Cord-like aggregates of retinal pigment epithelial cells, device and production method for producing same, and therapeutic agent comprising said cord-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

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

[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.

[0002] The use of ES / iPSC-derived retinal pigment epithelial cells (RPE) for cell-based regenerative therapy for ocular diseases has been a major focus of clinical interest in ophthalmology over the past decade (Non-Patent Document 1). These clinical studies have demonstrated the safety of this therapeutic approach, along with some potential efficacy. To date, these clinical studies have focused on two approaches: cell suspension-based approaches and RPE sheet-based approaches, each with its own advantages and disadvantages. Cell suspension-based transplantation can utilize ready-to-use cell stocks from tubes or short-term cultures and can be transplanted with minimal surgical intervention. However, controlling cell placement at the transplant site is difficult, and transplanted cells often form epiretinal membranes (ERMs) within the vitreous (Non-Patent Documents 1, 2). In contrast, RPE sheet transplantation allows visual confirmation of the targeted and precise placement of the transplanted sheet, but sheet preparation requires time, cost, and an invasive surgical procedure involving large incisions in the sclera and neural retina (Non-Patent Document 3). Therefore, the problem of providing a transplant material for retinal pigment epithelial cells that can be prepared in a short time and at low cost, that can be transplanted non-invasively, and that can be easily controlled at the transplant site remains unsolved.

[0003] Sugita, S et al., (2020) J. Clin. Med. 9, 2217.Schwartz, SD et al., (2015) Lancet 385, 509-516.Lyndon da Cruz et al., Nature Biotechnology Advance Online Publication, published online 19 March 2018.

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

[0005] We hypothesized that RPE cells, even in string-like aggregates that can be easily injected subretinal, could expand to cover a certain area, similar to small sheet transplants. Recently, the relationship between surface coverage, tissue contractility, and adhesive strength has been reported as a morphological mechanism of microtissue formation (Yamashita, T. et al., (2016). Acta Biomater. 45, 85-97). We confirmed that cells with a balanced adhesive and contractile strength spontaneously formed spheroid-like tissues on a small, concave surface. Based on this finding, we investigated the curvature of the lower edge of the concave surface and found that a grooved surface with a small curvature induced the formation of band-like tissues from cells. While the fabrication of small tissues typically required specialized techniques such as photolithography, we introduced a fabrication technique combining polydimethylsiloxane (PDMS) soft lithography and simple three-dimensional (3D) printing, making this tissue engineering approach widely applicable. In this invention, we fabricated a PDMS-based culture device with fine grooves and investigated whether hiPSC-RPE cells could form string-like aggregates. We then plated hiPSC-RPE cells on a dish, expanded them from the string-like aggregates, and confirmed whether the expanded hiPSC-RPE cells could exhibit the same RPE characteristics as before the formation of string-like aggregates. We also verified whether the hiPSC-RPE cells that formed string-like aggregates could be injected into animal eyes. Specifically, the present invention relates to the following:

[0006] [1] A string-like aggregate of retinal pigment epithelial cells. [2] The string-like aggregate of [1] above, characterized by a ratio of total length to outer diameter of body of 2 to 1,000. [3] The string-like aggregate of [2] above, characterized by a circular or elliptical cross-sectional shape. [4] The string-like aggregate of any of [1] to [3] above, in which the retinal pigment epithelial cells are cells induced to differentiate from pluripotent stem cells. [5] A device for producing the string-like aggregate of any of [1] to [4] above, comprising a base member, the base member having an upper surface with one or more grooves formed therein, the grooves having a cavity portion with the length and width of the string-like aggregate as a mold for culturing seeded retinal pigment epithelial cells into string-like aggregates. [6] The device according to [5] above, 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 to the groove. [7] The device according to [5] or [6] above, wherein the cross-sectional shape of the groove is V-shaped, and the deepest part of the V-shape is rounded. [8] A method for producing string-like aggregates of retinal pigment epithelial cells, comprising the following steps: (1) 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, and (2) culturing the retinal pigment epithelial cells in the grooves to form string-like aggregates of retinal pigment epithelial cells. [9] The method according to [8] above, wherein the medium contains a ROCK inhibitor.

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

[11] The method according to [9] above, wherein the density of retinal pigment epithelial cells cultured in the grooves is 2.5 x 10 3 cells / mL ~ 5x10 5

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

[10] above, wherein the retinal pigment epithelial cells are cultured for 1 to 7 days.

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

[10] above, wherein the retinal pigment epithelial cells are cultured for 1 to 7 days.

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

[11] above, wherein the retinal pigment epithelial cells are cultured for 1 to 7 days.

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

[14] A therapeutic agent for a disease associated with a disorder of the retinal pigment epithelium, comprising the string-like aggregates of retinal pigment epithelial cells according to any one of [1] to [4] above.

[0007] According to the present invention, it is possible to produce a transplant material for retinal pigment epithelial cells that can be prepared in a short time and at low cost, and that can be transplanted non-invasively and easily controlled at the transplant site.

[0008] FIG. 1 is a perspective view illustrating the structure of the device of the present invention and a method for producing string-like aggregates using the same. The figure shows an embodiment in which a single groove is provided on the upper surface of a base member to clearly illustrate the groove. Also, only a portion of the entire length of the groove is shown to illustrate the cross section of the groove (a cross section of the groove cut perpendicular to its longitudinal direction). FIG. 1(a) shows a state in which retinal pigment epithelial cells have been seeded in the groove together with a liquid medium (not shown). FIG. 1(b) shows a state in which the seeded retinal pigment epithelial cells have grown into string-like aggregates within the groove. FIG. 2 is a diagram illustrating the shapes of the groove and cavity portion. FIG. 3 is a diagram showing an example of a preferred embodiment of the groove and cavity portion. FIG. 4 is a schematic diagram illustrating the dimensions of each part of the string-like aggregate, the configuration of the device of the present invention, and the relationship between the string-like aggregate and the device. FIG. 4(a) is a simplified diagram illustrating the overall shape of the string-like aggregate for illustrative purposes. FIG. 4(b) is a diagram showing an example of the dimensional relationship between the groove and the string-like aggregate formed within the groove. FIG. 5 is a diagram showing a preferred embodiment of the device of the present invention. FIG. 5(a) is a perspective view showing the entire device, and FIG. 5(b) is a cross-sectional view of the device shown in FIG. 5(a) cut in the thickness direction of the device along the cutting line X1-X1 extending in the width direction of the groove. Hatching on the cross section is omitted. FIG. 6 is a diagram showing an example of a mold for producing the device of the present invention and also shows an example of a method for manufacturing the mold. FIG. 6(b) is a perspective view of the mold parts shown in FIG. 6(a). FIG. 6(d) is a perspective view of the mold shown in FIG. 6(c). FIG. 7 is a cross-sectional view showing an example of a manufacturing process for the device of the present invention, schematically showing the state in which the device is resin-molded using the molds shown in FIGS. 6(c) and (d). FIG. 8 is a photograph (FIG. 8(a)) showing a mold actually produced in an example of the present invention, and a photograph (FIG. 8(b)) showing the vise actually molded using the mold. FIG. 9 is a schematic diagram showing a method for manufacturing string-like aggregates according to the present invention. Figure 9(a) shows the state in which retinal pigment epithelial cells were seeded in the grooves of the device shown in Figure 5 (liquid medium not shown), and Figure 9(b) shows a schematic diagram of the string-like aggregates formed in the grooves being removed from the grooves. Figure 10 shows the measurement results of the PDMS groove structure.A: Photograph of a PDMS-coated mold with five fins, each 19.5 mm long and 1.6 mm high. B: Photograph of a PDMS-based culture device for measuring groove structure, with five grooves, each 19.5 mm long, 1 mm wide, and 1.6 mm deep. 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 are the base curvature radius of 0.21 mm, groove width of 1.16 mm, and groove depth of 1.60 mm. Figure 11 shows that the string-like aggregates replaced the damaged RPE defect area and partially restored its function. A: In vitro disease model and transplantation experimental schedule. Pathological RPE degeneration / damage was mimicked by treating RPE cells with MMC to create a wound. The transplantation process was then mimicked in vitro by plating either a hiPSC-RPE cell suspension or string-like aggregates (201B7modFucci strain) onto the defect area. B: The expansion of plated hiRPE cells on the RPE defect area was monitored by mCherry expression by cells in the G0-G1 cell cycle of the 201B7-Fucci hiPSC line. The initial scratched area and the defect area in the control well are indicated by dotted lines. B': The panels show the RPE defect area after scratching and the area refilled by RPE cell expansion from RPE cell suspensions or string-like aggregates. C: RPE cells expanding from string-like aggregates appeared to stop expanding upon contact with resident RPE, which showed increased mCherry expression along their borders. D: The border between pre-existing MMC-treated RPE and newly plated RPE was reasonably distinguishable by mCherry expression and RPE cell size (arrows). MMC-treated RPE were generally larger than RPE expanded from string-like aggregates. ZO-1 expression was observed between cells within the resident MMC-treated population and newly plated / expanded RPE cell populations, as well as between cells at the border. E and F: PEDF and VEGF secretion in each well at the initial stage (-d3), after MMC (-d2), after scratching (-d1), and after plating with or without hiPSC-RPE cells or string-like aggregates (n=3). Both VEGF and PEDF secretion decreased after MMC treatment and scratching.Without RPE cell supplementation, PEDF continued to decrease, but with RPE cell supplementation, either in suspension or as string-like aggregates, PEDF was more rapidly obtained. Figure 12 shows the optimization of string-like aggregate formation. A: hiPSC-RPE cells were plated into the grooves of a mold using three different media containing 10 μM Y-27632 (sheet medium, mixed medium, and maintenance medium). B: Appearance of string-like aggregates within the mold (mixed medium, maintenance medium). C: 4.5x10 hiPSC-RPE cells were plated into the grooves. 5 , 1.5x10 5 , 5x10 5 The cells were plated at 1000 x 1000 cells and incubated for 3 and 7 days. D: Appearance of string-like aggregates formed with 150,000 or 450,000 cells after 19 days on the plate. The string-like aggregates formed with 150,000 cells lost their initial shape more rapidly than the 450,000 cells, and had almost completely lost their initial shape by 19 days. Scale bar, 200 μm. Figure 13 shows the effects of starting cell number and Y-27632 concentration on string-like aggregate formation. A: String-like aggregates formed with 150,000 or 450,000 RPE cells were placed on a dish on day 4 and photographed the following day and 14 days later. RPE cells expanded from each string-like aggregate, and the string-like aggregates formed with 15,000 cells lost their shape after 14 days, while the string-like aggregates formed with 450,000 cells maintained their initial shape. A': Fragmented string-like aggregates lost most of their original shape after cell expansion. B: RPE (2 × 10 5Appearance of string-like aggregates on day 2 after seeding with either 10 μM or 2.5 μM Y-27632. C: Cross-sections of string-like aggregates formed with either 10 μM or 2.5 μM Y-27632. Expression of ezrin and laminin is fragmented, but ZO-1 expression is present between cells, and apical-basal polarity is not distinguished. The right panel shows control staining of RPE sheets. D, E, F, G: String-like aggregates formed with 2.5 μM Y-27632 adhered better to the plate, covered a larger area, and began to spread more rapidly on the plate compared to those formed with 10 μM Y-27632 (D, E: Day 2 string-like aggregates; F, G: Day 3 string-like aggregates). Figure 14 shows the optimization of Y-27632 concentration for string-like aggregate formation. A: String-like aggregate formation under different Y-27632 concentrations. B: Day 2 string-like aggregates (M8 strain) were plated in 24-well plates and the expansion of coverage area by hiPSC-RPE cells was monitored. String-like aggregates supplemented with 2-2.5 μM Y-27632 were most adherent to the plate and tended to lie flat when placed on the well. C: Expansion of hiPSC-RPE cells (M8 strain). Coverage area after placement of string-like aggregates in each well (n=6 for each Y-27632 concentration). D: Day 2 string-like aggregates (201B7modFucci strain) were plated in 24-well plates and the expansion of coverage area by hiPSC-RPE cells was monitored. String-like aggregates supplemented with 2-2.5 μM Y-27632 were consistently adherent to the plate and tended to lie flat when placed on the well. E: Expansion of hiPSC-RPE cells (201B7modFucci strain). Coverage area after string-like aggregates were placed in each well (n=2 for each Y-27632 concentration). Figure 15-1 shows the reproducibility of string-like aggregate formation using different hiPSC lines (M8 and 201B7modFucci L). A: String-like aggregates were similarly formed using other hiPSC lines, but optimization of Y-27632 was required for each line. The 201B7modFucci line required 5 μM Y-27632 to stably form string-like aggregates.B: String-like aggregates formed with each of the M8 and 201B7modFucci lines adhered to plates, expanded well, and exhibited the characteristic cobblestone appearance and pigmentation of RPE. C: RPE cells expanded from string-like aggregates expressed the tight junction marker ZO-1 and the RPE marker MiTF. D: RPE marker gene expression in hiPSC-RPE cells before string-like aggregate formation, RPE cells in string-like aggregates, and cells expanded from string-like aggregates derived from each hiPSC line. E: RPE cells migrated and proliferated from the string-like aggregates to form a monolayer. Figure 15-2 shows the reproducibility of string-like aggregate formation using different hiPSC lines (M8 and 201B7modFucci L). F: Number of RPE cells in string-like aggregates and number of RPE cells expanded from string-like aggregates. G: RPE cells expanded from string-like aggregates secreted VEGF and PEDF. Scale bars, 50 μm (C, E). Figure 16 shows the transplantation of string-like aggregates in nude rats. A: Fundus photograph of a nude rat eye after string-like aggregate transplantation. B: Fundus photograph of a nude rat eye 93 days after string-like aggregate transplantation. SLO-OCT revealed a strong RPE-like signal at the transplant site. C: Hematoxylin and eosin staining of the transplanted eye. D: Human nuclear antigen (HuNu) was positive only in the pigment sheet layer. E: The transplanted RPE showed well-oriented alignment, as indicated by the polarity markers human-specific ezrin and collagen type IV. Scale bars, 50 μm (B) and 20 μm (E). Figure 17 shows string-like aggregates loaded into a 24G intravenous cannula. Figure 18 shows the transplantation of string-like aggregates into two rabbit eyes. The practical handling of string-like aggregates during the transplantation procedure was tested in two rabbit eyes (A and B). Top: Loading of string-like aggregates. Center: The string-like aggregates were slowly injected inside the retinal hemorrhage. The retinotomy site is indicated by the white arrow. Bottom: Injected string-like aggregates. Figure 19 shows the COP groove structure. 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 with a cannula. Bar: 1 mm. Figure 21 shows the transplantation of string-like aggregates into a monkey's eyeball.

[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 string-like aggregates of the present invention).

[0010] In the present 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 cell morphology (intracellular melanin pigmentation, polygonal and flat epithelial-like cell morphology, formation of polygonal actin bundles, etc.). Furthermore, retinal pigment epithelial cell progenitor cells refer to cells that are induced to differentiate into retinal cells, and whether the 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)). Furthermore, the function of retinal pigment epithelial cells can be assessed using indicators such as the ability to secrete cytokines (VEGF, PEDF, etc.) or phagocytosis. Those skilled in the art can carry out these functional evaluations and confirmation operations by setting appropriate conditions.

[0011] RPE cells can be obtained from any animal (e.g., human) that possesses RPE cells, or can be obtained by inducing differentiation from pluripotent stem cells using known methods. However, cells induced to differentiate from pluripotent stem cells are more preferred because they can provide sufficient quantities of cells or cells appropriate for specific diseases. Pluripotent stem cells are not particularly limited as long as they have the pluripotency to differentiate into all cell types present in the body and also have the ability to proliferate. Examples of pluripotent stem cells 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). A preferred pluripotent stem cell is an 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., and most preferably humans, can be used.

[0012] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (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 any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs and oocytes or totipotent cells. Somatic cells include, but are not limited to, fetal somatic cells, neonatal somatic cells, and mature, healthy or diseased somatic cells, as well as primary cultured cells, subcultured cells, and established cell lines. Specifically, 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 (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0014] The reprogramming factors may be composed 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 low-molecular-weight compounds. Examples of genes included in the 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, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include 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. 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, Maekawa M, et al. (2011), Nature. 474:225-9.

[0015] RPE cells can be differentiated 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 also 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-cell adhesion" refers to the surface adhesion of RPE cells to each other in the string-like aggregates of the present invention. "Plane attachment" between cells refers to the surface adhesion of cells to each other. More specifically, "plane attachment" between cells refers to the proportion of the surface area of ​​a cell that is attached to the surface of another cell being, 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 immunostaining for cell adhesion factors (e.g., E-cadherin or N-cadherin).

[0017] 2. Device for Producing 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, the device of the present invention, or the device itself). As shown in FIG. 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 formed on the upper surface 110a of the base member 110. For the sake of explanation, FIG. 1 depicts the base member 110 as having only one groove, but in a preferred embodiment, as described below, multiple grooves are arranged adjacent to each other, and the grooves have a V-shaped cross section so that there is no upper surface (flat surface) between the grooves. The groove 120 has a cavity portion having a length corresponding to the length of the string-like aggregate to be formed and a width corresponding to the outer diameter of the string-like aggregate. In the example shown in FIG. 1 , the bottom of the groove 120 is the cavity portion. The cavity portion is a concave mold in which retinal pigment epithelial cells seeded in the cavity portion can grow into string-like aggregates. With this configuration, when an appropriate amount of retinal pigment epithelial cells a1 are seeded and cultured in the grooves 120 of the device together with a liquid medium (not shown) as shown in Figure 1(a), a string-like aggregate A1 having a shape corresponding to the length and width of the cavity portion is obtained as shown in Figure 1(b). The string-like aggregate obtained by this device has a body surface (the lower surface of string-like aggregate A1 in Figure 1(b)) that is smoothly shaped along the cavity portion, and a freely growing, raised surface (the upper surface of string-like aggregate A1 in Figure 1(b)).

[0018] (Base Member) 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 in which the string-like aggregates to be produced 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 of Figure 5(a), the overall shape of the base member 110 is disk-like. When the base member is plate-like, the outer peripheral shape is not limited to a circle (i.e., a disk-like shape) and may be a square, a rectangle, or the like. A disk-like shape is a preferred shape because it can be placed in a commercially available petri dish, which is generally circular, without creating dead space. Depending on the application, various additional parts such as walls surrounding the outer periphery, flanges extending out to the sides, handles for handling, feet on the back side, printing, engraving (concave or convex) or labeling of symbols to identify the cavity (letters, numbers, QR Code (registered trademark), etc.), and printing, engraving or labeling of dimensional scales to determine the length of the cultured string-like aggregates may be provided by integral molding or by attaching separate parts.

[0019] (Groove and Cavity Portion) As shown in Figure 2(a), the entire groove 120 may be the cavity portion, or as shown in Figures 2(b), (c), and (d), the groove 120 may have a structure in which the bottom portion is the cavity portion 121 and an upper portion 122 is provided above 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 protrude upward.

[0020] As illustrated in Figures 2(b) to (d), when the internal structure of the groove 120 has a cavity portion 121 as a bottom portion and an upper portion 122 located above it, the cavity portion 121 and the upper portion 122 may be integrally formed with each other, as in the examples of these figures, or may be separable into two upper and lower portions (combined as separate components) as illustrated in Figures 2(e) and 3. The embodiment of Figure 2(e) is a modification of the embodiment of Figure 2(d), but the cavity portion and the upper portion in various grooves, such as those illustrated in Figures 2(b) and 2(c), may be separable. Furthermore, the boundary surface of separation does not necessarily have to coincide with the boundary surface between the cavity portion and the upper portion; the cavity portion and the upper portion may be integrally formed, and the upper portion may be separable into two upper and lower portions. Such a structure that can be separated into two upper and lower portions allows only the cavity portion 121 to be exposed, as illustrated in Figure 3(b), making it easier to extract the string-like aggregate A1 after culture. Furthermore, such a structure that can be separated into two parts, top and bottom, does not require the formation of deep grooves, and there is an advantage in manufacturing that the degree of freedom in the groove profile is increased.

[0021] 3A and 3B are diagrams showing a specific example of a device for separating a cavity portion and an upper portion. In this example, as shown in an assembled state in FIG. 3A , a base portion 110 includes a first base portion 111 and a second base portion 112 disposed thereon. As shown in a separated state in FIG. 3B , the second base portion 112 is detachable from the first base portion 111. The first base portion 111 is provided with a cavity portion 121 of a 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, the first base portion 111 and the second base portion 112 are preferably provided with a positioning structure so that they can be 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 with each other to form one groove 120). Such a positioning structure is not particularly limited, and any conventionally known positioning structure can be appropriately adopted, such as a positioning protrusion and a positioning recess that receives it, or an outer peripheral shape (a shape other than a cylinder and a round hole) that fits together without misalignment.

[0022] When the base portion 110 can be separated into multiple parts (e.g., the first base portion 111 and the second base portion 112) as described above, the materials of the respective parts may be different from each other. For example, the first base portion 111, in which the cavity is provided, is preferably made of a material that has biocompatibility (GCP: Good Clinical Practice compliant) that allows string-like aggregates to be favorably grown, and that is difficult for cells to adhere to, so that the string-like aggregates can be easily peeled and removed from the cavity (such a material may also be used for the second base portion 112). Furthermore, both the first base portion 111 and the second base portion 112 are preferably made of a material that can be sterilized (e.g., a material that is heat-resistant enough to be heated in an autoclave, radiation-resistant enough to be irradiated with gamma rays, and compatible with EOG sterilization), or that is inexpensive and easy to process.

[0023] (Shape of the String-Like Aggregate) To explain the shape of the cavity portion, the shape of the string-like aggregate will be described here. In the present invention, a string-like aggregate is an aggregate in which RPE cells are randomly aggregated to form an elongated string shape. It does not need to have a clear apical-basal polarity like the retinal pigment epithelium in vivo, and there are no particular limitations on the aggregate, as long as it has sufficient strength to efficiently transfer the desired number of cells as a single mass using a needle or tubular transplantation tip during transplantation. Furthermore, in the present invention, the "string-like" shape of the string-like aggregate refers to a clearly elongated shape with a longitudinal direction, i.e., a shape that extends long in one direction, compared to the shapes seen in known cell aggregates or cell sheets. This elongated shape is such that, as shown as an explanatory model in Figure 4(a), the ratio (L1 / d1) of the total length L1 to the outer diameter d1 of the body when the string-like aggregate is linear 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. A shape having the above-mentioned ratio (L1 / d1), particularly the lower limit ratio of 2, allows for a more defined direction (i.e., the longitudinal direction of the string-like aggregate) than a simple cell mass. That is, in tissues where orientation is important, appropriately defining the direction in advance has the effect of producing a more desirable tissue. Furthermore, a string-like aggregate having the above-mentioned ratio can be transported as a single elongated structure by passing it through the inside of a syringe needle, and the effect of efficiently transporting and positioning cells over a wide area with a single operation is more pronounced. Furthermore, since it can pass through narrow areas (e.g., tiny through-holes) that are difficult for known cell sheets to pass through, the effect of reducing invasiveness to the living body is more pronounced.

[0024] When the ratio (L1 / d1) is below the lower limit 2, the above effect becomes less pronounced. Furthermore, there is no particular limitation on the upper limit of the ratio (L1 / d1), and, for example, even if the total length L1 is longer than the area of ​​the target site into which the string-like aggregate is to be transplanted, the aggregate can be cut as appropriate. Considering the need to avoid excessively long grooves in the device, the dimensions of the aggregate calculated from the number of cells to be transplanted, and transportation limitations (e.g., the length of the syringe needle), the upper limit of the ratio (L1 / d1) is, for example, about 1000, more preferably about 200, and particularly preferably about 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 to 1000, 5 to 1000, 10 to 1000, 20 to 1000, 50 to 1000, 10 to 200, 20 to 200, 50 to 200, 2 to 100, 5 to 100, 10 to 100, 20 to 100, or 50 to 100. The ratio (L1 / d1) of the string-like aggregates obtained using the devices fabricated in the examples of the present invention was approximately 2 to 100. The lower limit of the ratio is the minimum value required for the aggregates to have a "string-like" shape, but the upper limit of the ratio is not particularly limited, and the upper limit of the ratio can be appropriately determined depending on the space limitations of the culture equipment, the range of the outer diameter d1, the effort required to divide the aggregates into lengths suitable for cell transplantation, and the like.

[0025] (Outer diameter d1 of the body of the string-like aggregate) When a string-like aggregate is produced using this device, the cross-sectional shape of the string-like aggregate (the cross-sectional shape when the string-like aggregate is cut perpendicular to its longitudinal direction) is such that the lower portion during culture conforms to the cavity portion, and the upper portion conforms to a shape resulting from free growth, as illustrated in Figures 2(a) to (d). Therefore, the outer diameter d1 of the body of the string-like aggregate is variable depending on the direction of measurement. While such outer diameter d1 may be calculated by taking the average value of outer diameter values ​​measured in multiple directions, it is convenient to observe the string-like aggregate together with the device while it is still grown in the cavity portion (i.e., by observing the inside of the groove from above in Figure 4(b)), as illustrated 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 an observation, the string-like aggregate will be linear along the cavity portion, 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, but a shape close to circular or elliptical is preferred because it is desirable for the distance from the tissue surface to the inside of the tissue to be constant from the standpoint of supplying nutrients and oxygen and excreting waste products, it can pass more smoothly through narrow parts such as syringe needles, and a more smoothly curved cross-sectional shape of the cavity part of the groove is advantageous for cell proliferation and detachment.

[0027] (Example of Dimensions of String-Like Aggregates When Applied to a Living Body) As described above, there is no limitation on the length of the string-like aggregates, and if they are excessively long, they may be cut as appropriate when applied to a living body. Preferred dimensions when applied to a living body include, for example, a total length L1 of about 20 μm to 200 mm, preferably about 20 μm to 40 mm, more preferably about 1 mm to 40 mm, and even more preferably about 5 mm to 40 mm, and an example of a length compatible with the length of a 24-gauge needle is about 10 mm to 20 mm. There is no single most preferred range or value for these values ​​of the total length L1, and preferred ranges and values ​​can be selected as appropriate depending on the size of the device, the allowable groove length, the required number of cells, the length of the duct of the instrument for transplantation (e.g., a syringe needle), and the like. Furthermore, the outer diameter d1 can be about 10 μm (outer diameter corresponding to about one cell) to about 300 μm (outer diameter corresponding to the inner diameter of a 24-gauge needle), preferably about 20 μm to 300 μm, and more preferably about 100 μm to 250 μm. There is also no single most preferred range or value for these values ​​of the outer diameter d1, and 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, the inner diameter of the duct of the instrument for transplantation (e.g., a syringe needle), and the like.

[0028] When the string-like aggregate is transplanted by puncturing the eyeball with an injection needle and feeding the string-like aggregate through the injection needle to a target site such as the fundus, the preferred inner diameter of the injection needle is about 10 to 300 μm. In this case, the outer diameter d1 of the string-like aggregate that can preferably move within the injection needle is, for example, 80 to 95%, preferably about 90%, of the inner diameter of the injection needle. Therefore, in such a case, the outer diameter d1 of the string-like aggregate is, for example, about 100 μm to 270 μm as a preferred size range. In this case, the preferred length L1 of the string-like aggregate is, from the viewpoint of being effective for transplantation and compatible with the length of the injection needle, about 20 μm to 200 mm, preferably about 20 μm to 40 mm, more preferably about 1 mm to 40 mm, and even more preferably about 5 mm to 40 mm. An example of a length compatible with the length of a 24-gauge needle is about 10 mm to 20 mm. Therefore, the preferred ratio (L1 / d1) when sending string-like aggregates using an injection needle is, for example, 40 to 200, preferably about 80. In addition, the number of cells at that time is 1.5 × 10 ５ ~4.5 x 10 ５ The extent is exemplified.

[0029] As described above, the string-like aggregates 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. Accordingly, the string-like aggregates of the present invention may be characterized in that the ratio (L1 / d1) of the total length L1 to the outer diameter d1 of the body is 2 to 1,000. Furthermore, L1 / d1 may typically be 2 to 1,000, but may also be 5 to 1,000, 10 to 1,000, 20 to 1,000, 50 to 1,000, 10 to 200, 20 to 200, 50 to 200, 2 to 100, 5 to 100, 10 to 100, 20 to 100, or 50 to 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 be characterized in that their cross-sectional shape is circular or elliptical.

[0031] (Cross-sectional shape of cavity portion) The cavity portion in 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 a 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 determined appropriately taking into consideration cell culturing properties, mold releasability, etc. Preferred cross-sectional shapes include an arc shape, a semicircle, a rectangle with an open top, a U-shape, and a V-shape. The bent portions (corners) of the rectangle or V-shape are preferably rounded, for example, because it is easy to remove the string-like aggregate using tweezers or a syringe needle, and because it is preferable that the cross-sectional shape of the string-like aggregate be 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, the width W1 of the cavity portion shown in FIG. 4(b) is preferably the same as the outer diameter d1 of the string-like aggregate.

[0032] (Preferred embodiment of the 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, with the cross-sectional shape of the upper portion 122 widening from the bottom side toward the opening side. The cross-sectional shape of the entire groove is V-shaped, with the deepest portion being rounded as the above-mentioned cavity portion. This V-shaped cross-sectional shape guides cells seeded in suspension into the cavity portion at the bottom, facilitating the seeding operation and also facilitating the release of string-like aggregates from within the groove, facilitating the removal operation.

[0033] The depth of the entire groove (B1 shown in FIG. 4(b)) is not particularly limited, but is preferably about 5 to 5,000 μm (a dimension that is about 5 mm shorter than the depth of a typical Petri dish), and more preferably about 5 to 1,600 μm. The depth of the cavity portion (dimension b1 shown in FIG. 4(b)) is a dimension corresponding to the outer diameter d1 of the string-like aggregate to be formed, and is preferably about 5 to 300 μm, and more preferably about 5 to 250 μm. The width of the opening of the groove (W2 shown in FIG. 4(b)) is not particularly limited, but is preferably about 560 μm (outer diameter of a 24-gauge needle) to 5,000 μm (tip diameter of a 1 mL pipette tip), and more preferably about 1,000 μm to 3,000 μm (approximate value for the device), and more preferably about 1,500 μm to 2,500 μm. When the cross-sectional shape of the entire 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, more preferably about 40 to 70 degrees. The cross-sectional shape of the entire groove can be determined appropriately depending on the amount of cell suspension required for culture, the number of cells, the size of the container to be stored, etc. The length of the groove is preferably the same as the length of the string-like aggregate to be formed, but may be longer.

[0034] The upper portion of the groove in cross section may be a straight line as shown in Fig. 4(b) or may be a curved line that widens from the bottom toward the opening as shown in Fig. 5(b). Such a curve can also be interpreted as a rounded edge of the straight V-shaped opening portion as shown in Fig. 4(b).

[0035] (Groove Arrangement Pattern) In a preferred embodiment, as shown in FIG. 5, multiple grooves 120 (five in the illustrated example) are arranged in parallel stripes 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 may be determined appropriately along with the size of the base member depending on the scale of production, from experimental to commercial use. In the example shown in FIGS. 5(a) and (b), the upper part of the cross-sectional shape of the groove 120 is curved so that its width increases from the bottom side toward the opening side, and the groove 120 smoothly connects to the upper surface of the base member and adjacent openings. In FIG. 5(b), the boundary between adjacent grooves and the boundary 130 between the groove and the upper surface 110a of the base member 110 are represented by dashed lines for ease of understanding.

[0036] When multiple grooves are arranged in parallel stripes, in a preferred embodiment, as shown in Figure 5, the walls separating adjacent grooves do not have flat portions on the upper surface of the base member, and the openings of adjacent grooves contact each other. This configuration is preferably achieved when the cross-sectional shape of the entire groove is V-shaped. This configuration allows the grooves to be arranged more densely. When culturing using this device, culture medium and cells may be injected only into the grooves, or the entire device may be immersed in culture medium and cells seeded therein. Immersing the entire device in culture medium and then seeding cells is preferred because the seeded cells are less likely to accumulate on the flat surfaces between the grooves, allowing more cells to fall into the grooves.

[0037] (Material of Base Member) The material of the base member (i.e., the material of the device) is not particularly limited, and is preferably a material that is suitable for cell culture, inexpensive, and easy to manufacture, such as a metal or a polymer. Preferred examples include glass and various polymer materials (e.g., polystyrene, polycarbonate, acrylic, silicone, cycloolefin polymer (COP), etc.) that have traditionally been used as materials for cell culture vessels. Among these, polydimethylsiloxane (PDMS), a type of silicone, is exemplified as a more preferred material because it has advantages such as ease of molding using a mold, ability to be heated in an autoclave, flexibility, high chemical resistance, and applicability to microfabrication processes.

[0038] (Method for manufacturing the device) The method for manufacturing 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 member is made of PDMS, a preferred manufacturing method involves using a mold having a convex shape corresponding to the shape of the groove, pouring fluid PDMS before hardening into the mold (or bringing it into contact with the convex shape), and hardening the PDMS in that state to obtain a molded product (the device). PDMS hardens when heated, for example, at 80°C for about 3 hours, to become PDMS.

[0040] (Preferred Method for Manufacturing a Molding Mold) An example of a preferred method for manufacturing a molding mold is shown. FIG. 6 is a diagram showing an example of a preferred method for manufacturing a molding mold. In this example of the manufacturing method, first, as shown in FIGS. 6( a) and 6(b), protrusions 220 that serve as the cores of a convex mold corresponding to the shape of the grooves are formed on one surface 210a of a mold substrate 210 in accordance with the center-to-center pitch of the grooves. Next, as shown in FIGS. 6(c) and 6(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 utilized to shape the cross-sectional shape of the coating layer 230 into the shape of a V-shaped convex mold corresponding to the shape of the grooves to be molded, as shown in FIG. 6(c). In this way, a V-shaped convex mold can be easily obtained from a core of a simple shape.

[0041] Conditions for controlling the liquid polymer raw material into a V-shape as shown in FIG. 6( c) include, for example, applying uncured PDMS having a viscosity of approximately 3.5 (Pa s) at 25°C so as to cover the protrusions, leaving the mixture to stand for 30 minutes while degassing in a vacuum desiccator at room temperature (approximately 23 to 27°C), and then curing the mixture at 80°C for 3 hours.

[0042] In the embodiment of the present invention, the part shown in Figures 6(a) and (b) (a mold substrate 210 with protrusions 220) was produced using a 3D printer (the material was polylactic acid (PLA resin)). However, a molding die 200 with a V-shaped convex die 240 as shown in Figure 6(c) may also be produced using a 3D printer, and the surface may be further finished.

[0043] FIG. 7 is a cross-sectional view illustrating the molding of the device using the above-described mold. As shown in FIG. 7( a), a mold 200 is placed on the bottom surface of a molding container (not shown), or a molding container is formed by surrounding the mold 200 with wall members, and a pre-cured, fluid polymer material is poured into the mold. The polymer material is cured in this state to obtain a molded product (the device) 100. Next, as shown in FIG. 7( b), the molded product is peeled from the mold 200 to obtain the device 100. FIG. 8( a) is a photograph showing the substrate surface of a mold produced in an example of the present invention, in which protrusions 220 are provided on one main surface of the mold substrate 210. FIG. 8( b) is a photograph showing the substrate surface of a mold produced in an example of the present invention, which was molded using the mold of FIG. 8( a), in which protrusions 220 are 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 retinal pigment epithelial cells aggregated in strings (hereinafter referred to as the production method of the present invention). The production method of the present invention comprises the following steps: (1) As illustrated in Figure 9(a), seeding retinal pigment epithelial cells a1 suspended in a liquid medium into grooves 120 of device 100 of the present invention. (2) As illustrated in Figure 9(a), culturing retinal pigment epithelial cells a1 to form string-like aggregates A1 of retinal pigment epithelial cells (the figure shows one string-like aggregate being removed from the groove).

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

[0046] The liquid medium in which the retinal pigment epithelial cells are suspended in step (1) can be prepared using a medium commonly used for culturing animal cells as the basal medium. 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's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof, which can be used for culturing animal cells. The medium used for culturing may be either serum-containing or serum-free.

[0047] As used herein, "serum-containing medium" refers to a medium containing unconditioned or unpurified serum. The serum concentration of the serum-containing medium is typically 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, pyruvic acid, buffers, inorganic salts, etc.

[0048] As used herein, serum-free medium refers to a medium that does not contain unconditioned or unpurified serum. In the present invention, serum-free medium also includes media contaminated with purified blood-derived components or animal tissue-derived components (e.g., growth factors) as long as they do not contain unconditioned or unpurified serum.

[0049] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those that appropriately contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc., 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. Examples of such commercially available serum substitutes include Knockout TMSerum Replacement (Life Technologies; now ThermoFisher; hereafter sometimes referred to as KSR), Chemically-defined Lipid concentrated (Life Technologies), Glutamax TM (Life Technologies), B27 (Life Technologies), N2 Supplement (Life Technologies), and ITS Supplement (Life Technologies).

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

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

[0052] In the present invention, ROCK inhibitors are not limited to substances that inhibit the activity of Rho kinase (ROCK). Rho kinase (ROCK) was discovered as a serine / threonine kinase downstream of the small G protein Rho. The Rho / ROCK signaling pathway is involved in various cellular functions, including actin cytoskeleton and cell adhesion. Dissociation is required for subculture of iPS cells, but these stem cells are known to undergo apoptosis when cultured in a dissociated state. Dissociation-induced apoptosis is mediated by the Rho / ROCK signaling pathway, and it has been reported that the addition of ROCK inhibitors suppresses apoptosis (apoptosis-inhibiting effect). Furthermore, it has been reported that the addition of ROCK inhibitors during cryopreservation of cells increases cell survival after thawing (cell viability-improving effect). Based on these reports, ROCK inhibitors have become the preferred choice for the expansion and culture of iPS cells. On the other hand, regenerative medicine products typically use basic media such as balanced salt solution or DMEM / F12 medium, and do not contain any exogenous components. However, the inventors have discovered that in addition to the above-mentioned effects, when string-like aggregates are prepared using an appropriate concentration of a ROCK inhibitor, as described in the examples below, the string-like aggregates become more stable and have the new effect of promoting the engraftment of RPE cells and the growth of cells at the transplant site.

[0053] In the present invention, the ROCK inhibitor may be any molecule as long as it has the same or substantially the same effect as the newly discovered effect described above (the effect of promoting string-like aggregate formation). "Substantially the same" means that the effects are qualitatively (e.g., physiologically or pharmacologically) the same. Therefore, it is preferable that the effects are equivalent, but the magnitude of these effects (e.g., about 0.1 to about 10 times, preferably about 0.5 to about 2 times) may differ. The effect can be measured according to a method known per se. 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. 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 and the like are included, and preferred are Y-27632 dihydrochloride and Y-27632.

[0054] ROCK inhibitors can be produced by known methods. Alternatively, commercially available ROCK inhibitors can be purchased and used. For example, Y-27632 can be purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Ripasudil is commercially available under the trade name Glanatec (registered trademark) (Kowa), and Fasudil Hydrochloride is commercially available under the trade name Eril (registered trademark) (Asahi Kasei Pharma).

[0055] The concentration of the ROCK inhibitor contained in the culture medium is not particularly limited as long as it allows RPE cells to form string-like aggregates in the grooves of the device of the present invention, but 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 may become too hard, which may affect the spread of the 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-like aggregates in the grooves of the device of the present invention. 3 cells / mL or more, preferably 2.5x10 3 cells / mL ~ 5x10 5 cells / mL, more preferably 1x10 5 cells / mL ~ 2x10 5 If the cell density in the groove is higher than this, the outer diameter of the body of the string-like aggregates that are formed will become larger, raising concerns that the cells will not spread after transplantation and that the transplanted cells will form a multilayer 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-like aggregates of retinal pigment epithelial cells are obtained by step (2), but is usually 1 to 30 days, preferably 2 to 7 days. If the culture period is longer or shorter than this, there is a concern that the aggregates of the present invention may detach from the device of the present invention during culture medium replacement.

[0059] The culture conditions in step (2), such as culture temperature and CO2 concentration, can be set appropriately. 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. Therapeutic Agent for Diseases Caused by Retinal Tissue Damage The string-like aggregates of retinal pigment epithelial cells of the present invention obtained as described above are useful in transplantation therapy for diseases caused by retinal tissue damage, such as diseases caused by damage to the retinal pigment epithelium. Thus, the present invention provides a therapeutic agent for diseases caused by damage to the retinal pigment epithelium (the therapeutic agent of the present invention), which comprises string-like aggregates of retinal pigment epithelial cells.

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

[0062] Pharmaceutically acceptable carriers include physiological aqueous solvents (e.g., physiological saline, buffer solutions, serum-free media, etc.) If necessary, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc. may be added to pharmaceuticals containing tissues or cells to be transplanted in transplantation medicine.

[0063] The therapeutic agent of the present invention can be produced as a suspension by suspending string-like aggregates of retinal pigment epithelial cells in an appropriate physiological aqueous solvent. If necessary, a cryopreservative may be added, followed by cryopreservation, and the resulting suspension may be thawed at the time of use, washed with a buffer solution, and then used in transplantation therapy.

[0064] The string-like aggregates of retinal pigment epithelial cells of the present invention can be used as a therapeutic agent for diseases caused by retinal pigment epithelial damage, or to replenish the corresponding atrophied / damaged areas of retinal pigment epithelium in atrophied or damaged states. By transplanting the string-like aggregates of retinal pigment epithelial cells of the present invention into patients who require transplantation and have diseases caused by retinal pigment epithelial damage or atrophied or damaged retinal pigment epithelium, the diseases caused by retinal pigment epithelial damage or atrophied or damaged states of retinal pigment epithelium can be treated. Examples of diseases caused by retinal pigment epithelial damage or diseases accompanied by atrophy or damaged states of retinal pigment epithelium include ophthalmic diseases such as age-related macular degeneration, retinitis pigmentosa, and retinal pigment epithelial tears.

[0065] The amount of retinal pigment epithelial cell string-like aggregates contained in the therapeutic agent of the present invention is not particularly limited, as long as a therapeutically effective amount of retinal pigment epithelial cell string-like aggregates containing RPE cells is contained in the suspension (e.g., 50-500 μL, preferably 100-300 μL) injected into the diseased site, i.e., the site of retinal pigment epithelial defect in macular degeneration or retinitis pigmentosa. For example, a suspension of retinal pigment epithelial cell string-like aggregates can be injected so that the RPE cells are present at a concentration of 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 transplant site, as occurs with RPE cell suspensions, and the transplanted cells do not form an epiretinal membrane (ERM) in the vitreous. Furthermore, the therapeutic agent of the present invention does not require the time and cost required for sheet preparation, as occurs with RPE sheets, and does not require invasive surgical procedures involving large incisions.

[0066] The therapeutic agent of the present invention can be transplanted by subretinal injection into a mammal (e.g., human, mouse, rat, etc., preferably human) suffering from a retinal disease such as macular degeneration (e.g., age-related macular degeneration, retinitis pigmentosa, etc.) using a transplantation device including an appropriate syringe and needle (e.g., MedOne0® Poly Tip® Cannula 25g / 31g, etc.). A medium prepared by diluting a viscoelastic substance (e.g., Shellgan, Viscoat, etc.) 4-8 times with OptiMEM can be used as the medium used for transplanting the therapeutic agent of the present invention. The use of a transplantation medium allows for smooth suction and ejection 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 in any way.

[0068] Experimental Procedures All animal experimental protocols were approved by the Animal Care Committee of the RIKEN Biosystems Dynamics Research (BDR) Center and were conducted in accordance with local guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. hiPS cells were generated with the approval of the RIKEN Biosystems Dynamics Research (BDR) Center Ethics Committee and from volunteers with informed consent.

[0069] Example 1: Fabrication of a PDMS-Based Culture Device for the Generation of String-Like Aggregates of hiPSC-RPE. A PDMS-based culture device with grooves 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 this device was created by 3D printing using polylactic acid (PLA) filament. The 3D-printed mold was milled, and PDMS was applied to adjust the groove shape. A release agent (Novec 1720, Sumitomo 3M) was then applied to the mold. PDMS and a PDMS curing agent (Sypot 184W / C, Dow Corning Toray) were mixed in a 10:1 ratio and poured onto the 3D-printed mold. The mixture was then degassed for approximately 1 hour. The 3D-printed mold and PDMS mixture was then placed in an oven at 80°C for 3 hours. The cured PDMS was then removed 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 (FIG. 10).

[0070] Preparation of human iPSCs (hiPSCs) and hiPSC-derived RPE cells (hiPSC-RPE). We established the M8 human iPSC line in our laboratory by introducing six reprogramming factors (OCT3 / 4, SOX2, KLF4, L-MYC, LIN28, and a p53 carboxy-terminal dominant-negative fragment) into peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Briefly, episomal vectors (pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mp53DD, and pCXB-EBNA1, purchased from Addgene) were electroporated into PBMCs, and iPSC-like colonies were subsequently isolated 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 episomal vectors were detectable using plasmid-specific primers. The 201B7 and 253G1 hiPSC lines (Nakagawa et al., 2008; Takahashi et al., 2007) were obtained from the RIKEN BioResource Center. To visualize the G1 phase of the cell cycle and the entire cell body, the modified Fucci 201B7 line (201B7modFucci) was generated in our laboratory. Briefly, the mCherry-hCdt1(30 / 120)-P2A-mVenus region was excised from the tFucci(CA)2 / pCSII-EF vector (a gift from Dr. A. Miyawaki; https: / / cfm.brc.riken.jp / lentiviral-vectors / plasmid-list / ) and ligated 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 and passaged, and single clone-derived colonies were isolated. 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 iPS cells the day before differentiation induction. On day 0, iPS cells were suspended in growth factor-free chemically defined medium (gfCDM) containing 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 Industries, Ltd.), and penicillin-streptomycin, and cultured in a 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 Ultra Low Culture Dishes (Corning Incorporated) in 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-antimycotic (Gibco).Between days 30 and 60, pigmented colonies were picked and plated onto iMatrx511-coated 12-well plates in a 1:1 mixture of "RPE adhesion medium" and "RPE maintenance medium" (hereafter referred to as "mixed medium"). "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 medium contained 50X supplements (Thermo Fisher Scientific Inc.), and gentamicin solution (Sigma-Aldrich). Once cells attached, the medium was replaced with RPE maintenance medium containing 10 ng / mL basic fibroblast growth factor (bFGF). Cell cultures were expanded in RPE maintenance medium at passage 1 and stored at -150°C using a STEM-CELL BANKER (Nippon Zenyaku Kogyo Co., Ltd.). For each experiment, stock cells were thawed, seeded in mixed medium, and then replaced with RPE maintenance medium supplemented with 10 ng / mL bFGF and 0.5 μM SB431542. The medium was replaced every few days until the cells were used. Differentiated RPE cells from the 253G1 line were prepared as described in Kitahata et al., 2019.

[0071] Preparation of string-like aggregates. After thawing, hiPSC-RPE cells were cultured for 2 weeks, then collected and seeded into each groove of the PDMS-based culture device at the indicated cell number for each experiment using 15 μl RPE maintenance medium containing the optimal concentration of Y-27632 (concentration indicated for each experiment).

[0072] Analysis of RPE expansion area from plated string-like aggregates. String-like aggregates were released from the device grooves and seeded onto uncoated 24-well plates (Corning Incorporated) containing mixed medium. After the string-like aggregates adhered (2-3 days later), the medium was replaced with RPE maintenance medium (containing bFGF and SB431542). Images of the cultured dishes were taken using an IncuCyte Zoom system (Essen BioScience). Expanded cells were manually marked using Fiji from the resulting image data, and the expansion area was measured.

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

[0074]

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

[0076]

[0077] In vitro RPE injury model: RPE cells cultured at confluence in a 24-well plate were treated with 6 μg / mL Mitomycin C (KIRIN, Kyowa Hakko). The next day, a portion of the RPE cells was removed by scraping with a cell scraper approximately 6 mm wide. The cell suspension (2 × 10 5 ) or string-like aggregates were plated ("transplanted") onto or into the injured area of ​​RPE. Cells were maintained in 0.5 ml of medium and incubated for 24 hours before medium changes and cell collection for ELISA at each intervention. The experimental design and timing of medium collection are shown in Figure 11A.

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

[0079] Immunodeficient F344 / NJc1-rnu / rnu female nude rats (6 weeks old) were obtained from CLEA Japan. Disposable micropipettes (Drummond, 1-000-0500) were pulled using a micropipette puller (Sutter, P-97 / IVF Puller), and the tips were cut and sharpened using a microgrinder (Narishige, EG-400). The micropipettes were 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. Animals were anesthetized by inhalation of 5% isoflurane, and the pupils were dilated with 0.4% tropicamide. The string-like aggregates were loaded into the micropipettes and implanted subretinally.

[0080] Transplantation into Rabbit Eyes: kbl:JW rabbits (9 weeks old) were obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan). Animals were anesthetized with an intramuscular injection of 60 mg / kg ketamine and 10 mg / kg xylazine. After routine vitrectomy with posterior vitreous detachment, the string-like aggregate was loaded into a 24-gauge indwelling needle cannula (TOP, SS-6) with a custom-made flat-cut 25G blunt needle core. After creating a local 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 attach the retina, allowing for fluid-gas exchange.

[0081] Results Preparation of hiPSC-RPE with string-like aggregate formation We outlined a scheme for preparing string-like aggregates using a PDMS-based culture device. First, human iPSC-RPE cells (253G1 line) 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 into each groove. While the string-like aggregates appeared to collapse easily in the sheet medium, the string-like aggregate structure was well maintained in the maintenance medium and mixed medium (Figure 12A and B). Next, different numbers of cells (4.5 × 10 5 , 1.5×10 5 , 5×10 4 ) were seeded, and it was observed that string-like aggregates could be formed under the former two conditions (Fig. 12C). 5 The cell-seeded hiPSC-RPE was 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 1.5x10 cells 5 The string-like aggregates formed from the cells were plated into wells. Both starting cell numbers showed expansion of RPE cells from the string-like aggregates onto uncoated plates, with 1.5x10 5 The string-like aggregates formed by the cells still retained most of their initial shape after 2 weeks (4.5 x 10 cells). 5 Compared with the string-like aggregates formed with cells, the initial shape was lost more quickly (Fig. 13A and Fig. 12D). Therefore, we set the initial cell number to 1.5-2 x 10 5 The cells were fixed with string-like aggregates (1.5x10 5When hiPSC-RPEs (cells) were fragmented, most of the initial string-like aggregate morphology was lost (Figure 13A'). Our preliminary experiments (Figure 14A) indicated that the concentration of Y-27632 seemed to affect the texture of the string-like aggregates, with higher concentrations resulting in tighter string-like aggregates. We tested Y-27632 concentrations of 0, 1, 2, 2.5, and 10 μM on string-like aggregate formation. hiPSC-RPEs (M8 strain) were able to form string-like aggregates at concentrations of 2 μM or higher, but string-like aggregate formation was poor at 0 and 1 μM Y-27632. Thread-like aggregates formed with 10 μM Y-27632 readily detached after placement on uncoated wells and took longer to attach and spread on 24-well plates compared with 2-2.5 μM Y-27632 (Figures 14B and 14C). Next, we directly compared thread-like aggregates formed with either 2.5 μM or 10 μM Y-27632 (Figures 13B–G). Thread-like aggregates formed with 2.5 μM Y-27632 appeared flat and loose, whereas those formed with 10 μM Y-27632 appeared more compact (Figure 13B). Thread-like aggregate sections were stained for the gap junction marker Z0-1, the polarity markers Ezrin (apical), and laminin and collagen type IV (basal) (Figure 13C). In 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 fragmented cell clusters. Collagen IV, another basal marker, was not clearly expressed, indicating that cell polarity was not clearly determined in these string-like aggregates. Next, we compared the cell spreading behavior of string-like aggregates formed with 2.5 and 10 μM Y-27632 on days 2 (Figures 13D and E) and 3 (Figures 13F and G) after plating onto uncoated wells. String-like aggregates formed with 2.5 μM Y-27632 tended to attach to the plate and begin spreading soon after plating.In contrast, the string-like aggregates formed with 10 μM Y-27632 tended to coil and remained mobile for several days before settling down and beginning to expand.

[0083] Reproducibility of String-like Aggregate Formation Using Other hiPSC-RPE Lines We tested whether other hiPSC lines could similarly form string-like aggregates. We induced RPE differentiation using the 201B7modFucci line and compared it with hiPSC(M8)-RPE. While hiPSC(201B7modFucci)-RPE formed string-like aggregates similar to hiPSC(M8)-RPE, the 201B7modFucci line required Y-27632 concentrations of 5 μM or higher to form stable string-like aggregates (Figure 15-1A). The optimal Y-27632 concentration for the adhesion / spreading assay was also 5 μM for hiPSC(201B7modFucci)-string-like aggregates. The optimal Y-27632 concentration for the desired adhesion / spreading effect varied depending on the cell line used, and optimization was required (Figures 14D and E). Cells expanded from string-like aggregates of both hiPSC lines exhibited the characteristic pigmentation and cobblestone-like appearance of RPE cells and expressed ZO-1 and MITF (Figures 15-1B and 1C). Expression of RPE-specific marker genes (BEST1, RPE65, and RLBP1) was also confirmed by polymerase chain reaction (PCR) at each stage of hiPSC-RPE development before, during, and after string-like aggregate formation (Figure 15-1D). RPE cells were observed expanding from the string-like aggregates into a monolayer on plates (Figure 15-1E). To determine whether expansion from string-like aggregates was accompanied by RPE proliferation, the total number of cells was increased to 2 × 10 14 days after plating the string-like aggregates. 5 The total number of cells in the expanded and remaining string-like aggregates was 5.28 × 10 on average, compared to the initial number of cells in the string-like aggregates formed with hiPSC-RPE cells on day 2. 5 This is the average cell number of the day-2 string-like aggregates (1.36 × 10 5), indicating that RPE proliferation also contributed to cell expansion from the string-like aggregates (day 2 string-like aggregates, n = 4; expansion from hiPSC(M8)-string-like aggregates, n = 6; expansion from hiPSC(201B7modFucci)-string-like aggregates, n = 6) (Figure 15-2F). VEGF and PEDF secretion was examined by the combined expanded cells and the remaining string-like aggregates at the same time point (14 days after the string-like aggregates were deposited). Expanded hiPSC-RPE secreted similar levels of VEGF and PEDF compared to the cells originally used to form the string-like aggregates (hiPSC(M8)-RPE, n = 6 and hiPSC(201B7modFucci)-RPE, n = 6; pre-string-like aggregate RPE cells of both lines served as controls, n = 4) (Figure 15-2G).

[0084] Replacement of RPE by Mimicking hiPSC-RPE Transplantation in an In Vitro RPE Damage Model with Cell Suspension or String-Like Aggregates. To investigate how transplantation of string-like aggregates or cells can compensate for damaged REEs, we 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 uncoated 24-well plates were treated with MMC to inhibit cell proliferation, and the center of the well was scratched the following day. The following day, string-like aggregates or suspension cells (201B7modFucci strain) were placed or seeded on top of the scratched area, and filling or replacement by the "transplanted" RPE cells was monitored using an Incucyte™ system (Figure 11B). In control wells (unfed wells), the existing MMC-treated RPE gradually expanded and migrated, slowly reducing the defect area. Transplanted RPE cells in suspension rapidly occupied the defect area. When using string-like aggregates, RPE expanded from the string-like aggregates until they contacted pre-existing RPE, but contact inhibition appeared to halt cell expansion from the string-like aggregates. The average defect area and the area occupied by transplanted RPE were measured and quantitatively shown in Figure 11B' (string-like aggregates and suspension; n = 5, control; n = 3) at days 1, 7, and 28 after in vitro transplantation. In string-like aggregates, the boundary between pre-existing and transplanted RPE was reasonably well identified by mCherry expression by transplanted 201B7modFucci cells and RPE cell size; MMC-treated, non-dividing RPE were generally larger than those expanded from transplanted RPE (Figure 11C). Interestingly, the gap junction marker ZO-1 was observed throughout both the pre-existing and transplanted RPE populations without any interference across the boundary between the two populations (Figure 11D). Furthermore, VEGF (Fig. 11E) and PEDF (Fig. 11F) secretions were also monitored as parameters to estimate overall RPE function in each well. VEGF secretion significantly decreased after scratching and then increased in all groups by 28 days.In the cell suspension transplantation group, VEGF secretion was almost the same as before MMC treatment. PEDF secretion decreased in the transplantation group after scratching. In the string-like aggregate transplantation group, PEDF secretion decreased once after scratching, but by 28 days there was no difference compared to before MMC treatment. In the cell transplantation group, PEDF secretion increased significantly after scratching, but without cell replacement, PEDF secretion continued to decrease (control n=4, string-like aggregate n=8, RPE cell n=7).

[0085] Transplantation of string-like aggregates into nude rats Next, we performed a 2x10 5 String-like aggregates formed from the cells were transplanted into albino nude rats to test surgical operability and post-transplant survival. The transplanted string-like aggregates were successfully stably implanted subretinally (n = 10, 10 μM Y-27632; n = 11, 2.5 μM Y-27632) onto normal RPE in the host eye. Although the presence of healthy RPE appeared to inhibit the expansion of the transplanted string-like aggregates, the aggregates survived stably for up to 10 months without unexpected tumor formation or graft loss (Figure 16A and Table 3). Optical coherence tomography imaging (Envisu R2200 VHR, Bioptigen, Inc.) revealed a clear RPE-like reflection on the graft surface (Figure 16B). Histological analysis of the same eyes revealed positive staining for the human marker Ku80 / XRCC5 in the pigment cell layer of the string-like aggregates (Figures 16C and D). These cells clearly expressed apical (ezrin) and basal (collagen type IV) markers and displayed correct polarity after transplantation (Fig. 16E).

[0086]

[0087] Finally, we used two rabbit eyes to confirm the feasibility of the surgical technique for transplanting string-like aggregates in a clinical setting. A 24G intravenous cannula was loaded with string-like aggregates (Figure 17). After standard vitreous surgery, retinal detachment was performed, and the string-like aggregates were successfully injected into the area of ​​retinal hemorrhage under good visual control (Figure 18).

[0088] Example 2: Preparation of a COP-based culture device for generating string-like aggregates of hiPSC-RPE A cycloolefin polymer (COP) plate (manufactured by Zeon Corporation) was machined to prepare a COP-based culture device with a groove measuring 20 mm in length, 1 mm in width, and 2 mm in depth (groove bottom radius: 0.5 mm) (Figure 19).

[0089] Preparation of hiPSC-derived RPE cells (hiPSC-RPE) The M8 hiPSC line (RIKEN) was differentiated using the SFEBq method, and the QHJI01s04 hiPSC line (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 stored at -150°C using the same method as in Example 1.

[0090] Preparation of string-like aggregates. After thawing and culturing for 2 weeks, hiPSC-RPE cells (M8 and QHJI01s04 strains) were harvested and then mixed with 10 μl of maintenance medium containing 5 μM Y-27632 at 2 × 10 5 The cells were seeded into each groove of the COP-based culture device at a cell number of 1000. After 2 days, the formation of string-like aggregates was confirmed.

[0091] Immediately after thawing, hiPSC-RPE cells were seeded into the grooves of the COP-based culture device, and string-like aggregates were formed two days later. This means that the time from cell thawing to string-like aggregate formation can be shortened to just two days.

[0092] Cryopreservation of string-like aggregates After production, the medium containing the string-like aggregates was replaced with STEMCELLBANKER, placed in cryotubes and temporarily stored at -80°C, after which they were frozen and stored at -150°C. After 4 months, the string-like aggregates were thawed, suspended in mixed medium and plated onto dishes. The plated string-like aggregates were confirmed to adhere to the dishes and expand.

[0093] Transplantation into Rabbit or Monkey Eyes: We confirmed that string-like aggregates could be aspirated and ejected using 25g / 31g, 25g / 33g, and 25g / 38g PolyTip cannulae (MedOne). Using a viscoelastic material (e.g., Shellgan or Viscoat) diluted 4-7 times with OptiMEM as a medium, string-like aggregates could be smoothly aspirated and ejected (Figure 20). Using these, we confirmed that string-like aggregates could be inserted into the subretinal space of rabbits and monkeys using a 38g MedOne cannula and then inserted using a 25g / 31g or 25g / 33g cannula. Similarly, after creating a detachment in a monkey eyeball, we aspirated string-like aggregates of hiPSC-RPE cells measuring 20 mm in length using approximately 2 μl of medium and inserted them into the retinal detachment site. The survival of the string-like aggregates was confirmed by fundus observation after surgery (Figure 21).

[0094] This invention makes it possible to produce a retinal pigment epithelial cell transplant material that can be prepared in a short time, at low cost, and that can be easily controlled at the transplant site while being non-invasive.This application is based on Patent Application No. 2021-078154 filed in Japan (filing date: April 30, 2021), the contents of which are incorporated in their entirety herein.

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

Claims

1. String-like aggregates of retinal pigment epithelial cells.

2. The string-like aggregate according to claim 1, characterized in that the ratio of total length to outer diameter of the body is 2 to 1,000.

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

4. The string-like aggregate according to any one of claims 1 to 3, wherein the retinal pigment epithelial cells are cells induced to differentiate from pluripotent stem cells.

5. A device for producing the string-like aggregates of claim 1, comprising: A base member is provided. the base member having an upper surface with one or more grooves; The groove has a cavity portion having the length and width of the string-like aggregate as a mold for culturing the seeded retinal pigment epithelial cells into the string-like aggregate. The device.

6. 6. The device of claim 5, wherein said groove has a cavity portion as a bottom of said groove and a top portion located above said cavity portion, said top portion comprising an opening to said groove.

7. The device of claim 5 , wherein the cross-sectional shape of the groove is V-shaped, the deepest portion of the V-shape being rounded.

8. A method for producing string-like aggregates of retinal pigment epithelial cells, comprising the steps of: (1) seeding retinal pigment epithelial cells suspended in a liquid medium into the grooves of the device according to claim 5; (2) culturing retinal pigment epithelial cells in the groove to form string-like aggregates of retinal pigment epithelial cells.

9. The method of claim 8 , wherein the medium contains a ROCK inhibitor.

10. The method of claim 9, wherein the ROCK inhibitor is Y-27632.

11. The density of retinal pigment epithelial cells cultured in the groove was 2.5x10 3 cells / mL ~ 5x10 5 The method according to any one of claims 8 to 10, wherein the concentration is cells / mL.

12. The method according to any one of claims 8 to 10, wherein the culture period of the retinal pigment epithelial cells is 1 to 7 days.

13. A pharmaceutical composition comprising the string-like aggregates of retinal pigment epithelial cells according to any one of claims 1 to 3.

14. A therapeutic agent for a disease caused by damage to the retinal pigment epithelium, comprising the string-like aggregates of retinal pigment epithelial cells according to any one of claims 1 to 3.