1 day before subconfluency were cultured feeder-free for 1 day in the presence of SB431542 (5 μM) and SAG (300 nM) (Preconditioning treatment).

[0289] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) as a cell dispersion solution and further dispersed into single cells by pipetting. The dispersed human iPS cells were then cultured at 1.3 x 10 per well in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) 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 the third day after the start of suspension culture, 50 μl of the above serum-free medium was added to the medium containing human recombinant BMP4 (R&D Co., Ltd.) without Y27632 or SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0290] Four days later (seven days after the start of suspension culture), the medium was replaced with the above serum-free medium that did not contain Y27632 or human recombinant BMP4. The medium replacement procedure consisted of discarding 60 μl of the medium in the culture vessel and adding 90 μl of the above serum-free medium, making the total medium volume 180 μl. Thereafter, half of the medium was replaced with the above serum-free medium that did not contain Y27632 or human recombinant BMP4 once every 2 to 4 days. The half-medium replacement procedure consisted of discarding half the volume of the medium in the culture vessel, i.e., 90 μl, and adding 90 μl of the above serum-free medium, making the total medium volume 180 μl.

[0291] The cell masses thus obtained, 17 days after the start of suspension culture, were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM) and SU5402 (5 μM) for 3 days, i.e., until the 20th day after the start of suspension culture.

[0292] The resulting cell aggregates, 20 days after the start of suspension culture, were cultured under 5% CO2 conditions using the serum medium shown in [1], [2], and [3] below, until the 84th day after the start of suspension culture. [1] From 20 to 40 days after the start of suspension culture: Medium A. [2] From the 40th to 60th day after the start of suspension culture: Medium A and Medium B mixed in a 1:3 ratio. [3] After 60 days from the start of suspension culture: Medium B.

[0293] The cell aggregates obtained on the 84th day after the start of the suspension culture were observed with an inverted microscope (EVOS, Thermo Fisher Scientific) and were found to contain a continuous epithelial structure of neural tissue. The storage conditions were examined using the cell aggregates on the 84th day after the start of the suspension culture.

[0294] The following four types of preservation solutions were examined (see Tables 4, 8-9). -B medium mentioned above ·Optisoll (contains 2.5% chondroitin sulfate) 1% chondroitin sulfate-containing medium (a 3:1 mixture of DMEM, low glucose, and Discovisc. DMEM and low glucose were manufactured by Thermo Fisher Scientific (product number: 11885-084), and Discovisc was manufactured by Alcon Japan (sales code: 1319818Q1025).) - 0.5% chondroitin sulfate-containing medium (a 7:1 mixture of DMEM low glucose and Discovisc) [Table 8] [Table 9]

[0295] The following two storage temperatures were considered: 37℃ (incubator, 5% CO2, Panasonic) 17℃ (thermostat, atmospheric pressure, sealed conditions, manufactured by Wakembee Tech Co., Ltd.)

[0296] The storage containers considered were the following two: 60mm low adhesion plate (low adhesion plate, manufactured by Sumitomo Bakelite Co., Ltd.) 2.0mL cryovial (Thermo Fisher Scientific)

[0297] Specifically, the following storage conditions A to D were examined. Storage condition A (normal culture conditions). Storage solution is medium B, storage temperature is 37℃ (incubator), storage container is 60mm low-attachment plate (5% CO2) Storage condition B: Storage solution is Optisol, storage temperature is 17℃ (thermostat), storage container is 2.0mL cryovial (sealed at atmospheric pressure) Storage condition C: Storage solution is 1% chondroitin sulfate-containing medium, storage temperature is 17°C (thermostat), storage container is 2.0 ml cryovial (sealed at atmospheric pressure) Storage condition D: Storage solution is 0.5% chondroitin sulfate-containing medium, storage temperature is 17°C (thermostat), storage container is 2.0 ml cryovial (sealed at atmospheric pressure)

[0298] The cell aggregates on day 84 after the start of suspension culture were stored for 72 hours under the above storage conditions A to D. Immediately after storage, bright field images (phase contrast images) of the cell aggregates were observed using an inverted microscope (Olympus, IX83) (Figure 20, top). As a result, the morphology of three-dimensional tissue was maintained in most of the cell aggregates under all storage conditions.

[0299] Furthermore, as a recovery culture treatment, the cell aggregates after storage under storage conditions A to D were subjected to suspension culture in medium B using a non-cell-adhesive 96-well culture plate (PrimeSurface 96 slit well plate, manufactured by Sumitomo Bakelite Co., Ltd.) in an incubator at 37°C and 5% CO2 (recovery culture).

[0300] The resulting cell aggregates after recovery culture were observed as bright-field images (phase contrast images) using an inverted microscope (Olympus, IX83) (Figure 20, second row from the top). As a result, regardless of the storage conditions, the morphology of three-dimensional tissue was maintained in most of the cell aggregates, and neuroepithelial structures were observed.

[0301] The obtained cell aggregates after the recovery culture were fixed with 4% paraformaldehyde to prepare frozen sections. These frozen sections were immunostained for Crx (anti-Crx antibody, Takara, rabbit) and Chx10 (anti-Chx10 antibody, Exalpha, sheep), which are markers for photoreceptor precursor cells. These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images (Fig. 20, second row from the bottom and bottom row). Then, using the method described in Example 1, it was examined whether Crx-positive photoreceptor precursor cells exist in layers on the surface and whether a continuous epithelial structure of neural retinal tissue exists. As a result, it was found that Crx-positive photoreceptor precursor cells exist in layers on the surface (Fig. 20, second row from the bottom). Furthermore, it was found that neural retinal precursor cells of Chx10-positive cells exist in layers inside the Crx-positive cell layer on the surface (Fig. 20, bottom row). As a result, it was found that a continuous epithelial structure of neural retinal tissue exists at a high frequency under all storage conditions.

[0302] These results indicate that the morphology of three-dimensional tissues was maintained in most cell aggregates under all preservation conditions. In other words, three-dimensional retinas could be cryopreserved in solutions with chondroitin sulfate concentrations of 0.5-2.5%.

[0303] Furthermore, the cell aggregates could be preserved for 48 hours and 120 hours, respectively, using HBSS in Example 1 and DMEM in Example 9. Since these solutions do not contain chondroitin sulfate, it was demonstrated that the chondroitin sulfate concentration capable of maintaining the morphology of a three-dimensional retina is 0 to 2.5%.

[0304] Example 13: Cell aggregates containing retinal tissue prepared from human iPS cells were preserved and then transplanted into rats with retinal degeneration Human iPS cells (DSP-SE line, established by Sumitomo Dainippon Pharma) were established using Sendai virus vectors (four factors: Oct3 / 4, Sox2, KLF4, and c-Myc; CytoTune Kit, IDPharma) according to the protocol published by Thermo Fisher Scientific (iPS 2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and the protocol published by Kyoto University (establishment and maintenance of human iPS cells in a feeder-free environment; CiRA_Ff-iPSC_protocol_JP_v140310, http: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html) using StemFit medium (AK03N; Ajinomoto Co.) and Laminin511-E8 (Nippi Co.).

[0305] The human iPS cells (DSP-SE line) were cultured in StemFit medium under feeder-free conditions, using the method described in Example 1, until 1 day before the cells became subconfluent.

[0306] As a differentiation induction procedure, human iPS cells (DSP-SE line) that had reached subconfluency one day prior were cultured in the presence of SB431542 (5 μM) and SAG (300 nM) in a feeder-free environment for an additional day (preconditioning treatment).

[0307] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) as a cell dispersion solution and further dispersed into single cells by pipetting. The dispersed human iPS cells were then cultured at 1.3 x 10 per well in a non-cell-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite). 4The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× chemically defined lipid concentrate. At the start of suspension culture (day 0 after the start of suspension culture), IWR-1e (final concentration 3 μM), Y27632 (final concentration 20 μM), and SAG (final concentration 10 nM) were added to the serum-free medium. Three days after the start of suspension culture, 50 μl of fresh serum-free medium described above was added to the medium containing human recombinant BMP4 (manufactured by R&D) and IWR-1e (final concentration 3 μM) but not containing Y27632 or SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0308] Three days later (six days after the start of suspension culture), the medium was replaced with the above serum-free medium that did not contain Y27632, SAG, human recombinant BMP4, and IWR-1e. The operation of medium replacement was performed by discarding 100 μl of the medium in the culture vessel and adding 130 μl of the above serum-free medium so that the concentration of exogenous IWR-1e was 10% or less compared to before the medium replacement, and then discarding 130 μl of the medium in the culture vessel and adding 130 μl of the above serum-free medium, so that the total amount of the medium was 180 μl. Thereafter, half of the medium was replaced with the above serum-free medium that did not contain IWR-1e, Y27632, SAG, and human recombinant BMP4 once every 2 to 4 days. The operation of half of the medium replacement was performed by discarding half the volume of the medium in the culture vessel, i.e., 90 μl, and adding 90 μl of the above serum-free medium, so that the total amount of the medium was 180 μl.

[0309] The cell masses thus obtained, 13 days after the start of suspension culture, were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM) and SU5402 (5 μM) for 3 days, i.e., until the 16th day after the start of suspension culture.

[0310] The resulting cell aggregates on the 16th day after the start of suspension culture were cultured under 5% CO2 conditions using the serum medium shown in [1], [2], and [3] below, until the 82nd day after the start of suspension culture. [1] From 21 to 40 days after the start of suspension culture: Medium A. [2] From the 40th to 60th day after the start of suspension culture: Medium A and Medium B mixed in a 1:3 ratio. [3] After 60 days from the start of suspension culture: Medium B.

[0311] The obtained cell aggregates 82 days after the start of suspension culture were stored at 17°C for 96 hours using Optisol as a storage solution and a 17°C incubator (atmospheric pressure, sealed conditions, manufactured by Wakembie Tech Co., Ltd.) as a storage device.

[0312] The storage container examined was a 1.5 mL Eppendorf tube (manufactured by Eppendorf).

[0313] The above cell aggregates were transplanted subretinaly into nude rats with retinal degeneration (SD-Foxn1 Tg(S334ter)3LavRrrc nude rats), a photoreceptor degeneration model, using a syringe according to the method described in a publicly known literature (Shirai et al., PNAS 113, E81-E90).

[0314] Eye tissues aged 294 days after the start of suspension culture were fixed with paraformaldehyde (PFA) and sucrose substituted. The fixed eye tissues were frozen using a cryostat. These frozen sections were immunostained for rhodopsin (anti-RetP1 antibody, Sigma, mouse), a marker for rod photoreceptors, cone arrestin (anti-cone arrestin antibody, Novus Biological, goat), PRPH2 (anti-Peripherin-2 antibody, Proteintech, rabbit), a marker for photoreceptor outer segments, and human nuclei (anti-Ku80 antibody, R&D systems, goat), a marker for human nuclei. The immunostained frozen sections were observed for fluorescence using a confocal laser microscope (trade name: TCS SP8, Leica) (Figure 21 AE).

[0315] First, in the rats, almost no photoreceptors were observed in the areas where the transplanted cell aggregate-derived human cells were not present. On the other hand, in the retina of the rat transplanted with the cell aggregate stored at 17°C for 96 hours, it was observed that Rhodopsin-positive rod photoreceptors had formed rosette structures, engrafted, and matured. Furthermore, Cone arrestin-positive cone photoreceptors and PRPH2-positive photoreceptor outer segments were also detected, indicating that the transplanted retinal tissue had engrafted and matured.

[0316] These results demonstrated that retinal tissues stored at low temperatures for 96 hours survived and matured after transplantation, demonstrating that the method of this invention can preserve neural tissue suitable for transplantation for up to four days. [Industrial Applicability]

[0317] According to the present invention, it is possible to store neural tissues extracted from living bodies and neural tissues prepared from pluripotent stem cells for periods not exceeding two weeks without freezing, thereby making the treatment of neurodegenerative diseases by transplantation therapy more feasible.

Claims

1. A method for preserving forebrain or midbrain tissue, comprising preserving forebrain or midbrain tissue having a neuroepithelial structure in a preservation solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 12°C to 22°C.

2. The method for preserving the food product according to claim 1, wherein the preservation temperature is 17°C.

3. 3. The method for preserving tissue according to claim 1 or 2, wherein the forebrain tissue or midbrain tissue is derived from a human.

4. The method for preserving tissue according to any one of claims 1 to 3, wherein the forebrain tissue or midbrain tissue is a tissue derived from pluripotent stem cells.

5. The method for preserving tissue according to any one of claims 1 to 4, wherein the forebrain tissue or midbrain tissue is a tissue to be used for transplantation.

6. The method for preserving tissue according to any one of claims 1 to 5, wherein the forebrain tissue or midbrain tissue is a cell aggregate.

7. The preservation method according to claim 6, wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of the cell aggregate and / or a rosette structure present on the surface or inside the cell aggregate.

8. The method for preserving tissue according to any one of claims 1 to 7, wherein the forebrain tissue or midbrain tissue is a tissue containing Otx2-positive cells.

9. The method for preservation according to any one of claims 1 to 8, wherein the potassium ion concentration is greater than 0 mM and less than 60 mM.

10. The method for preservation according to claim 9, wherein the potassium ion concentration is greater than 0.1 mM and less than 10 mM.

11. The method according to any one of claims 1 to 10, characterized in that the storage is performed for a period of 14 days or less.

12. The method according to any one of claims 1 to 11, characterized in that the storage is performed for a period of 5 days or less.

13. The method for preservation according to any one of claims 1 to 12, wherein the preservation solution is an aqueous solution which contains a buffer substance and has a buffering effect to a pH in the range of 6.0 to 8.

6.

14. The method for preservation according to any one of claims 1 to 13, wherein the preservation solution is an aqueous solution further containing glycosaminoglycans.

15. The method for preservation according to claim 14, wherein the concentration of glycosaminoglycans is 0.1% (w / v) or more and 10% (w / v) or less.

16. The method for preservation according to claim 14 or 15, wherein the glycosaminoglycans are one or more glycosaminoglycans selected from the group consisting of chondroitin sulfate and hyaluronic acid.

17. A method for transporting forebrain or midbrain tissue, comprising transporting forebrain or midbrain tissue having a neuroepithelial structure in a storage solution having a potassium ion concentration greater than 0 mM and less than 115 mM at a storage temperature of 12°C to 22°C.

18. 18. The method of claim 17, wherein the storage temperature is 17°C.

19. The method of claim 17 or 18, wherein the forebrain tissue or midbrain tissue is tissue derived from human pluripotent stem cells.

20. The method according to any one of claims 17 to 19, wherein the forebrain tissue or midbrain tissue is a tissue to be used for transplantation.

21. The method of any one of claims 17 to 20, wherein the forebrain tissue or midbrain tissue is a cell aggregate.

22. The method of claim 21 , wherein the neuroepithelial structure is a continuous epithelial structure present on the surface of the cell aggregate and / or a rosette structure present on the surface or inside the cell aggregate.

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