String-like aggregates of retinal pigment epithelial cells, a device for producing the same, a method for producing the same, and a therapeutic agent containing the string-like aggregates.

Abstract

The present invention provides: a graft material of retinal pigment epithelial cells, which can be prepared within a short period of time and at low cost and can be controlled easily at a site at which the graft material is transplanted in a non-invasive transplantation method; and a method for producing the graft material.

Description

[Technical Field]

[0001] The present invention relates to string-like aggregates of retinal pigment epithelial cells, a device and method for producing the same, and a therapeutic agent containing the string-like aggregates. [Background technology]

[0002] The use of ES / iPSC-derived retinal pigment epithelial cells (RPEs) for cell-based regenerative therapy for eye diseases has been a major area of ​​interest in ophthalmic clinical practice over the past decade (Non-Patent Literature 1). These clinical studies have revealed the safety of this therapeutic approach, along with some potential efficacy. To date, these clinical studies have explored two approaches: one using cell suspensions and the other using RPE sheets, both of which have advantages and disadvantages. Cell suspension transplantation allows for the use of readily available cells from tubes or cell stocks cultured for a short period, and can be performed with minimal surgical intervention. However, controlling the placement of cells at the transplantation site is difficult, and transplanted cells often form an epiretinal membrane (ERM) within the vitreous humor (Non-Patent Literature 1, 2). In contrast, RPE sheets allow for visual confirmation that the transplanted sheet is precisely positioned as intended, but sheet preparation requires time, cost, and invasive surgical intervention involving large incisions in the sclera and neuroretina (Non-Patent Literature 3). Therefore, the challenge of providing a transplant material for retinal pigment epithelial cells that can be prepared quickly and at low cost, and whose transplantation method is non-invasive, while also being easily controllable at the transplantation site, remained unresolved. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Sugita, S et al., (2020) J. Clin. Med. 9, 2217. [Non-Patent Document 2] Schwartz, SD et al., (2015) Lancet 385, 509-516. [Non-Patent Document 3] Lyndon da Cruz et al., Nature Biotechnology Advance Online Publication, published online 19 March 2018. [Overview of the project] [Problems that the invention aims to solve]

[0004] The problem that the present invention aims to solve is a transplant material for retinal pigment epithelial cells that can be prepared in a short time and at low cost, is non-invasive, and is easily controllable at the transplant site, as well as a method for producing the transplant material. [Means for solving the problem]

[0005] The inventors hypothesized that even RPE cells, which are filamentous aggregates that can be easily injected under the retina, can be expanded to cover a certain area, similar to small sheet transplants. In recent years, the relationship between surface coverage, tissue contractility, and adhesive strength has been reported as a morphological mechanism of microtissue (Yamashita, T. et al., (2016). Acta Biomater. 45, 85-97.). The inventors confirmed that cells with a balanced adhesive strength and contractility spontaneously generated spheroidal tissue on a concave surface with low curvature. Based on this finding, they examined the curvature of the lower end of the concave surface and found that grooved surfaces with low curvature generate band-like tissue from cells. While the fabrication of small tissues basically required special techniques such as photolithography, the inventors introduced a fabrication technique that combines polydimethylsiloxane (PDMS) soft lithography and simple three-dimensional (3D) printing, making this tissue engineering approach widely possible. In this invention, a PDMS-based culture device with fine grooves was fabricated, and it was investigated whether hiPSC-RPE cells could form filamentous aggregates. Then, it was confirmed whether hiPSC-RPE cells expanded from the filamentous aggregates plated on a dish, and whether the expanded hiPSC-RPE cells could exhibit the same RPE characteristics as before the formation of the filamentous aggregates. Furthermore, it was verified whether the hiPSC-RPE cells that had formed filamentous aggregates could be substantially injected into the eyes of animals. In other words, the present invention relates to the following.

[0006] [1] A string-like aggregate of retinal pigment epithelial cells. [2] The string-like aggregate described in [1] above, characterized in that the ratio of total length to outer diameter of the body is 2 to 1,000. [3] The string-like aggregate described in [2] above, characterized in that its cross-sectional shape is circular or elliptical. [4] A string-like aggregate according to any of [1] to [3] above, wherein the retinal pigment epithelial cells are cells differentiated from pluripotent stem cells. [5] A device for manufacturing a string-like aggregate as described in any of [1] to [4] above, Having a base member, The base member has a top surface provided with one or more grooves, the grooves, serving as a mold for culturing seeded retinal pigment epithelial cells into string-shaped aggregates, have a cavity portion having the length and width of the string-shaped aggregate, The device. [6] The device according to [5] above, wherein the groove has a cavity portion as a bottom of the groove, and has an upper portion positioned above the cavity portion, and the upper portion has an opening of the groove. [7] The device according to [5] or [6] above, wherein the cross-sectional shape of the groove is a V-shape, and the deepest portion of the V-shape is rounded. [8] A method for producing a string-shaped aggregate of retinal pigment epithelial cells, comprising the following steps; (1) a step of seeding retinal pigment epithelial cells suspended in a liquid medium into the grooves of the device according to any one of [5] to [7] above, (2) a step of culturing retinal pigment epithelial cells in the grooves to form a string-shaped aggregate of retinal pigment epithelial cells. [9] The method according to [8] above, wherein the medium comprises a ROCK inhibitor.

[10] The method according to [9] above, wherein the ROCK inhibitor is Y-27632.

[11] The density of retinal pigment epithelial cells cultured in the grooves is 2.5x10 3 cells / mL to 5x10 5 cells / mL, the method according to any one of [8] to

[10] above.

[12] The method according to any one of [8] to

[11] above, wherein the culture period of the retinal pigment epithelial cells is 1 day to 7 days.

[13] A pharmaceutical composition comprising the string-shaped aggregate of retinal pigment epithelial cells according to any one of [1] to [4] above.

[14] A therapeutic agent for a disease caused by a retinal pigment epithelium disorder, comprising the string-shaped aggregate of retinal pigment epithelial cells according to any one of [1] to [4] above. Effects of the Invention

[0007] According to the present invention, it is possible to produce a retinal pigment epithelial cell transplant material that can be prepared in a short time at low cost, and whose size is easily controllable at the transplant site even when the transplantation method is non-invasive. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] [Figure 1] Figure 1 is a perspective view illustrating the structure of the device of the present invention and a method for producing a string-shaped aggregate using the same. In this figure, to clearly explain the grooves, an embodiment in which one groove is provided on the upper surface of a base member is shown, and only a part of the full length of the groove is shown to illustrate the cross-section of the groove (the cross-section obtained by cutting the groove perpendicularly to its longitudinal direction). Figure 1(a) shows a state where retinal pigment epithelial cells are seeded in the groove together with a liquid medium (not shown). Figure 1(b) shows a state where the seeded retinal pigment epithelial cells have grown into string-shaped aggregates in the groove. [Figure 2] Figure 2 is a diagram illustrating an example of the shapes of a groove and a cavity portion. [Figure 3] Figure 3 is a diagram showing an example of a preferred embodiment of a groove and a cavity portion. [Figure 4] Figure 4 is a schematic diagram for explaining the dimensions of each part of a string-shaped aggregate, the configuration of the device of the present invention, and the relationship between the string-shaped aggregate and the device. Figure 4(a) is a diagram showing a simplified overall shape of the string-shaped aggregate for explanation. Figure 4(b) is a diagram showing an example of the dimensional relationship between the string-shaped aggregate formed in the groove and the groove. [Figure 5] Figure 5 is a diagram showing a preferred embodiment of the device of the present invention. Figure 5(a) is a perspective view showing the entire device, and Figure 5(b) is a cross-sectional view taken along the cutting line X1-X1 extending in the width direction of the groove, in the thickness direction of the device shown in Figure 5(a). Hatching on the cut surface is omitted. [Figure 6] Figure 6 is a diagram showing an example of a mold for producing the device of the present invention, and is also a diagram showing an example of a method for producing the mold. Figure 6(b) is a perspective view of the mold component shown in Figure 6(a). Figure 6(d) is a perspective view of the mold shown in Figure 6(c). [Figure 7] Figure 7 is a cross-sectional view showing an example of the manufacturing process of the device of the present invention, schematically illustrating how the device is molded in resin using the molds shown in Figures 6(c) and 6(d). [Figure 8] Figure 8 shows a photographic diagram (Figure 8(a)) of a mold actually manufactured in an embodiment of the present invention, and a photographic diagram (Figure 8(b)) of the vise actually molded using the mold. [Figure 9] Figure 9 is a schematic diagram illustrating a method for producing string-like aggregates according to the present invention. Figure 9(a) shows retinal pigment epithelial cells seeded in the groove of the device shown in Figure 5 (liquid culture medium is not shown), and Figure 9(b) schematically shows the process of removing string-like aggregates formed in the groove from the groove. [Figure 10] Figure 10 shows the measurement results of the PDMS groove structure. A: Photograph of a PDMS coated mold with five fins measuring 19.5 mm in length and 1.6 mm in height. B: Photograph of a PDMS-based culture device for groove structure measurement, which has five grooves measuring 19.5 mm in length, 1 mm in width, and 1.6 mm in depth. C: Laser microscope image of the groove bottom. D: Measurement of the groove shape of the PDMS-based culture device. The average values ​​for each measurement item were a bottom curvature radius of 0.21 mm, groove width of 1.16 mm, and groove depth of 1.60 mm. [Figure 11]Figure 11 shows that filamentous aggregates partially restored function as they replaced damaged RPE-deficient regions. A: In vitro disease model and transplantation experimental schedule. Pathological RPE degeneration / damage status was mimicked by treating RPE cells with MMC to create a wound, and then the transplantation process was mimicked in vitro by plating either a hiPSC-RPE cell suspension or filamentous aggregates (201B7modFucci strain) onto the defect region. B: Expansion of plated hiRPE cells on the RPE-deficient region was monitored by mCherry expression by cells in the G0-G1 cell cycle of the 201B7-Fucci hiPSC strain. The initially wounded region and the defect region in the control well are indicated by dotted lines. B': The panel shows the RPE-deficient region after scratching and the region refilled by RPE cell expansion from RPE cell suspension or filamentous aggregates. C: RPE cells expanded from filamentous aggregates appeared to cease expanding upon contact with resident RPE cells exhibiting increased mCherry expression along the boundary. D: The boundary between existing MMC-treated RPE cells and newly plated RPE cells was reasonably distinguishable by mCherry expression and RPE cell size (arrows). MMC-treated RPE cells were generally larger than RPE cells expanded from filamentous aggregates. ZO-1 expression was observed between cells within each population of resident MMC-treated and newly plated / expanded RPE cells, as well as between cells at the boundary. E and F: Secretion of PEDF and VEGF in each well at baseline (-d3), after MMC (-d2), after scratching (-d1), and with and without plating of hiPSC-RPE cells or filamentous aggregates (n=3). Secretion of both VEGF and PEDF decreased after MMC treatment and scratching. Without RPE cell supplementation, PEDF continued to decrease, but with RPE cell supplementation, either in suspension or filamentous aggregates, PEDF was obtained more rapidly. [Figure 12]Figure 12 shows the optimization of filamentous aggregate formation. A: hiPSC-RPE cells were plated into mold grooves using three types of media: 10 μM Y-27632 (sheet medium, mixed medium, and maintenance medium). B: Appearance of filamentous aggregates in the mold (mixed medium, maintenance medium). C: hiPSC-RPE cells were plated into grooves at 4.5 x 10⁵, 1.5 x 10⁵, and 5 x 10⁵ and incubated for 3 and 7 days. D: Appearance of filamentous aggregates formed with 150,000 or 450,000 cells after 19 days on a plate. Filamentous aggregates formed with 150,000 cells lost their initial shape much more rapidly than those formed with 450,000 cells, and after 19 days, their initial shape was almost completely lost. Scale bar, 200 μm. [Figure 13] Figure 13 shows the effect of starting cell number and Y-27632 concentration on filamentous aggregate formation. A: Appearance of filamentous aggregates formed with 150,000 or 450,000 RPE cells on day 4, placed on a dish and photographed the following day and 14 days later. RPE cells expanded from each filamentous aggregate, and the filamentous aggregate formed with 15,000 cells lost its shape after 14 days, while the filamentous aggregate formed with 450,000 cells maintained its initial shape. A': Fragmented filamentous aggregates lost almost their initial shape after cell expansion. B: Appearance of filamentous aggregates on day 2 after seeding RPE (2 × 10⁵). C: Cross-sectional view of filamentous aggregates formed with either 10 μM or 2.5 μM Y-27632. Ezrin and laminin expression is fragmented, but ZO-1 is expressed between cells, and apical-basal polarity is not distinguished. The panel on the right shows the control staining of the RPE sheet. D, E, F, G: String-like aggregates formed with 2.5 μM Y-27632 showed higher adhesion to the plate, covered a larger area, and began to expand on the plate more quickly compared to those formed with 10 μM Y-27632 (D, E: String-like aggregates on day 2, F, G: String-like aggregates on day 3). [Figure 14]Figure 14 shows the optimization of Y-27632 concentration for filamentous aggregate formation. A: Filamentous aggregate formation under different Y-27632 concentrations. B: Filamentous aggregates (M8 strain) on day 2 were plated in a 24-well plate, and the expansion of the coverage area by hiPSC-RPE cells was monitored. Filamentous aggregates with 2-2.5 μM Y-27632 added showed the highest adhesion to the plate and tended to adhere flat when placed on the wells. C: Expansion of hiPSC-RPE cells (M8 strain). Coverage area after placing filamentous aggregates in each well (n=6 for each Y-27632 concentration). D: Filamentous aggregates (201B7modFucci strain) on day 2 were placed in a 24-well plate, and the expansion of the coverage area by hiPSC-RPE cells was monitored. String-like aggregates treated with 2-2.5 μM Y-27632 consistently adhered to the plate and tended to adhere flat when placed on the wells. E: Magnification of hiPSC-RPE cells (201B7modFucci strain). Coverage area after placing string-like aggregates in each well (n=2 for each Y-27632 concentration). [Figure 15-1] Figure 15-1 shows the reproducibility of filamentous aggregate formation using different hiPSC strains (M8 and 201B7modFucci L). A: Similar filamentous aggregate formation was achieved using other hiPSC strains, but optimization of Y-27632 was required for each strain. The 201B7modFucci strain required 5 μM Y-27632 to stably form filamentous aggregates. B: Filamentous aggregates formed with each M8 and 201B7modFucci strain adhered to the plate, were well magnified, and exhibited the characteristic cobblestone appearance and pigmentation of RPE. C: RPE cells magnified from filamentous aggregates expressed the tight junction marker ZO-1 and the RPE marker MiTF. D: RPE marker gene expression in hiPSC-RPE cells before filamentous aggregate formation, RPE cells from filamentous aggregates, and cells magnified from filamentous aggregates derived from each hiPSC strain. E: RPE cells migrated and proliferated in a monolayer from the string-like aggregates. [Figure 15-2]Figure 15-2 shows the reproducibility of filamentous aggregate formation using different hiPSC strains (M8 and 201B7modFucci L). F: Number of RPE cells in the filamentous aggregate and number of RPE cells expanded from the filamentous aggregate. G: RPE cells expanded from the filamentous aggregate secreted VEGF and PEDF. Scale bar, 50 μm (C, E). [Figure 16] Figure 16 shows filamentous aggregate transplantation in nude rats. A: Fundus photograph of a nude rat eye after filamentous aggregate transplantation. B: Fundus photograph of a nude rat eye 93 days after filamentous aggregate transplantation. A strong RPE-like signal was observed at the transplantation site by SLO-OCT. C: Hematoxylin eosin staining of the eye after transplantation. D: Human nuclear antigen (HuNu) was positive only in the dye sheet-like layer. E: The transplanted RPE showed well orientation, as indicated by the polarity markers human-specific ezrin and collagen type IV. Scale bars, 50 μm (B) 20 μm (E). [Figure 17] Figure 17 shows a string-like aggregate loaded into a 24G intravenous cannula. [Figure 18] Figure 18 shows the implantation of filamentous aggregates into the eyes of two rabbits. The practical handling of filamentous aggregates during implantation surgery was tested in two rabbit eyes (A and B). Top: Loading of filamentous aggregates. Center: Slow injection of filamentous aggregates into the medial side of the retinal hemorrhage. The retinal incision site is indicated by a white arrow. Bottom: Injected filamentous aggregates. [Figure 19] Figure 19 shows the COP groove structure. [Figure 20] Figure 20 is a photograph showing the moment when string-like aggregates suspended in a medium consisting of a viscoelastic substance diluted 4-7 times with OptiMEM are aspirated using a cannula. Bar; 1 mm [Figure 21] Figure 21 shows the transplantation of string-like aggregates into the eyeball of a monkey. [Modes for carrying out the invention]

[0009] 1. String-like aggregates of retinal pigment epithelial cells The present invention provides string-like aggregates of retinal pigment epithelial cells (hereinafter referred to as "the string-like aggregates of the present invention").

[0010] In this invention, retinal pigment epithelial cells (hereinafter sometimes referred to as RPE cells) refer to epithelial cells that constitute the retinal pigment epithelium, and their progenitor cells. Whether a cell is a retinal pigment epithelial cell can be confirmed, for example, by the expression of cell markers (RPE65, CRALBP, MERTK, BEST1, etc.) or by the cell morphology (intracellular melanin pigment deposition, polygonal and flattened epithelial-like cell morphology, formation of polygonal actin bundles, etc.). Furthermore, progenitor cells of retinal pigment epithelial cells refer to cells that have been directed towards differentiation into retinal cells, and whether a cell is a progenitor cell can be confirmed by the expression of cell markers (Mitf (pigment epithelial cells, pigment epithelial progenitor cells), Pax6 (pigment epithelial progenitor cells), Rx (retinal progenitor cells), OTX2 (retinal progenitor cells), RPE65 (pigment epithelial cells), BEST1 (pigment epithelial cells)). In addition, the function of retinal pigment epithelial cells can be evaluated, for example, by indicators such as the secretion capacity and phagocytic capacity of cytokines (VEGF, PEDF, etc.). These functional evaluation and verification operations can be carried out by a person skilled in the art, by setting appropriate conditions.

[0011] RPE cells can be obtained from any animal that possesses RPE cells (e.g., humans), or by differentiating them from pluripotent stem cells using known methods. However, cells differentiated from pluripotent stem cells are preferred because they allow for a sufficient or appropriate supply of cells depending on the disease. Pluripotent stem cells are not particularly limited as long as they are stem cells that possess pluripotency, the ability to differentiate into all cells present in the body, and also have proliferative capacity. Examples include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). The preferred pluripotent stem cell is iPS cell. The origin of the pluripotent stem cells is not particularly limited; for example, any animal from which the establishment of any of the following pluripotent stem cells has been reported, preferably mammals, more preferably humans, mice, rats, etc., most preferably humans.

[0012] iPS cells are artificial stem cells derived from somatic cells that possess characteristics nearly identical to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal, and can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein (K. Takahashi and S. Yamanaka (2006), Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO2007 / 069666).

[0013] As used herein, the term "somatic cells" refers to all animal cells (preferably mammalian cells, including human cells) except germline cells such as oocytes and oocytes, or totipotent cells. Somatic cells include, non-limitingly, fetal somatic cells, neonatal somatic cells, and mature, healthy, or diseased somatic cells, as well as primary cultured cells, passaged cells, and established cell lines. Specifically, examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0014] Reprogramming factors may consist of genes specifically expressed in ES cells, their gene products or non-coding RNAs, genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or non-coding RNAs, or small molecule compounds. Examples of genes included in reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al.(2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. Examples of combinations are given in the following publications: 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, and Maekawa M, et al. (2011), Nature. 474:225-9.

[0015] It is possible to induce differentiation of RPE cells from iPS cells (e.g., Neurosci. Lett., 458: 126-131, 2009; PLoS One, 8: 409-412, 2011). Alternatively, methods described in WO2015 / 053375, WO2015 / 053376, WO2015 / 125941, WO2017 / 043605, etc., can be used.

[0016] The string-like aggregates of the present invention are formed by the adhesion of RPE cells to each other. In this case, cell adhesion means that RPE cells adhere to each other on a surface in the string-like aggregates of the present invention. Plane attachment means that cells adhere to each other on a surface. More specifically, plane attachment means that the proportion of the surface area of ​​one cell that is adhered to the surface of another cell is, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. The cell surface can be observed by staining with a membrane staining reagent (e.g., DiI) or by immunostaining of cell adhesion factors (e.g., E-cadherin or N-cadherin).

[0017] 2. Device for manufacturing string-like aggregates of retinal pigment epithelial cells The present invention also provides a device for producing string-like aggregates of retinal pigment epithelial cells (hereinafter referred to as the device of the present invention, or the said device). As shown in Figure 1, the device of the present invention has a base member 110, which is the main body of the device. One or more grooves 120 are provided on the upper surface 110a of the base member 110. In Figure 1, only one groove is shown for illustrative purposes, but in a preferred embodiment, as will be described later, multiple grooves are arranged adjacent to each other, and the cross-sectional shape of the grooves is V-shaped so that there is no upper surface (plane) between the grooves. The groove 120 has a cavity portion having a length corresponding to the string-like aggregate to be formed and a width corresponding to the outer diameter of the string-like aggregate. In the example in Figure 1, the bottom of the groove 120 is the cavity portion. The cavity portion is a concave shape in which retinal pigment epithelial cells seeded within the cavity portion can grow into string-like aggregates. With this configuration, as shown in Figure 1(a), when an appropriate amount of retinal pigment epithelial cells a1 are seeded and cultured in the groove 120 of the device together with a liquid culture medium (not shown), string-like aggregates A1 with a shape corresponding to the length and width of the cavity are obtained, as shown in Figure 1(b). The string-like aggregates obtained by this device have a body surface that is smoothly formed along the cavity (the lower surface of string-like aggregates A1 in Figure 1(b)) and a surface that grows freely and rises (the upper surface of string-like aggregates A1 in Figure 1(b)).

[0018] (Base component) The overall shape and dimensions of the base member are not particularly limited, and any shape and dimensions having an upper surface capable of forming one or more cavity portions capable of forming the string-like aggregates to be manufactured can be used. A plate-like shape is exemplified as a preferred shape because it does not take up unnecessary space, is easy to manufacture, and is easy to handle during aggregate production. In the example in Figure 5(a), the overall shape of the base member 110 is disc-shaped. When the base member is plate-shaped, the outer shape is not limited to a circle (i.e., disc-shaped), but may be a square or rectangle, etc. A disc-shaped shape is preferred because it can be placed in commercially available petri dishes, which are generally circular, without creating dead space. Furthermore, depending on the application, various ancillary parts may be provided, either by integral molding or by attaching separate parts. These include a wall surrounding the outer perimeter, flanges extending laterally, handles for handling, legs on the back side, markings (recessed or raised) or labels for identifying the cavity (letters, numbers, QR codes (registered trademarks), etc.), and markings (recessed or raised) or labels for determining the length of the cultured string-like aggregates.

[0019] (Grooves and cavity sections) As shown in Figure 2(a), the entire groove 120 may be a cavity portion, or as shown in Figures 2(b), (c), and (d), the bottom of the groove 120 may be a cavity portion 121, with an upper portion 122 on top of the cavity portion. In the embodiment shown in Figure 2(a), the string-like aggregates may protrude outward from the opening of the groove and bulge outwards.

[0020] As illustrated in Figures 2(b) to (d), if the internal structure of the groove 120 has a cavity portion 121 as the bottom and an upper portion 122 located above it, the cavity portion 121 and the upper portion 122 may be formed integrally with each other, as in the examples in these figures, or they may be separable into two parts, upper and lower, as illustrated in Figures 2(e) and 3 (combined from separate parts). The embodiment in Figure 2(e) is a modification of the embodiment in Figure 2(d), but the cavity portion and the upper portion may be separable in various grooves, such as in Figures 2(b) and (c). Furthermore, the separation interface does not necessarily have to coincide with the interface between the cavity portion and the upper portion; the cavity portion and the upper portion may be formed integrally, and the upper portion may be separable into two parts, upper and lower. With such a separable upper and lower structure, as illustrated in Figure 3(b), only the cavity portion 121 can be exposed, making it easier to remove the filamentous aggregate A1 after culturing. Furthermore, a structure that can be separated into two upper and lower parts offers manufacturing advantages, such as eliminating the need to form deep grooves and allowing for greater freedom in designing the groove profile.

[0021] Figure 3 shows a specific example of a device for separating the cavity portion and the upper portion. In this embodiment, as shown in the assembled state in Figure 3(a), the base portion 110 is configured to have a first base portion 111 and a second base portion 112 positioned on top of it. As shown in the separated state in Figure 3(b), the second base portion 112 can be removed from the first base portion 111. The first base portion 111 is provided with a cavity portion 121 of the groove 120, and the second base portion 112 is provided with an upper portion 122 of the groove 120. Although not shown in the figure, it is preferable that the first base portion 111 and the second base portion 112 are provided with a positioning structure so that they are connected to each other in a predetermined positional relationship (i.e., a positional relationship in which one cavity portion 121 and one upper portion 122 coincide to form one groove 120). Such a positioning structure is not particularly limited, and conventionally known positioning structures can be appropriately adopted, such as a positioning protrusion and a positioning recess that receives it, or an outer circumference shape (a shape other than a cylinder and a round hole) that fits together without misalignment.

[0022] If the base portion 110 is separable into multiple parts (for example, a first base portion 111 and a second base portion 112) as described above, the materials of each part may be different. For example, it is preferable to use a material for the first base portion 111, which is provided with a cavity, that has biocompatibility (compliant with GCP: Good Clinical Practice) that allows for the favorable growth of string-like aggregates, and that does not readily adhere to cells so that the string-like aggregates can be easily detached and removed from the cavity (such a material may also be used for the second base portion 112). Furthermore, it is preferable to use a material that can be sterilized (for example, a material that is heat-resistant enough to withstand autoclave heating, radiation-resistant enough to withstand gamma irradiation, and suitable for EOG sterilization), is inexpensive, and is easy to process for both the first base portion 111 and the second base portion 112.

[0023] (Shape of string-like aggregates) To explain the shape of the cavity, we will now describe the shape of the string-like aggregates. In the present invention, a string-like aggregate is an aggregate formed by randomly condensing RPE cells into an elongated shape to form a string-like structure. It does not need to have clear apical and basal polarity like retinal pigment epithelium in vivo, and is not particularly limited as long as it is an aggregate with sufficient strength to allow the target number of cells to be efficiently inserted and removed as a single mass using a needle or a tubular transplantation tip during transplantation. Furthermore, in the present invention, the "string-like" shape of a string-like aggregate is clearly an elongated shape with a longitudinal direction compared to the shapes seen in known cell aggregates or cell sheets, that is, a shape that extends long in one direction. As shown in Figure 4(a) as a model for explanation, this elongated shape is such that the ratio of the total length L1 to the outer diameter d1 of the body (L1 / d1) when the string-like aggregate is straightened is 2 or more, more preferably 5 or more, even more preferably 10 or more, even more preferably 20 or more, and particularly preferably 50 or more. If the shape has the aforementioned ratio (L1 / d1), particularly the lower limit ratio of 2, then the orientation (i.e., the longitudinal direction as a string-like aggregate) can be defined more precisely than in a simple cell aggregate. In other words, in tissues where orientation is important, appropriately defining the orientation in advance has the effect of obtaining a more desirable tissue. Furthermore, if the string-like aggregates have the aforementioned ratios, they can be transported as a single elongated structure by passing through the inside of a syringe needle, and the effect of efficiently transporting and positioning cells over a wide area in a single operation can be more pronounced. Moreover, since they can pass through narrow areas (e.g., minute through-holes) that are difficult to pass through with known cell sheets, the effect of reducing invasiveness to living organisms can be more pronounced.

[0024] If the ratio (L1 / d1) falls below the lower limit 2, the aforementioned effect becomes less pronounced. Furthermore, there is no particular upper limit to the ratio (L1 / d1); for example, even if the total length L1 is longer than the area of ​​the target site to which the string-like aggregate is to be transplanted, it can be appropriately cut. In terms of avoiding the groove of the device becoming excessively long, the dimensions of the aggregate calculated from the number of cells to be transplanted, and transport limitations (e.g., syringe needle length), an example upper limit for the ratio (L1 / d1) is approximately 1000, more preferably approximately 200, and particularly preferably approximately 100. The lower and upper limits of the ratio (L1 / d1) can be freely selected and combined to define the range of the ratio, for example, ratio (L1 / d1) = 2~1000, 5~1000, 10~1000, 20~1000, 50~1000, 10~200, 20~200, 50~200, 2~100, 5~100, 10~100, 20~100, or 50~100. In the embodiment of the present invention, the ratio (L1 / d1) in the string-like aggregates obtained by the fabricated device was approximately 2~100. The lower limit of the ratio is the minimum value required for the aggregates to be in the shape of a "string," but the upper limit of the ratio is not particularly limited and can be appropriately determined according to space limitations in the culture facility, the range of the outer diameter d1, the effort required to divide the cells into lengths suitable for cell transplantation, etc.

[0025] (Outer diameter d1 of the body of the string-like aggregate) When string-like aggregates are produced using this device, the cross-sectional shape of the string-like aggregate (the shape of the cross-section when the string-like aggregate is cut perpendicular to its longitudinal direction) is as illustrated in Figures 2(a) to (d), with the lower part following the cavity during cultivation and the upper part growing freely. Therefore, the outer diameter d1 of the string-like aggregate body is indeterminate depending on the direction of measurement. While the average value of the outer diameter measured in multiple directions may be used, it is convenient to observe the string-like aggregate together with the device while it is growing in the cavity (i.e., observe the inside of the groove from above in Figure 4(b)) and use the width dimension of the body visible at that time as the outer diameter d1 of the string-like aggregate. Furthermore, in such observation, the string-like aggregate becomes linear along the cavity, making it easy to measure the total length L1.

[0026] (Cross-sectional shape of string-like aggregates) The cross-sectional shape of the string-like aggregates is not particularly limited and is usually irregular. However, from the viewpoint of nutrient and oxygen supply and waste removal, it is desirable for the distance from the tissue surface to the tissue interior to be constant. Furthermore, a shape close to a circle or ellipse is preferable because it allows for smoother passage through narrow areas such as syringe needles, and a more smoothly curved cross-sectional shape of the groove cavity is advantageous for cell proliferation and detachment.

[0027] (Example dimensions of string-like aggregates when applied to living organisms) As mentioned above, there is no limit to the length of the string-like aggregates, and if they are excessively long, they may be cut as appropriate when applied to a living organism. Preferred dimensions when applied to a living organism include, for example, a total length L1 of approximately 20 μm to 200 mm, preferably 20 μm to 40 mm, more preferably 1 mm to 40 mm, and even more preferably 5 mm to 40 mm. An example of a length suitable for a 24-gauge needle is approximately 10 mm to 20 mm. There is not just one most preferred range or value for these total length L1 values; a preferred range or value can be appropriately selected depending on the size of the device, the allowable groove length, the required number of cells, and the length of the conduit for the transplantation instrument (e.g., the injection needle). Furthermore, the outer diameter d1 can range from approximately 10 μm (outer diameter corresponding to approximately one cell) to approximately 300 μm (outer diameter corresponding to the inner diameter of a 24-gauge needle), preferably 20 μm to 300 μm, and more preferably 100 μm to 250 μm. There is no single most preferable range or value for these outer diameter d1 values; rather, a preferred range or value can be appropriately selected depending on the size of the device, the allowable inner diameter of the groove cavity, the required number of cells, and the inner diameter of the conduit for the transplantation instrument (e.g., the injection needle).

[0028] When an injection needle is inserted into the eyeball and the string-like aggregate is delivered through the needle to a target site such as the fundus region for implantation, the preferred inner diameter of the injection needle used is approximately 10 to 300 μm. In this case, the outer diameter d1 of the string-like aggregate that can move favorably within the injection needle is, for example, approximately 80 to 95%, preferably 90%, of the inner diameter of the injection needle. Therefore, in such cases, the preferred dimensional range for the outer diameter d1 of the string-like aggregate is approximately 100 μm to 270 μm. The preferred length L1 of the string-like aggregate in this case is approximately 20 μm to 200 mm, preferably 20 μm to 40 mm, more preferably 1 mm to 40 mm, and even more preferably 5 mm to 40 mm, as it is effective for implantation and is suitable for the length of the injection needle. An example of a length suitable for the length of a 24-gauge needle is approximately 10 mm to 20 mm. Therefore, when delivering string-like aggregates by injection needle, a preferred ratio (L1 / d1) is 40 to 200, preferably around 80. The cell count at that time is 1.5 × 10⁻⁶. 5 ~4.5×10 5 The degree is illustrated by an example.

[0029] As described above, the string-like aggregate of the present invention can be characterized by the ratio (L1 / d1) of the total length L1 to the outer diameter d1 of the body. Therefore, the string-like aggregate of the present invention may have the characteristic that the ratio (L1 / d1) of the total length L1 to the outer diameter d1 of the body is between 2 and 1,000. In addition, L1 / d1 may usually be between 2 and 1,000, but may also be between 5 and 1,000, 10 and 1,000, 20 and 1,000, 50 and 1,000, 10 and 200, 20 and 200, 50 and 200, 2 and 100, 5 and 100, 10 and 100, 20 and 100, or 50 and 100.

[0030] The string-like aggregates of the present invention can be further characterized by their cross-sectional shape. As described above, the cross-sectional shape of the string-like aggregates is not particularly limited and is usually irregular, but a shape close to a circle or ellipse is preferred. Therefore, the string-like aggregates of the present invention may further have the characteristic of having a circular or elliptical cross-sectional shape.

[0031] (Cross-sectional shape of the cavity) The cavity portion within the groove of the device preferably has a cross-sectional shape and length corresponding to the shape of the string-like aggregate described above. The cross-sectional shape of the cavity portion is the shape of the cross section when the cavity portion is cut perpendicular to the longitudinal direction of the groove. The cross-sectional shape of the cavity portion can also be appropriately determined considering cell culture properties, release properties, etc., and examples of preferred cross-sectional shapes include arc shape, semicircle shape, rectangle with an open top, U-shape, and V-shape. The bent portion (corner) in the rectangle and V-shape is preferably rounded because it facilitates the removal of the string-like aggregate using tweezers or a syringe needle, and because it is preferable to have a cross-sectional shape of the string-like aggregate that is close to circular. The radius of curvature of the rounded corner is not particularly limited, but a preferred value is about half the outer diameter d1 of the string-like aggregate (for example, a radius of about 5 μm to 150 μm). Furthermore, it is preferable that the width W1 of the cavity portion shown in Figure 4(b) is the same as the outer diameter d1 of the string-like aggregate.

[0032] (Preferred form of groove) In a preferred embodiment, as shown in Figures 2(c), (d), and 4(b), the groove has a cavity portion 121 at its bottom and an upper portion 122 above the cavity portion, the cross-sectional shape of the upper portion 122 being wider from the bottom side towards the opening side. The overall cross-sectional shape of the groove is V-shaped, and its deepest part is rounded as the cavity portion described above. This V-shaped cross-sectional shape guides cells seeded in a suspension state into the cavity portion at the bottom, making the seeding operation easier, and also makes it easier for string-like aggregates to come out of the groove, making the removal operation easier.

[0033] The overall depth of the groove (B1 shown in Figure 4(b)) is not particularly limited, but is preferably about 5 to 5000 μm (about 5 mm shorter than the depth of a typical petri dish), and more preferably about 5 to 1600 μm. The depth of the cavity (dimension b1 shown in Figure 4(b)) is preferably about 5 to 300 μm, and more preferably about 5 to 250 μm, depending on the outer diameter d1 of the string-like aggregate to be formed. The width of the groove opening (W2 shown in Figure 4(b)) is not particularly limited, but is preferably about 560 μm (outer diameter of a 24 gauge needle) to 5000 μm (tip diameter of a 1 mL pipette tip), more preferably about 1000 μm to 3000 μm (approximate value of the device), and even more preferably about 1500 μm to 2500 μm. When the overall cross-sectional shape of the groove is V-shaped as described above, the angle of inclination of the inner wall (θ1 shown in Figure 4(b)) is preferably about 1 to 90 degrees, and more preferably about 40 to 70 degrees. The overall cross-sectional shape of the groove can be appropriately determined according to the amount of cell suspension required for culture, the number of cells, the size of the container to which it will be stored, etc. The length of the groove is preferably the same as the length of the string-like aggregate to be formed, but it may be longer.

[0034] The upper part of the groove's cross-sectional shape may be a straight line as shown in Figure 4(b), or it may be a curved shape that widens from the bottom towards the opening, as shown in Figure 5(b). Such curvature can also be interpreted as the edges of a straight V-shaped opening, as shown in Figure 4(b), being rounded.

[0035] (Groove arrangement pattern) In a preferred embodiment, as shown in Figure 5, multiple grooves 120 (five in the example shown) are arranged in a parallel stripe pattern on the upper surface 110a of the base member 110. This allows multiple string-like aggregates to be obtained in a single culture, thereby improving production efficiency. The number of grooves is not particularly limited and can be appropriately determined along with the size of the base member depending on the scale of production, from experimental to commercial use. In the examples shown in Figures 5(a) and (b), the upper part of the cross-sectional shape of the groove 120 is a curved shape that widens from the bottom side towards the opening side, and is smoothly connected to the upper surface of the base member and the adjacent opening. In Figure 5(b), the boundary lines between adjacent grooves and the boundary line 130 between the groove and the upper surface 110a of the base member 110 are represented by dashed lines for clarity.

[0036] When multiple grooves are arranged in a parallel stripe pattern, in a preferred embodiment, as shown in Figure 5, the walls separating adjacent grooves do not have a flat surface on the upper surface of the base member, and the openings of adjacent grooves are in contact with each other. This configuration is preferably achieved when the overall cross-sectional shape of the grooves is V-shaped. This configuration allows for a denser arrangement of grooves. When performing culture using the device, culture may be performed by injecting culture medium and cells only into the grooves, or the entire device may be immersed in culture medium and cells may be seeded and cultured. When the entire device is immersed in culture medium and then cells are seeded, it is preferable because the seeded cells are less likely to accumulate on the flat surfaces between the grooves, and more cells fall into the grooves.

[0037] (Material of the base component) The material of the base component (i.e., the material of the device) is not particularly limited, but is preferably a material suitable for cell culture, such as a metal or polymer, that is inexpensive and easy to manufacture. Preferred materials include glass and various polymer materials (e.g., polystyrene, polycarbonate, acrylic, silicone, cycloolefin polymer (COP)), which have been conventionally used as materials for cell culture containers. Among these, polydimethylsiloxane (PDMS), a type of silicone, is a particularly preferred material because it has advantages such as being easy to mold using a mold, being heatable by autoclaving, being flexible, having high chemical resistance, and being applicable to microfabrication processes.

[0038] (Method of manufacturing the device) The manufacturing method of the device may be determined appropriately depending on the material, and various methods are available, such as plastic deformation by pressing, resin molding using a mold, cutting, 3D printing, subtractive groove formation on the upper surface of the base member, additive groove processing on the upper surface of the base member, and laser processing.

[0039] When the base material is PDMS, a preferred manufacturing method involves using a mold having a convex shape corresponding to the groove shape, pouring (or bringing into contact with the convex shape) fluid PDMS before curing into the mold, and curing the PDMS in that state to obtain the molded product (the device). PDMS hardens, for example, by heating at 80°C for about 3 hours.

[0040] (Preferred method for manufacturing molds) An example of a preferred method for manufacturing a mold is shown. Figure 6 is a diagram illustrating an example of a preferred method for manufacturing a mold. In this example of the manufacturing method, first, as shown in Figures 6(a) and (b), a projection 220, which will serve as a convex core corresponding to the shape of the groove, is formed on one surface 210a of the mold substrate 210, according to the pitch between the centers of the groove. Next, as shown in Figures 6(c) and (d), a fluid, uncured polymer material (e.g., PDMS) is applied to cover the protrusions 220 and cured to obtain a V-shaped convex mold 240. Here, when applying the polymer material, the fluidity, viscosity, surface tension, etc., of the polymer material are used to shape the cross-sectional shape of the coating layer 230 into a V-shaped convex mold corresponding to the shape of the groove to be molded, as shown in Figure 6(c). This makes it easy to obtain a V-shaped convex mold from a core with a simple shape.

[0041] Examples of conditions for controlling the liquid polymer raw material into a V-shape as shown in Figure 6(c) include, for example, applying uncured PDMS with a viscosity of about 3.5 (Pa·s) at 25 degrees Celsius to cover the protrusions, letting it stand for 30 minutes in a vacuum desiccator at room temperature (about 23-27°C) while degassing, and then curing it at 80°C for 3 hours.

[0042] In the embodiments of the present invention, the parts shown in Figures 6(a) and (b) (a mold substrate 210 with protrusions 220) were manufactured by a 3D printer (the material being polylactic acid (PLA resin)). However, a mold 200 with a V-shaped convex mold 240, as shown in Figure 6(c), may also be manufactured by a 3D printer, and the surface may be further finished.

[0043] Figure 7 is a cross-sectional view illustrating the process of molding the device using the mold described above. As shown in Figure 7(a), the mold 200 is placed on the bottom surface of a molding container (not shown), or the molding container is formed by surrounding the mold 200 with wall members, and a polymer raw material with fluidity before curing is poured into it. The polymer raw material is then cured to obtain a molded product (the device) 100. Next, as shown in Figure 7(b), the molded product is peeled off the mold 200 to obtain the device 100. Figure 8(a) is a photograph showing the substrate surface of a mold manufactured in an embodiment of the present invention, with a projection 220 provided on one main surface of the mold substrate 210. Figure 8(b) is a photograph showing the substrate surface of a mold manufactured in an embodiment of the present invention using the mold shown in Figure 8(a), with a projection 220 provided on one main surface of the mold substrate 210.

[0044] 3. Method for producing string-like aggregates of retinal pigment epithelial cells The present invention also provides a method for producing string-like aggregated retinal pigment epithelial cells (hereinafter referred to as the "production method of the present invention"). The production method of the present invention includes the following steps. (1) A step of seeding retinal pigment epithelial cells a1 suspended in a liquid culture medium into the groove 120 of the device 100 of the present invention, as illustrated in Figure 9(a). (2) As illustrated in Figure 9(a), the process involves culturing retinal pigment epithelial cells a1 to form filamentous aggregates A1 of retinal pigment epithelial cells (the figure shows one filamentous aggregate being removed from the groove).

[0045] In step (1), retinal pigment epithelial cells suspended in a culture medium are seeded into the grooves of the device of the present invention.

[0046] In step (1), the liquid medium in which the retinal pigment epithelial cells are suspended can be prepared using a basal medium that is commonly used for culturing animal cells. Examples of basal media 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, Ham medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof, which are media that can be used for culturing animal cells. The culture medium may be serum-containing medium or serum-free medium.

[0047] In this specification, serum-containing medium means a medium containing unprocessed or unpurified serum. Examples of serum-containing mediums include those containing unprocessed or unpurified serum at a concentration of 5% or less, preferably 3% or less. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvate, buffers, inorganic salts, etc.

[0048] In this specification, serum-free medium means a medium that does not contain unprocessed or unpurified serum. In the present invention, a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) is also included in serum-free medium as long as it does not contain unprocessed or unpurified serum.

[0049] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Commercially available serum substitutes may also be used. An example of such a commercially available serum substitute is Knockout.TM Serum Replacement (manufactured by Life Technologies; now ThermoFisher; hereafter sometimes referred to as KSR), Chemically-defined Lipid concentrated (manufactured by Life Technologies), Glutamax TM Examples include (manufactured by Life Technologies), B27 (manufactured by Life Technologies), N2 supplement (manufactured by Life Technologies), and ITS supplement (manufactured by Life Technologies).

[0050] The serum-free culture medium used in the culture may, as appropriate, contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, etc.

[0051] The culture medium in which the retinal pigment epithelial cells are suspended in step (1) may further contain a ROCK inhibitor.

[0052] In this invention, a ROCK inhibitor is not limited to any substance that inhibits the action of Rho kinase (ROCK). Rho kinase (ROCK) was discovered as a serine / threonine kinase located downstream of the low molecular weight G protein Rho. The Rho / ROCK signaling pathway is involved in various cellular functions, such as the actin cytoskeleton and cell adhesion. In the subculturing of iPS cells, it is necessary to disperse the cells, but it is known that these stem cells undergo apoptosis (programmed cell death) when cultured in a dispersed state. The Rho / ROCK signaling pathway is involved in apoptosis caused by dispersion, and it has been reported that apoptosis is suppressed by adding a ROCK inhibitor (apoptosis inhibitory effect). Furthermore, it has been reported that adding a ROCK inhibitor when cryopreserving cells increases the cell viability after thawing (cell viability improvement effect). Based on these reports, ROCK inhibitors have come to be added during the proliferation culture of iPS cells. On the other hand, while regenerative medicine products typically use basic media such as equilibrium salt solutions or DMEM / F12 medium and do not contain exogenous components, the inventors have discovered that, in addition to the effects described above, using an appropriate concentration of ROCK inhibitor to produce filamentous aggregates, as shown in the examples described later, makes the filamentous aggregates more stable and further promotes the engraftment of RPE cell filamentous aggregates at the transplant site and the germination of cells, thus exhibiting a new effect.

[0053] In the present invention, the ROCK inhibitor may be any molecule, as long as it has an effect that is identical or substantially identical to the newly discovered effect described above (an effect that promotes the formation of string-like aggregates). "Substantially identical" means that their effects are qualitatively (e.g., physiologically or pharmacologically) the same. Therefore, it is preferable that the effects are equivalent, but the degree of these effects (e.g., about 0.1 to about 10 times, preferably about 0.5 to about 2 times) may differ. The effects can be measured according to methods that are already known. Examples of such ROCK inhibitors include Y-27632 dihydrochloride, Y-27632, Fasudil Hydrochloride, Chroman 1, SLx-2119, HSD1590, GSK269962A hydrochloride, Exoenzyme C3, Clostridium botulinum, Ripasudil, Afuresertib, Thiazovivin, GSK269962A, RKI-1447, Y-33075, GSK429286A, AT13148, H-1152 dihydrochloride, Y-33075 dihydrochloride, LX7101, SAR407899, ROCK-IN-2, and Afuresertib. Examples include hydrochloride, Hydroxyfasudil, GSK180736A, BDP5290, SR-3677, CCG-222740, CMPD101, Rho-Kinase-IN-1, SAR407899 hydrochloride, ROCK inhibitor-2, ZINC00881524, H-1152, Hydroxyfasudil hydrochloride, Fasudil, ROCK2-IN-2, Verosudil, SB-772077B dihydrochloride, GSK-25, CRT0066854 hydrochloride, Ripasudil free base, ROCK-IN-1, etc. Preferably, Y-27632 dihydrochloride and Y-27632 are used.

[0054] A ROCK inhibitor can be produced by a method known per se. Commercially available products can also be purchased and used as the ROCK inhibitor. For example, Y-27632 can be purchased from FUJIFILM Wako Pure Chemical Corporation, etc. Ripasudil is commercially available under the brand name Glanatec (registered trademark) (Kowa), and Fasudil Hydrochloride is commercially available under the brand name Eril (registered trademark) (Asahi Kasei Pharma), etc.

[0055] The concentration of the ROCK inhibitor contained in the medium is not particularly limited as long as it allows RPE cells to form string-shaped aggregates in the grooves of the device of the present invention, and is usually 0 μM to 20 μM, preferably 2 μM to 10 μM. If the concentration of the ROCK inhibitor is too high, the aggregates of the present invention become excessively hard and aggregated, which may raise a concern of affecting the spreading of cells after transplantation.

[0056] In step (2), the retinal pigment epithelial cells seeded in step (1) are cultured.

[0057] In step (2), the density of the retinal pigment epithelial cells cultured in the grooves is not particularly limited as long as the retinal pigment epithelial cells form string-shaped aggregates in the grooves of the device of the present invention, and is usually 2.5×10 3 cells / mL or more, preferably 2.5×10 3 cells / mL to 5×10 5 cells / mL, more preferably 1×10 5 cells / mL to 2×10 5 cells / mL. If the cell density in the grooves is higher than this range, the outer diameter of the body of the formed string-shaped aggregate increases, which raises concerns such as failure of the transplanted cells to spread after transplantation, and formation of multilayers by the transplanted cells at the surgical site.

[0058] The culture time for the retinal pigment epithelial cells in step (2) is not particularly limited as long as string-shaped aggregates of retinal pigment epithelial cells can be obtained in step (2), and is usually 1 day to 30 days, preferably 2 days to 7 days. If the culture period is longer or shorter than this range, there is a concern that the aggregates of the present invention may detach from the device of the present invention during medium exchange.

[0059] The culture conditions in step (2), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0060] 4. Treatments for diseases based on retinal tissue damage The string-like aggregates of retinal pigment epithelial cells obtained as described above are useful in transplantation therapy for diseases based on damage to retinal tissue, such as diseases based on damage to the retinal pigment epithelium. Therefore, the present invention provides a therapeutic agent for diseases based on damage to the retinal pigment epithelium (the therapeutic agent of the present invention) that includes string-like aggregates of retinal pigment epithelial cells.

[0061] The therapeutic agent of the present invention comprises an effective amount of filamentous aggregates of retinal pigment epithelial cells and a pharmaceutically acceptable carrier. The filamentous aggregates of retinal pigment epithelial cells contained in the therapeutic agent of the present invention may be those produced by the production method of the present invention.

[0062] Acceptable carriers for pharmaceuticals include physiological aqueous solvents (such as physiological saline, buffer solutions, and serum-free culture media). If necessary, preservatives, stabilizers, reducing agents, isotonic agents, etc., commonly used in transplant medicine, may be added to the pharmaceutical product containing the tissue or cells to be transplanted.

[0063] The therapeutic agent of the present invention can be prepared as a suspension by suspending filamentous aggregates of retinal pigment epithelial cells in a suitable physiological aqueous solvent. If necessary, a cryopreservative may be added, the suspension may be frozen for storage, thawed before use, washed with a buffer solution, and used in transplant medicine.

[0064] The string-like aggregates of retinal pigment epithelial cells of the present invention can be used as a therapeutic agent for diseases based on damage to the retinal pigment epithelium, or to replenish the affected atrophied / damaged areas in cases of retinal pigment epithelial atrophy or damage. By transplanting the string-like aggregates of retinal pigment epithelial cells of the present invention into patients with diseases based on damage to the retinal pigment epithelium, or in cases of retinal pigment epithelial atrophy or damage requiring transplantation, the retinal pigment epithelium can be replenished, thereby treating diseases based on damage to the retinal pigment epithelium, or in cases of retinal pigment epithelial atrophy or damage. Examples of diseases based on damage to the retinal pigment epithelium or in cases of retinal pigment epithelial atrophy or damage include ophthalmic diseases such as age-related macular degeneration, retinitis pigmentosa, and retinal pigment epithelial tears.

[0065] The content of filamentous aggregates of retinal pigment epithelial cells in the therapeutic agent of the present invention is not particularly limited as long as the suspension (e.g., 50-500 μL, preferably 100-300 μL) injected into the disease site, i.e., the site of retinal pigment epithelial defect in macular degeneration or retinitis pigmentosa, contains filamentous aggregates of retinal pigment epithelial cells containing a therapeutically effective amount of RPE cells. For example, the suspension of filamentous aggregates of retinal pigment epithelial cells can be injected so that the RPE cell content is 100-20,000 cells / μL, preferably 1,000-10,000 cells / μL. The therapeutic agent of the present invention does not involve the difficulty of controlling cells at the transplantation site, as is the case with RPE cell suspensions, and the transplanted cells do not form an epiretinal membrane (ERM) in the vitreous humor. Furthermore, the therapeutic agent of the present invention does not require the time and cost of preparing sheets, as is the case with RPE sheets, and does not require invasive surgical procedures involving large incisions.

[0066] The therapeutic agent of the present invention can be implanted by injecting it subretinate into mammals (e.g., humans, mice, rats, preferably humans) with retinal diseases such as macular degeneration (e.g., age-related macular degeneration, retinitis pigmentosa, etc.) using an implantation device including a suitable syringe and needle (e.g., MedOne0® Poly Tip® Cannula 25g / 31g, etc.). As a medium used when implanting the therapeutic agent of the present invention, a medium obtained by diluting a viscoelastic substance (such as Shellgun or Viscoat) 4-8 times with OptiMEM can be used. The use of an implantation medium allows for smooth aspiration and dispensing of the string-like aggregates of retinal pigment epithelial cells of the present invention.

[0067] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0068] Experimental Procedure All animal experiment protocols were approved by the Animal Care Committee of the RIKEN Biosystems Dynamics Research Center (BDR) and conducted in accordance with local guidelines and the ARVO statement on the use of animals in eye and vision research. hiPS cells were prepared with the approval of the Ethics Committee of the RIKEN Biosystems Dynamics Research Center (BDR) and with informed consent from volunteers.

[0069] Example 1 Fabrication of a PDMS-based culture device for the production of string-like aggregates of hiPSC-RPE A PDMS-based culture device with a groove measuring 19.5 mm in length, 1 mm in width, and 1.6 mm in depth was fabricated. The fabrication process for this device is summarized below. First, a mold for the device was fabricated using polylactic acid (PLA) filament 3D printing. The 3D printed mold was trimmed, PDMS was applied to adjust the groove shape, and then a release agent (Novec 1720, manufactured by Sumitomo 3M) was applied to the mold. PDMS and a PDMS hardener (Cypot 184 W / C) (manufactured by Dow Corning Toray) were mixed in a 10:1 ratio and poured onto the 3D printed mold, and degassed for approximately 1 hour. Next, the 3D printed mold and PDMS mixture were placed in an 80°C oven for 3 hours. The hardened PDMS was recovered from the 3D printed mold and trimmed. The radius of curvature of the groove bottom was measured using a confocal scanning laser microscope (KEYENCE, VK-8710) and found to be R 0.2 mm (Figure 10).

[0070] Preparation of human iPSCs (hiPSCs) and hiPSC-derived RPE cells (hiPSC-RPEs) In our laboratory, we established the M8 human iPSC strain by introducing six reprogramming factors (OCT3 / 4, SOX2, KLF4, L-MYC, LIN28, and the p53 carboxy-terminal dominant-negative fragment) into peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Simply put, we electroporated PBMCs with episomatic vectors (pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1, purchased from Addgene), and then extracted iPSC-like colonies as previously described (Okita et al., 2011). After several passages, we performed PCR analysis using DNA extracted from the iPSC-like cells and confirmed that no residual episomatic vectors could be detected using plasmid-specific primers. We obtained the 201B7 strain and the 253G1 hiPSC strain (Nakagawa et al., 2008; Takahashi et al., 2007) from the RIKEN BioResource Center. To visualize the G1 phase of the cell cycle and the entire cell body, we created a modified Fucci 201B7 strain (201B7modFucci) in the inventors' laboratory. Briefly, we extracted the mCherry-hCdt1(30 / 120)-P2A-mVenus region from the tFucci(CA)2 / pCSII-EF vector (donated by Professor A. Miyawaki; https: / / cfm.brc.riken.jp / lentiviral-vectors / plasmid-list / ) and ligated it into the pAAVS1-Nst-CAG-DEST vector obtained from Addgene (Plasmid #80489). 201B7 was co-transfected with a modified Fucci construct encoding a gRNA designed to target the AAVS1 locus and a Cas9 vector (Addgene Plasmid #62988) using DNA-in CRISPR delivery medium (MTI-GlobalStem, Gaithersburg, MD) (Oceguera-Yanez et al., 2016). After transfection, mVenus-positive 201B7 colonies were dissociated, passaged, and colonies derived from a single clone were extracted. hiPSC-RPE cells were differentiated using the SFEBq method (Kuwahara et al., 2015, 2019; Nakano et al., 2012). Briefly, 5 μM SB431542 (Sigma-Aldrich) and 300 nM SAG (Enzo Life Sciences, Inc.) were added to the iPS cells the day before differentiation induction. On day 0, iPS cells were cultured in growth factor-free chemically defined medium (gfCDM) suspended with IMDM:F12 (1:1, Life Technologies), 10% Knockout Serum Replacement (KSR), 1% Chemically Defined Lipid Concentrate (Life Technologies), 30 w / v % BSA (fatty acid-free), 450 μM monothioglycerol, 30 nM SAG, 20 μM Y-27632 (Fujifilm Wako Pure Chemical Corporation), and penicillin-streptomycin, in PrimeSurface 96V (Sumitomo Bakelite Co., Ltd.). On day 6, 1.5 nM BMP4 was added, and half of the gfCDM was replaced every 3 days. On day 18, cells were transferred to an Ultra Low Culture Dish (Corning Incorporated) using DMEM / F12-Glutamax medium containing 1% N2 supplement (Life Technologies), 3 μM CHIR99021 (Stemgent, Cambridge), and 5 μM SU5402. From day 22 onward, cells were cultured in DMEM / F12-Glutamax medium (Life Technologies) containing 1% N2 supplement, 10% fetal bovine serum (Biosera), 0.5 μM retinoic acid (sigma), 0.1 mM taurine (sigma), and 1x antibiotic antifungal agent (gibco). Between days 30 and 60, pigmented colonies were picked and plated in a 1:1 mixed medium of "RPE Adhesion Medium" and "RPE Maintenance Medium" (hereinafter referred to as "Mixed Medium") on 12-well plates coated with iMatrx511. "RPE Adhesion Medium" contained DMEM / F-12 (Sigma-Aldrich), 10% fetal bovine serum (SAFC Biosciences Inc.), and gentamicin solution (Sigma-Aldrich). "RPE Maintenance Medium" contained DMEM-low glucose (Sigma-Aldrich), 30% F-12 (Sigma-Aldrich), 2% L-glutamine solution (Sigma-Aldrich), and 2% B-27 TM The culture medium included supplement (50X) (Thermo Fisher Scientific Inc.) and gentamicin solution (Sigma-Aldrich). After cell attachment, the medium was replaced with RPE maintenance medium containing 10 ng / mL basic fibroblast growth factor (bFGF). After one passage, the cell culture medium was spread into RPE maintenance medium and the cells were stocked at -150°C using a STEM-CELL BANKER (Nippon Zenyaku Kogyo Co., Ltd.). In each experiment, stock cells were thawed, seeded in mixed medium, and then the medium was replaced with RPE maintenance medium supplemented with 10 ng / mL bFGF and 0.5 μM SB431542. The medium was changed every few days until the cells were used. RPE cells differentiated from strain 253G1 were prepared as described in Kitahata et al., 2019.

[0071] Preparation of string-like aggregates After thawing, hiPSC-RPE cells were cultured for two weeks, then collected and seeded in 15 μl RPE maintenance medium containing the optimal concentration of Y-27632 (at the concentration indicated in each experiment) in the cell counts described in each experiment, into each groove of a PDMS-based culture device.

[0072] Analysis of the expanded area of ​​RPE from plated string-like aggregates The filamentous aggregates were released from the device grooves and seeded onto an uncoated 24-well plate (Corning Incorporated) containing mixed culture medium. After the filamentous aggregates adhered (2-3 days later), the medium was replaced with RPE maintenance medium (containing bFGF and SB431542). The culture dish was imaged using the IncuCyte Zoom system (Essen BioScience). From the obtained image data, the expanded cells were manually marked using Fiji, and the expanded area was measured.

[0073] immunohistochemistry Cultured filamentous aggregates or cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. The transplanted eyes were fixed with 4% paraformaldehyde at 4°C for 1 hour, and the cornea was removed after fixation. Samples for frozen sections were immersed in 30% sucrose for more than 1 day, frozen in OCT compound (Sakura Finetek Japan Co., Ltd.), and 10 μm thick cryo-sections were prepared. The samples were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 30 minutes, blocked with Blocking One (Nacalai Tesque) at room temperature for 1 hour, and incubated overnight at 4°C with primary antibody diluted with antibody diluent (Dako). The antibodies used are listed in Table 1. Secondary antibodies were treated with secondary antibody at room temperature for 1 hour, and images were obtained using a confocal microscope (LSM700; Carl Zeiss) and a fluorescence microscope (BZ-X810; KEYENCE).

[0074] [Table 1]

[0075] PCR of RPE marker gene Total RNA was extracted from cells using the RNeasy Micro Kit (Qiagen), and cDNA was synthesized using the SuperScript III Reverse Transcriptase Kit (Invitrogen). The sequences of the RPE markers (BEST1, RPE65, CRALBP) and housekeeping gene (GAPDH) primers are shown in Table 2. PCR reactions were performed using Blend Taq-Plus- (TOYOBO Co.,Ltd.). The thermal cycling conditions were as follows: 32 cycles were performed with 180 seconds per cycle at 94°C, denaturation at 94°C for 30 seconds, priming at 58°C for 30 seconds, and extension at 72°C for 60 seconds, followed by one cycle of 60 seconds at 72°C.

[0076] [Table 2]

[0077] In vitro RPE injury model RPE cells cultured at confluence in 24-well plates were treated with 6 μg / mL Mitomycin C (Kyowa Hakko KIRIN), and the following day, a portion of the RPE cells were removed by scraping with a cell scraper approximately 6 mm wide. Cell suspension (2 x 10 5 ) or filamentous aggregates were plated ("transplanted") on or into the damaged portion of the RPE. Cells were maintained in 0.5 ml of medium and incubated for 24 hours before medium change, and cells were harvested for ELISA at the time of each intervention. The experimental design schedule and timing of medium collection are shown in Figure 11A.

[0078] ELISA for VEGF and PEDF ELISA for VEGF and PEDF was performed according to the manufacturer's protocols for the VEGF Human ELISA Kit (Life Technologies) and the Human PEDF ELISA Kit (Biovendor), respectively.

[0079] transplantation into rat eyes Immunodeficient F344 / NJc1-rnu / rnu female nude rats (6 weeks old) were obtained from CLEA Japan. A disposable micropipette (Drummond, 1-000-0500) was pulled with a micropipette puller (Sutter, P-97 / IVF Puller), and the tip was cut and sharpened using a microgrinder (Narishige, EG-400). The micropipette was then attached to an electrode holder (WPI, MPH310) on a 6.3 mm electrode handle (WPI, 2505) and connected to a 10 μl microsyringe (Hamilton, 1701LT) using an extension tube. The animals were anesthetized by inhalation of 5% isoflurane, and the pupils were dilated with 0.4% tropicamide. String-like aggregates were loaded into the micropipette and implanted subretinally.

[0080] Transplants into rabbit eyes kbl:JW rabbits (9 weeks old) were obtained from Oriental Yeast Co., Ltd. (Tokyo). The animals were anesthetized by intramuscular injection of 60 mg / kg ketamine and 10 mg / kg xylazine. After routine vitrectomy with posterior vitreous detachment, the filamentous aggregates were loaded into a 24-gauge indwelling cannula (TOP, SS-6) with a custom-made flat-cut 25G blunt needle core. After preparing a localized retinal detachment with a PolyTip cannula 25g / 38g (MedOne, 25g / 38g), the graft was slowly injected into the detached retinal bleb using a 50 μl microsyringe (Hamilton, 1705LT). Perfluorocarbon liquid (Alcon, 8065900111) was then injected onto the detachment to compress and adhere the retina, followed by fluid-gas exchange.

[0081] result Preparation of string-like aggregated hiPSC-RPE A scheme for producing filamentous aggregates using a PDMS-based culture device was summarized. First, human iPSC-RPE cells (253G1 strain) were suspended in "RPE maintenance medium," "RPE cell sheet medium (F10 medium containing 10% fetal bovine serum)," or "mixed medium" containing 10 μM Y-27632, and seeded in each furrow. The filamentous aggregates appeared to break down easily in the sheet medium, but the filamentous aggregate structure was well maintained in the maintenance medium and mixed medium (Figures 12A and B). Next, different numbers of cells (4.5 × 10⁶) were used. 5 , 1.5×10 5 , 5×10 4 ) was sown, and it was observed that string-like aggregates could be formed under the former two conditions (Figure 12C). In the example, 4.5 x 10 5 The hiPSC-RPE cells, which had been seeded with cells, were detached from the groove and loaded into a 24G intravenous cannula.

[0082] Optimization of Y-27632 concentration for string-like aggregates Next, 4.5x10 5 Cells and 1.5 x 10 5 Stringy aggregates formed by cells were plated into wells. Both starting cell counts of stringy aggregates show expansion of RPE cells from the stringy aggregates onto an uncoated plate, 1.5 x 10⁶. 5 The string-like aggregates formed by the cells still retained most of their initial shape after two weeks (4.5 x 10⁻⁶). 5 Compared to string-like aggregates formed from cells, the initial shape was lost more quickly (Figures 13A and 12D). Therefore, the inventors increased the number of starting cells to 1.5-2 x 10⁶. 5 Fixed to cells. String-like aggregates (1.5 x 10) 5 When the cells were fragmented, most of the initial stringy aggregate morphology was lost (Figure 13A'). In the inventors' preliminary experiments (Figure 14A), the concentration of Y-27632 appeared to affect the texture of the filamentous aggregates, with higher concentrations resulting in tighter filamentous aggregates. The inventors tested Y-27632 concentrations of 0, 1, 2, 2.5, and 10 μM in filamentous aggregate formation. hiPSC-RPEs (M8 strain) were able to form filamentous aggregates at concentrations of 2 μM and above, but filamentous aggregates were not well formed with 0 and 1 μM of Y-27632. Filamentous aggregates formed with 10 μM of Y-27632 easily detached after being placed on uncoated wells, took longer to adhere to the 24-well plate, and expanded more slowly compared to filamentous aggregates formed with 2-2.5 μM of Y-27632 (Figures 14B and C). Next, the inventors directly compared filamentous aggregates prepared with either 2.5 μM or 10 μM Y-27632 (Figure 13B-G). The filamentous aggregates formed with 2.5 μM Y-27632 appeared flat and loose. On the other hand, the filamentous aggregates formed with 10 μM Y-27632 appeared more compact (Figure 13B). Sections of filamentous aggregates were stained for the gap junction marker Z0-1, the polarity marker Ezrin (apical), laminin, and collagen type IV (basal) (Figure 13C). Under both conditions, the gap junction marker ZO1 was expressed between cells, the basal marker laminin was distributed throughout the cell surface, and the apical marker Ezrin appeared to be localized to the surface of the fragmented cell aggregates. Collagen IV, another basal marker, was not clearly expressed, and the cell polarity was not clearly determined in these filamentous aggregates. Next, using filamentous aggregates from day 2 (Figures 13D and E) and day 3 (Figures 13F and G), we compared the cell expansion after plating to uncoated wells between filamentous aggregates formed with 2.5 μM and 10 μM Y-27632. Filamentous aggregates formed with 2.5 μM Y-27632 tended to adhere to the plate and begin expanding early after placement. On the other hand, filamentous aggregates formed with 10 μM Y-27632 tended to become coil-like, settled down, and remained mobile for several days before beginning to expand.

[0083] Reproducibility of string-like aggregate formation using a different hiPSC-RPE strain. The inventors tested whether other hiPSC strains could similarly form filamentous aggregates. They induced differentiation of RPE using the 201B7modFucci strain and compared it with hiPSC(M8)-RPE. While hiPSC(201B7modFucci)-RPE also formed filamentous aggregates similarly to hiPSC(M8)-RPE, the 201B7modFucci strain required a Y-27632 concentration of 5 μM or higher to stably form filamentous aggregates (Figure 15-1A). The optimal Y-27632 concentration for the adhesion / expansion assay was also 5 μM for hiPSC(201B7modFucci)-filamentous aggregates, indicating that the optimal Y-27632 concentration for the desired adhesion / expansion effect differed depending on the cell line used, requiring optimization (Figures 14D and E). Cells expanded from filamentous aggregates of both hiPSC strains exhibited the characteristic pigmentation and cobblestone appearance of RPE cells and expressed ZO-1 and MITF (Figure 15-1B and C). Expression of RPE-specific marker genes (BEST1, RPE65, RLBP1) was also confirmed by polymerase chain reaction (PCR) at each stage of hiPSC-RPE: before filamentous aggregate formation, during filamentous aggregate formation, and after expansion from filamentous aggregates (Figure 15-1D). RPE cells were observed to expand from filamentous aggregates in a monolayer on the plate (Figure 15-1E). To see if expansion from filamentous aggregates was accompanied by RPE proliferation, the total number of cells 14 days after placing filamentous aggregates on the plate was measured at 2 × 10⁶. 5 The number of cells was compared to the initial number of cells in the filamentous aggregates formed on day 2 using hiPSC-RPE cells. The total number of cells in the expanded cells and the remaining filamentous aggregates averaged 5.28 × 10⁶. 5 This is the average number of cells in the day-2 stringy aggregates (1.36 × 10⁻¹⁰). 5This was approximately four times greater, indicating that RPE proliferation also contributed to cell expansion from the filamentous aggregates (day 2 filamentous aggregates n=4; expansion from hiPSC(M8)-filamentous aggregates n=6; expansion from hiPSC(201B7modFucci)-filamentous aggregates n=6) (Figure 15-2F). At the same time, the secretion of VEGF and PEDF from the expanded cells and the remaining filamentous aggregates was tested (14 days after the filamentous aggregates were placed). The enlarged hiPSC-RPE cells secreted VEGF and PEDF to similar levels compared to the cells originally used for filamentous aggregate formation. (hiPSC(M8)-RPE n=6 and hiPSC(201B7modFucci)-RPE n=6; pre-filamentous aggregate formation RPE cells of both strains as control; n=4) (Figure 15-2G).

[0084] Replacement of the RPE by mimicking hiPSC-RPE transplantation into an in vitro RPE injury model using cell suspension or string-like aggregates. To investigate how filamentous aggregates or cell transplantation can compensate for damaged REE, the inventors designed an in vitro model of hiPSC-RPE transplantation in an RPE-damaged environment, as shown in Figure 11A. Briefly, hiPSC-RPE (M8 strain) cultures in an uncoated 24-well plate were treated with MMC to inhibit cell proliferation, and the center of the wells was scratched the following day. The next day, filamentous aggregates or suspension cells (201B7modFucci strain) were placed or seeded on the scratched area, respectively, and the filling or replacement by the "transplanted" RPE cells was monitored using Incucyte (Figure 11B). In the control well (non-supply well), existing MMC-treated RPE cells gradually expanded and migrated, slowly reducing the defect area. Transplanted RPE cells in suspension rapidly occupied the defect area. When using filamentous aggregates, RPE cells expanded from the filamentous aggregates until they came into contact with existing RPE cells, but contact inhibition appeared to halt cell expansion from the filamentous aggregates. The mean areas of the defect and areas occupied by transplanted RPE cells were quantitatively measured at 1, 7, and 28 days post-in vitro transplantation in Figure 11B' (filamentous aggregates and suspension; n=5, control; n=3). In filamentous aggregates, the boundary between existing RPE cells and transplanted RPE cells was reasonably identified by mCherry expression by transplanted 201B7modFucci cells and the size of the RPE cells; MMC-treated, non-dividing RPE cells were generally larger than those expanded from transplanted RPE cells (Figure 11C). Interestingly, the gap junction marker ZO-1 was observed throughout both the existing and transplanted RPE populations without interfering across the boundary between the two populations (Figure 11D). Furthermore, VEGF (Figure 11E) and PEDF (Figure 11F) secretion were also monitored as parameters to estimate the overall RPE function in each well. VEGF secretion significantly decreased after scratching and then increased in all groups until 28 days later. In cell suspension transplantation, VEGF secretion was almost the same as before MMC treatment. PEDF secretion decreased in the transplantation group after scratching. In filamentous aggregate transplantation, PEDF secretion initially decreased after scratching, but after 28 days there was no difference compared to before MMC treatment. In cell transplantation, PEDF secretion increased significantly after scratching, but without cell replacement, PEDF secretion continued to decrease (control n=4, filamentous aggregate n=8, RPE cells n=7).

[0085] Transplantation of string-like aggregates into nude rats Next, the inventors used 2 x 10⁻¹⁶ Y-27632 in 2.5 μM or 10 μM concentrations. 5String-like aggregates formed from cells were transplanted into albino nude rats, and their surgical handling and graft survival rates were tested. The transplanted string-like aggregates were successfully and stably implanted subretinically on normal RPE (retinal pleoplasm) in the host eye (n=10, 10 μM Y-27632; n=11, 2.5 μM Y-27632). While the presence of healthy RPE appeared to inhibit the growth of the transplanted string-like aggregates, they survived stably for up to 10 months without unexpected tumor formation or graft loss (Figure 16A and Table 3). Cross-sectional images obtained using optical coherence tomography (Envisu R2200 VHR, Bioptigen, Inc.) showed clear RPE-like reflections on the graft surface (Figure 16B). Histological analysis of the same eye revealed positivity for the human marker Ku80 / XRCC5 in the pigment cell layer of the filamentous aggregates (Figures 16C and D). These cells clearly expressed apical (ezrin) and basal (collagen type IV) markers and exhibited correct polarity after transplantation (Figure 16E).

[0086] [Table 3]

[0087] Injection of string-like aggregates into rabbit eyes Finally, the practicality of the surgical technique for implanting filamentous aggregates in a clinical setting was confirmed using two rabbit eyes. Filamentous aggregates were loaded into a 24G intravenous cannula (Figure 17). After a standard vitrectomy, retinal detachment was performed, and the filamentous aggregates were successfully injected into the retinal hemorrhage area under good visual control (Figure 18).

[0088] Example 2 Fabrication of a COP-based culture device for the production of filamentous aggregates of hiPSC-RPE A COP-based culture device with grooves measuring 20 mm in length, 1 mm in width, and 2 mm in depth (with a groove bottom radius of 0.5 mm) was fabricated by cutting a cycloolefin polymer (COP) plate (manufactured by Nippon Zeon Co., Ltd.) (Figure 19).

[0089] Preparation of hiPSC-derived RPE cells (hiPSC-RPE) The M8 hiPSC strain (RIKEN) was differentiated using the SFEBq method, and the QHJI01s04 hiPSC strain (Kyoto University) was differentiated using the method described in N Engl J Med 2017;376:1038-46 to prepare hiPSC-derived RPE cells (hiPSC-RPE). The cells were then stocked at -150°C using the same method as in Example 1.

[0090] Preparation of string-like aggregates: After harvesting hiPSC-RPE cells (M8 strain, QHJI01s04 strain) cultured for 2 weeks after thawing, 2x10⁶ cells were mixed with 10 μl of maintenance medium containing 5 μM Y-27632. 5 The cells were seeded in each groove of a COP-based culture device at the specified cell count. After 2 days, the formation of string-like aggregates was confirmed.

[0091] Preparation of string-like aggregates using cells immediately after thawing. When thawed hiPSC-RPE cells were seeded into each groove of a COP-based culture device, filamentous aggregates were formed after two days. Therefore, it was possible to shorten the time from cell thawing to filamentous aggregate formation to two days.

[0092] Cryopreservation of string-like aggregates The culture medium containing the prepared filamentous aggregates was replaced with STEMCELLBANKER, placed in a cryotube, and temporarily stored at -80°C before being frozen at -150°C. After 4 months, the filamentous aggregates were thawed, suspended in a mixed culture medium, and plated onto a dish. The plated filamentous aggregates were confirmed to adhere to the dish and expand.

[0093] Transplants into the eyes of rabbits or monkeys We confirmed that the string-like aggregates could be aspirated and extruded using 25g / 31g, 25g / 33g, and 25g / 38g PolyTip cannulas (MedOne). When using viscoelastic materials (such as Shellgun and Viscoat) diluted 4-7 times with OptiMEM as a medium, the string-like aggregates could be smoothly aspirated or extruded (Figure 20). Using these methods, we confirmed that after creating detachment subretinal lesions in rabbits and monkeys using a 38g MedOne cannula, the string-like aggregates could be inserted using 25g / 31g and 25g / 33g cannulas. Similarly, after creating detachment in the eyeballs of monkeys, string-like aggregates of hiPSC-RPE cells 20mm in length were aspirated using approximately 2μl of medium and inserted into the detachment site. Postoperatively, the engraftment of the string-like aggregates was confirmed by fundus observation (Figure 21). [Industrial applicability]

[0094] The invention makes it possible to produce a transplant material of retinal pigment epithelial cells that can be prepared in a short time and at low cost, and whose transplantation method is non-invasive, while also being easily controllable at the transplantation site. This application is based on Japanese Patent Application No. 2021-078154 (filing date: April 30, 2021), the contents of which are fully incorporated herein by reference. [Explanation of symbols]

[0095] 100: Device 110: Base component 110a: Top surface of the base member 111: First base section 112: Second base section 120: Groove 121: Cavity portion of the groove 122: Top of the groove 130: Boundary line between the groove and the upper surface of the base member 200: Molding mold 210-type circuit board 210a: One side of the type substrate 220:Protrusion 230: Coating layer 240: V-shaped convex type a1: Retinal pigment epithelial cells A1: String-like aggregates b1: Depth of the cavity B1: Overall depth of the groove d1: Outer diameter of the body of the string-like aggregate L1: Total length of the string-like aggregate W1: Width of the cavity W2: Width of the groove opening X1: Cutting line extending in the width direction of the groove θ1: Angle of inclination of the inner wall of the groove

Claims

1. A string-like aggregate of retinal pigment epithelial cells, The ratio of overall length to outer diameter of the fuselage is between 2 and 1,000. A string-like aggregate formed by the surface adhesion between retinal pigment epithelial cells.

2. The string-like aggregate according to claim 1, characterized in that its cross-sectional shape is circular or elliptical.

3. The string-like aggregate according to claim 1 or 2, wherein the retinal pigment epithelial cells are cells differentiated from pluripotent stem cells.

4. A device for manufacturing the string-like aggregate described in claim 1, Having a base member, The base member has an upper surface provided with one or more grooves, The groove has a cavity portion with the length and width of the filamentous aggregate, serving as a mold for culturing seeded retinal pigment epithelial cells into filamentous aggregates. The aforementioned device.

5. The device according to claim 4, wherein the groove has a cavity portion as the bottom of the groove and an upper portion located above the cavity portion, the upper portion having an opening in the groove.

6. The device according to claim 4, wherein the cross-sectional shape of the groove is V-shaped, and the deepest part of the V-shape is rounded.

7. A method for producing string-like aggregates of retinal pigment epithelial cells, comprising the following steps; (1) A step of seeding retinal pigment epithelial cells suspended in a liquid culture medium into the groove of the device according to claim 4, (2) A step of culturing retinal pigment epithelial cells in the groove to form string-like aggregates of retinal pigment epithelial cells.

8. The method according to claim 7, wherein the culture medium contains a ROCK inhibitor.

9. The method according to claim 8, wherein the ROCK inhibitor is Y-27632.

10. The density of retinal pigment epithelial cells cultured in the groove is 2.5 x 10⁻⁶. 3 cells / mL ~ 5x10 5 The method according to any one of claims 7 to 9, wherein the amount is cells / mL.

11. The method according to any one of claims 7 to 9, wherein the culture period of retinal pigment epithelial cells is 1 to 7 days.

12. A pharmaceutical composition containing string-like aggregates of retinal pigment epithelial cells as described in claim 1 or 2.

13. A therapeutic agent for diseases based on disorders of the retinal pigment epithelium, comprising string-like aggregates of retinal pigment epithelial cells as described in claim 1 or 2.

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