WO2007032224A1 - Cultured cell sheet and method for preparation thereof - Google Patents

Abstract

Disclosed is a cultured cell sheet which is expected to have a high therapeutic effect. Also disclosed is a method for preparation of the cultured cell sheet. The cultured cell sheet can be prepared by culturing an iris pigmented epithelial cell or retinal pigmented epithelial cell on a collagen gel thin film which has a thickness of 20 μm or less and is previously vitrificated.

Description

 Specification

 Cultured cell sheet and method for producing the same

 Technical field

 [0001] The present invention relates to a cultured cell sheet and a method for producing the same. Specifically, the present invention relates to a cultured cell sheet having a cell layer derived from iris pigment epithelial cells or retinal pigment epithelial cells and a method for producing the same. The cultured cell sheet provided by the present invention is used in the treatment of retinal diseases that require reconstruction of the retinal pigment epithelium, such as age-related macular degeneration, retinal pigment degeneration, diabetic retinopathy, myopic retina choroidal atrophy, and retinal pigment tumor. It can be used as a transplant material. Background art

 In animal experiments, it has been reported that fibroblast growth factor (bFGF), one of the cell growth factors, accelerates bone regeneration. This is one example of artificially controlling self-renewal using cell growth factors. For ischemic heart disease, clinical applications using similar methods have also begun. On the other hand, regenerative medicine has been introduced in the field of ophthalmology. For example, attempts have been made to treat corneal epithelial diseases using corneal epithelial stem cells and amniotic membrane (Non-patent Document 1). With the advancement of regenerative medicine, aggressive treatment has been tried for diseases that have not been treated before. On the other hand, for intractable retinal diseases, there are still no established treatments, and many of the conditions are unknown. There are many retinal diseases that become severe once they develop. Although it is not at the stage where clinical results can be evaluated, regenerative medicine using cell transplantation has been developed for retinal chromatic degeneration and age related macular degeneration (AMD), which are representative of intractable retinal diseases. Application is being attempted.

[0003] Among age-related macular degeneration (AMD), wet AMD, which is said to be common among Japanese, causes severe visual impairment due to the formation of the choroidal neovascular plate in the macular region. Or vitreous hemorrhage causes sudden visual impairment. Traditionally, it was said to be more common among whites, but it often occurs after middle age, and the number of Japanese people is increasing as the population ages. In recent years, the subretinal neovascularization that causes it is removed together with the vitreous, surgical treatment to move the macular retina, and special compounds and laser light are used. Attempts have been made to eliminate new blood vessels.

 [0004] Research on the function of the retina and retinal pigment epithelium (retinalpigment mark ithelium: RPE) in the 1970s, research on intraocular fatigue developed in the 1980s, and further on the photoreceptor protection effect by RPE and retina transplantation based on them Research has been actively conducted, and transplantation of non-self-pigmented epithelial cells and undifferentiated fetal neural retina has been attempted for patients. Comparison of RPE and iris pigment epithelial cells (irispigment sign ithelium: IPE) using molecular biology techniques, and observation of the inhibitory effect of RPE by subretinal transplantation on photoreceptor degeneration. These basic studies, global clinical studies, and clinical courses of patients with simple removal of submacular neovascular membranes were collected, and the patient's own IPE was collected and cultured with the patient's own serum. At the same time as the removal operation, a method of transplanting under the macula was performed, but although no recurrence or side effects were observed after 2 years of follow-up, the visual recovery was not satisfactory. Transplanting own IPE to the RPE countermeasures that fall out with the removal of CNV, based on the results of previous surgery on AMD and simply removing choroidal neovascularizatjon (CNV) Regenerative medicine has been tried. As a result, it was found that it is safe to culture their own IPE and transplant it under the retina. However, from the viewpoint of visual acuity prognosis, it was considered that further innovation was necessary. The method of transplanting autologous IPE after CNV removal (Non-Patent Document 2) uses IPE as a surrogate cell for retinal pigment epithelium that has been impaired after CNV removal, but its effect is far from clinical application. .

 [0005] Degeneration and loss of retinal pigment epithelium (RPE) has been shown to cause degeneration and atrophy of choroidal capillaries or directly to degeneration of photoreceptor cells. Therefore, after CNV removal due to age-related macular degeneration (AMD), attempts have been made to prevent the degeneration and disappearance of photoreceptor cells by supplementing RPE or a substitute for RPE in the area where RPE is lost or atrophyed. For this, eyeball RPE, fetal RPE, or own IPE provided by Eye Bank were used (Non-Patent Documents 3 to 7).

The iris itself is derived from the mesoderm and neuroectoderm, but IPE is derived from the neuroectoderm and is embryologically identical in origin to RPE. As described above, IPE, which is clinically easy to collect, has also been transplanted into the RPE deficient part as a substitute for the dropped RPE. However, as a therapeutic effect, there is an upper limit of 0.4 for the final visual acuity so far. There is no significant difference from the patient group who simply removed CNV. The cultured IPE shows almost no pigment and it is difficult to confirm the transplanted cells directly. Therefore, when transplanting cultured cells, it is difficult to discuss the relationship between postoperative visual function and transplanted cells. On the other hand, if IPE is isolated during surgery and transplanted directly under the retina without culturing (iris translocation), a pigmented IPE mass can be confirmed after surgery. In addition, histological examination after transplantation of cultured autologous IPE to monkeys confirmed that the cultured autologous IPE phagocytosed the outer segment of photoreceptor cells, and the similarities in the functions of cultured IPE and RPE. From the reported results, it is considered that the transplanted IPE has a possibility of having a function similar to RPE. Despite IPE transplantation, there is no significant difference in postoperative visual acuity from patients with CNV removal. Reasons for neuroretinal problems, subretinal distribution of transplanted cells, survival rate, Problems such as the viability of transplanted cells are considered.

 [0006] Non-Patent Document 1: Takahiro Nakamura, Shigeru Kinoshita: Position of amniotic membrane in eye regenerative medicine, ophthalmic surgery

 15: 5-9,2002

 Non-Patent Document 2: Abe T. et al., Tohoku J Exp Med 191: 7-20,2000

 Non-Patent Document 3: Peyman GA. Et al., Ophthalmic Surg 22: 102-108, 1991

 Non-Patent Document 4: Algvere PV. Et al., Graefes Arch Clin Exp Ophthalmol 235: 149-158,

1997

 Non-Patent Document 5: Weisz JM. Et al, Retina 19: 540-545, 1999

 Non-Patent Document 6: Abe T. et al., Tohoku J Exp Med 189: 295-305, 1999

 Non-Patent Document 7: Thumann G. et al., Arch Ophthalmol 118: 1350-1355, 2000

 Non-Patent Document 8: Del Priore LV. Et al "Am J Ophthalmol 131: 472-480, 2001

 Disclosure of the invention

 Problems to be solved by the invention

As described above, various attempts have been made to reconstruct the retinal pigment epithelium (RPE).

 However, the current situation is that a satisfactory therapeutic effect has not been obtained. Accordingly, an object of the present invention is to provide a transplant material that can be used as an alternative to RPE and that exhibits a high therapeutic effect (good reconstruction of RPE) and a method for producing the same.

Means for solving the problem The present inventors have conducted intensive studies in view of the above problems. First, based on previous reports, I thought that iris pigment epithelial cells (IPE cells) or retinal pigment epithelial cells (RPE cells) were appropriate as cells used for the reconstruction of the retinal pigment epithelium. Next, focusing on the engraftment rate, operability (easy to handle and transplant), and the function expected after transplantation, pre-organized cells, similar to living RPE, That is, it was considered preferable to transplant monolayered cells (cell sheet). In addition, the substrate as a carrier is indispensable for the construction of the cell layer. The substrate for constructing a cell sheet that can be expected to have a high therapeutic effect has the following characteristics: (1) (2) High strength, (3) Good adhesion between cells to be used, and good adhesion between cells to be used and substrate, and (4) Cells seeded on them We thought that it would be required to be able to maintain the functions of Looking at these characteristics and searching for a substrate, we found that a vitrified thin-film collagen gel (collagen gel thin film) was optimal. In particular, the collagen gel thin film has a feature that there is almost no swelling at the time of transition from a dry state to a wet state, as compared to a product made from a commonly distributed collagen such as natural collagen or collagen. It has been found that if this is used as a substrate, an extremely thin cultured cell sheet can be constructed. As the investigation proceeded and the construction of cell layers was attempted using IPE cells and RPE cells, the usefulness of the substrate was confirmed. On the other hand, as a result of devising a method for producing a collagen gel thin film, we succeeded in stably supplying a collagen gel thin film of about 10 μm or less. The present invention is mainly based on the above knowledge and provides the following configurations.

 [1] A cultured cell sheet comprising a vitrified collagen gel thin film having a thickness of about 20 zm or less, and an iris pigment epithelial cell or retinal pigment epithelial cell-derived cell layer formed thereon. .

 [2] The cultured cell sheet according to [1], wherein the collagen gel thin film has a thickness of about 5 μπι to about 10 zm.

 [3] The cultured cell sheet according to [1] or [2], wherein the collagen gel thin film is made of type I collagen.

[4] The collagen gel thin film has a support for maintaining its shape. The cultured cell sheet according to any one of [1] to [3].

 [5] The cultured cell sheet according to any one of [1] to [4], comprising one or more features selected from the group consisting of:

 (1) the cell layer has a single layer structure;

(2) the cell density of the cell layer is about 1000-10000 cells / mm ² ;

 (3) The planar view shape of the cells constituting the cell layer is a polygonal shape;

 (4) The cells are arranged almost uniformly and regularly in the cell layer;

 (5) Tight junctions are observed between cells in the cell layer;

 (6) the cell layer has phagocytic ability;

 (7) melanin deposition is observed in the cell layer;

 (8) Expression of a retinal pigment epithelial cell marker is observed in the cell layer.

 [6] The cultured cell sheet according to [5], comprising all of the features (1) to (8).

 [7] A method for producing a cultured cell sheet comprising the following steps:

 (1) providing a vitrified collagen gel thin film having a thickness of about 20 / m or less;

(2) preparing iris pigment epithelial cells or retinal pigment epithelial cells;

 (3) seeding and culturing the cells on the collagen gel thin film;

 (4) A step of recovering the collagen gel thin film and the cell layer formed thereon after culturing.

 [8] The production method according to [7], wherein the collagen gel thin film has a thickness of about 5 μπι to about 10 zm.

 [9] The production method according to [7] or [8], wherein the collagen gel thin film is made of type I collagen.

 [10] The production method according to any one of [7] to [9], wherein the collagen gel thin film is provided with a support for maintaining the shape thereof.

 [11] Substrate consisting of a vitrified collagen gel thin film with a thickness of about 10 μm or less

[Fig. 1] Schematic representation of the support used to produce a vitrified collagen gel film FIG.

 FIG. 2 is a stereomicrograph of an IPE cell sheet constructed on a collagen gel thin film.

 FIG. 3 is a stereomicrograph of an RPE cell sheet constructed on a collagen gel thin film.

FIG. 4 is a diagram showing the results (stereomicrograph) of an experiment in which beads were phagocytosed on an IPE cell sheet constructed on a collagen gel thin film.

FIG. 5 is a diagram showing the results of an experiment in which beads were phagocytosed on an RPE cell sheet constructed on a collagen gel thin film (photomicrograph).

BEST MODE FOR CARRYING OUT THE INVENTION

(the term)

 The term “cultured cell sheet” refers to a sheet-like construct comprising a cell layer formed by culturing predetermined cells collected from a living body in vitro. Similarly, the term “cultured IPE sheet” refers to a sheet-like construct comprising a cell layer formed by culturing iris pigmented epithelial cells (IPE cells) collected from a living body in vitro, The term “cultured RPE sheet” refers to a sheet-like construct comprising a cell layer formed by culturing retinal pigment epithelial cells (RPE cells) collected from a living body in vitro.

 The term “iris pigment epithelial cells (IPE cells)” is used as a comprehensive expression of cells derived from the iris pigment epithelium unless otherwise specified, ie, includes iris pigment epithelial stem cells. Similarly, unless otherwise specified, the term “retinal pigment epithelial cell (RPE cell)” is used as a comprehensive expression of cells derived from the retinal pigment epithelium, that is, includes retinal pigment epithelial stem cells.

 The term “irid pigment epithelial cell-derived cell layer” refers to a cell layer constructed by culturing iris pigment epithelial cells. Similarly, the term “cell layer derived from retinal pigment epithelial cells” refers to a cell layer constructed by culturing retinal pigment epithelial cells.

 The term “collagen gel” refers to collagen having a network structure containing a large amount of water, and is prepared by gelling a collagen sol.

The term “collagen gel dry body” means that collagen gel is thinned under vitrified conditions. The substance obtained by drying into a shape is added.

 The term “collagen gel thin film” refers to a sheet-like structure obtained by rehydrating a dried collagen gel.

 The term “vitrification” refers to changing to a hard and transparent substance by drying (Takush i E., Edible eyeballs from fish. Nature 345, 298, 1990).

(Cultivated cell sheet)

 The first aspect of the present invention relates to a cultured cell sheet. The cultured cell sheet of the present invention comprises (1) a vitrified collagen gel thin film having a thickness of about 20 μm or less, and (2) iris pigmented epithelial cells (IPE cells) or Cell layer derived from retinal pigment epithelial cells (RPE cells). Thus, the cultured cell sheet of the present invention is characterized by using a specific collagen gel thin film as a substrate for a cell layer derived from IPE cells or RPE cell vesicles.

 On the other hand, the cell layer formed on the collagen gel thin film is constructed of cells derived from IPE or RPE. This is confirmed by examining the morphology of cells contained in the cell layer and the presence or absence of expression of markers characteristic of IPE cells (or RPE cells) (eg cytokeratin, RPE65, CRALBP, pP344, Bestro phin). be able to.

 The cell layer preferably has some of the following characteristics, particularly preferably all the following characteristics.

 (1) The cell layer has a single layer structure (single layer structure). This is one of the features that biological IPE and RPE have in common.

(2) a cell density of RPE biological is about 3000 to about 5000 cells ZMM ^2. In view of this, the cell density in the cell layer of the present invention is preferably about 1000 to about 100,000 cells Zmm ² , more preferably about 2000 to about 8000 cells / mm ² .

 (3) The planar view shape of the cells constituting the cell layer is a polygonal shape. This is one of the features of RPE cells in the body. By observing this feature, the cell layer of the present invention is expected to exhibit a function similar to that of RPE, similar to the retinal pigment epithelium of a living body.

(4) Cells are almost uniformly and regularly aligned in the cell layer. For living body RPE In this case, the cells constituting the cell are uniformly and regularly arranged, thereby serving as a barrier between the retina and the choroid. Therefore, by providing such morphological features, the cell layer of the present invention is expected to exhibit the same function as RPE in a living body.

 (5) Tight junctions are observed between cells in the cell layer. The presence or absence of tight junctions can be confirmed by examining the expression of tight junction-related protein ZO-1.

 (6) The cell layer has phagocytic ability. Living RPE cells exert an important function of metabolism of photoreceptors by their phagocytic ability. As the cell layer has phagocytic ability, it is expected to exhibit the same functions as RPE.

 (7) Deposition of melanin pigment is observed in the cell layer. Melanin in retinal pigment epithelial cells protects the retina by absorbing light that could not be absorbed by the retina. Since the cell layer has melanin, RPE's retinal protective function is expected.

 (8) Expression of a retinal pigment epithelial cell marker is observed in the cell layer. The characteristics of the cultured cell sheet of the present invention and the production method thereof will be described in detail below.

 [0012] (Method for producing cultured cell sheet)

 In the method for producing a cultured cell sheet according to the present invention, the following steps (i) to (4) are performed.

 (1) Step of preparing a vitrified collagen gel thin film with a thickness of about 20 μm or less

(2) Step to prepare iris pigment epithelial cells (IPE cells) or retinal pigment epithelial cells (RPE cells)

 (3) seeding and culturing the cells on the collagen gel thin film

 (4) A step of recovering the collagen gel thin film and the cell layer formed thereon after culturing.

 Details of each step will be described below.

[0013] (1) Step of preparing a collagen gel thin film

The collagen gel thin film can be prepared according to the method described in International Publication No. 014774 (WO2005 / 014774A1) or Cell transplantation, Vol. 13, 卯 .463-473, 2004. The outline of the preparation method is shown below (for details, see the above publication and literature). First, a collagen aqueous solution (for example, type I collagen aqueous solution) having a predetermined concentration (for example, 0.5% (w / v)) and an equal amount of buffer solution (for example, phosphate buffer) are mixed. The collagen sol thus obtained is transferred to a suitable container (for example, a culture dish) and allowed to gel by allowing it to stand in a moisturizing incubator at 37 ° C. under 5% CO for 2 hours. Subsequently, the container is transferred to a clean bench under conditions of 10 ° C and 40% humidity, and left for 2 days with the container open to dry completely. As a result, the collagen gel is vitrified. The dried collagen gel thus obtained is left as it is or after rinsing with PBS or the like and subsequent drying (for example, drying under the same conditions as the above drying treatment), and then aseptic at room temperature. Store and maintain as a dried collagen gel. By securing sufficient storage and maintenance time, the strength can be increased. A collagen gel thin film can be obtained by returning the dried collagen gel to a wet state (rehydration) with an appropriate liquid (for example, a physiological saline, a buffer solution such as PBS, or a culture solution).

 In the present invention, a vitrified collagen gel thin film is used. A vitrified collagen gel thin film has excellent strength and high transparency. In addition, it can be made extremely thin during use (wet state) with very little swelling during the transition from the dry state to the wet state. In contrast, a normal collagen sheet that has not been vitrified (for example, a type I collagen sheet disclosed in JP-A-2005-229869) swells about 3 to 5 times. Considering this and the fact that it is the limit to produce a normal collagen sheet with a thickness of about 10 μm at the current technical level, conventional collagen sheets can be dried at the time of use. 3 to 5 times the thickness of the body, ie 30 μπ! ~ 50 μm. Such a thick collagen sheet lacks qualification as a substrate for cultured cell sheets that are transplanted under the retina where physical space is scarce. As is clear from the above explanation, the swelling at the time of transition from the dry state to the wet state is very small. Therefore, the extremely thin state can be obtained at the time of use when used as a substrate for a cultured cell sheet. This is the biggest advantage of the collagen gel thin film.

On the other hand, as shown in the examples described later, as a result of the study by the present inventors, both the IPE cells and the RPE cells adhere well to the vitrified collagen gel thin film, and the adhesion between cells is also improved. It was good. In addition, high cell growth efficiency can be obtained and seeded. It was found that the function of the cells retained was retained. As described above, it was revealed that the vitrified collagen gel thin film has many characteristics preferable as a substrate for constructing a cell layer derived from IPE cells or a cell layer derived from RPE cells. A cultured cell sheet (cultured IPE sheet or cultured RPE sheet) obtained using a vitrified collagen gel thin film retains strength suitable for transplantation, and can be expected to have a high therapeutic effect that is easy to handle. It will be a thing.

 [0015] The thickness of the collagen gel thin film used in the present invention is about 20 zm or less. By using such a very thin collagen gel thin film, an extremely thin cultured cell sheet suitable for RPE transplantation is constructed. Here, the intimal part of the eyeball to which the cultured cell sheet is transplanted is scarce in physical space. Therefore, the substrate as a carrier should be as thin as possible. On the other hand, the substrate is required to have sufficient strength to support the cell layer. Considering these points, the thickness of the collagen gel thin film as a substrate is preferably about 5 μm to 7j¾¾20 μm, more preferably about 5 μm to about 15 μm, and still more preferably about 5 μm to about 10 μm.

 [0016] The type and origin of collagen constituting the collagen gel thin film are not particularly limited. Collagen types include type I collagen, type III collagen, type IV collagen, type VIII collagen and the like. A collagen gel thin film is constructed in a state where multiple types of collagen are mixed.

 It is preferable that the collagen gel thin film is composed of type I collagen (or main component is type I collagen). This is because very good growth of IPE cells and RPE cells is observed on a collagen gel thin film made of type I collagen (see Examples below), and type I collagen is easy to supply stably.

 Examples of the origin of collagen include horse, ushi, pig, hidge, monkey, chimpanzee, and human. You can also use (recombinant) human collagen prepared by gene recombination technology. Of these, collagen from recombinant human collagen, horse, ushi, or pig is preferred. This is because it is easy to obtain.

[0017] A physiologically active substance may be added to the collagen gel thin film. Physiologically active substances here include cell growth factor, differentiation-inducing factor, cell adhesion factor, site force in, angiogenesis Inhibitors, analgesic anti-inflammatory agents and the like can be exemplified. Different physiologically active substances may be added in combination.

 [0018] In principle, the higher the strength of the collagen gel thin film, the better from the viewpoint of handling the cultured cell sheet and maintaining the shape after transplantation. Therefore, preferably, a collagen gel thin film having a compressive fracture strength of ¾ times to 20 times (about 100 g to about 800 g when measured under the following measurement conditions) as compared to the state before vitrification (collagen gel). used. A collagen gel thin film that satisfies this condition can be obtained by keeping it for about 40 days or more when it is dried at room temperature. In order to homogenize the gelation of the collagen contained, it is desirable to control the gelling process at 15 ° C or 4 ° C. In addition, air drying at normal temperature is desirable for drying, but a method performed in this field such as reduced pressure drying or freeze drying can be used. Detailed conditions can be changed as appropriate using the strength, transparency, uniformity, cell adhesion, and ease of peeling of container force during the production of the collagen gel thin film as indices.

 <Measurement conditions for compressive fracture strength>

 Contact area with Nidec Kogyo's strength measuring machine (digital force gauge) 1.1

Attach a 3 cm ² circular adapter, press the thin film at a speed of 9 mm / min, and measure the maximum load when the thin film breaks.

 [0020] On the other hand, the higher the transparency of the collagen gel thin film, the better, in order to increase the transparency of the cultured cell sheet to exert a high therapeutic effect. Therefore, preferably, a collagen gel thin film having an absorbance at 400 nm of 10% to 70% (absorbance of about 0.1 to 0.2) as compared to the state before vitrification (collagen gel) is used. A collagen gel thin film satisfying this condition can be obtained by storing and maintaining after drying for about 40 days or more. The gelation process can be controlled at 15 ° C or 4 ° C. In addition, although air drying at normal temperature is desirable, a method performed in this field such as reduced pressure drying or freeze drying can be used.

[0021] Here, in the present invention, it is preferable to prepare a collagen gel thin film with a support. By using the support, the morphology of the collagen gel thin film can be maintained. At the same time, peeling from the container during the production of the thin film becomes easy. As a result, it is possible to ensure and maintain the flatness of the collagen gel thin film at the time of cell seeding or during the following culture. Cell adhesion, proliferation, and organization are improved, and a high-quality cell layer is constructed. Further, when the formed cell layer is recovered together with the collagen gel thin film, the collage gel thin film can be peeled off from the culture vessel relatively easily by grasping the support with tweezers. That is, by using the support, the operability when collecting the prepared cultured cell sheet is improved. Furthermore, since the cultured cell sheet can be collected while maintaining its shape by the support, it can be transferred to a transplantation device without damaging the formed cell layer. However, a highly flat cultured cell sheet suitable for transplantation can also be obtained.

[0022] The collagen gel thin film with the support can be prepared, for example, by the following procedure.

 First, a nylon membrane molded into a donut shape is prepared as a support. Next, put the nylon membrane into the culture dish. Thereafter, collagen sol is prepared and added to the culture dish, gelled, and vitrified by the procedure described above. With the above steps, a collagen gel thin film having a support attached on one side of the outer peripheral portion is completed.

 Examples of the material of the support include synthetic fibers (synthetic resins) such as nylon, natural fibers such as cotton, bioabsorbable materials such as polylactic acid, and metals. On the other hand, the shape of the support is preferably circular (circle, ellipse, square, etc.).

 In the case of a cultured cell sheet formed using a collagen gel thin film with a support, the support is used before transplanting the cultured cell sheet to the patient (from the preparation of the cultured cell sheet to immediately before transplantation of the cultured cell sheet). Is removed.

[0023] (2) Step of preparing cells (IPE cells or RPE cells)

 (a) IPE cells

 (a_l) IPE cell collection

IPE cells can be collected routinely from the recipient himself or from the iris of a suitable donor. For example, first, a peripheral iris excision is performed under local anesthesia, and an iris piece is collected. Epithelial cells are separated from the collected iris pieces by trypsinization or manual methods. The separated epithelial cells (IPE cells) are cultured in an appropriate culture medium in which IPE cells can grow as necessary. As the culture solution, for example, a commercially available F12 medium supplemented with serum, antibiotics (amphotericin, gentamicin, penicillin, streptomycin, etc.) and the like can be used. [0024] As serum to be added to the medium, human serum, fetal bovine serum, sheep serum and the like can be used. Among them, the ability to use serum derived from the same species (human serum), it is preferable to use autologous serum (ie, the recipient's own serum). Of course, if possible, it is most preferable to use autologous serum that eliminates the risk of causing immune rejection. For example, protein components derived from heterologous animals such as commercially available Epi Life ™ (Cascade), MCDB153 medium (Nissui Pharmaceutical Co., Ltd.), and media prepared by modifying the amino acid composition of these mediums are completely removed. A serum-free medium containing no serum can also be used. That is, a serum-free culture method may be employed as the culture method in the present invention. In such an embodiment, problems such as rejection of immunity due to contamination of serum-derived components can be avoided.

 [0025] It is preferable to use a culture container (culture dish) whose surface is coated with type I collagen, type IV collagen, fibronectin, laminin or the like. This is because adhesion of IPE cells to the surface of the culture container is promoted, and good growth is performed. In particular, it is preferable to use a culture vessel coated with type I collagen, type IV collagen, or fibronectin (see Examples below). Alternatively, the surface of a synthetic polymer or the like is treated with a nanotechnology technique and a material having an interface structure suitable for IPE cell growth is used.

 [0026] The temperature conditions for culturing IPE cells are not particularly limited as long as the IPE cells grow. For example, about 25 ° C to about 45 ° C, preferably about 30 ° C in consideration of the growth efficiency. To about 40 ° C, more preferably about 37 ° C. The culture time (that is, the time of primary culture) for subsequent subculture is, for example, 1 to 60 days, although it varies depending on the state of the cells used.

 Here, when available, it is preferable to use the recipient's own IPE cells. This is because it is possible to produce a cultured IPE sheet that does not cause immune rejection when transplanted, that is, transplantation without immune rejection becomes possible. If the recipient's own IPE cells are not available or difficult to obtain, non-recipient IPE cells can be used, but in this case the donor should be selected in consideration of immunocompatibility. preferable.

[0027] (a-2) Subculture, cell suspension preparation Subculture can be performed after the IPE cells subjected to culture have proliferated. Preferably, subculture is performed when subconfluent and confluent. Subculture can be performed as follows. First, after removing the cell culture medium, the cells are washed with PBS (−) solution, treated with trypsin-EDTA or the like to peel the cells from the surface of the culture container, and the cells are collected in the next step. A culture solution is added to the collected cells to prepare a cell suspension.

 The cell suspension is seeded in a culture vessel in the same manner as in the primary culture described above and used for culture. Subculture can be performed under the same culture conditions as the above primary culture. The culture time varies depending on the state of the cells used and is, for example, 1 to 60 days. The above subculture can be performed multiple times as necessary. By repeating subculture, the number of cells can be increased and a cell suspension with a high cell density can be prepared. It is preferable to finally prepare a cell suspension having a cell density of about 20,000 cells / ml to 100,000 cells / ml.

[0028] (b) RPE cells

 (b-l) RPE cell collection

 RPE cells can be collected in a conventional manner from the recipient itself or from the subretinal area of a suitable donor. For example, a scleral incision is first performed under local anesthesia, and the retinal pigment epithelium under the peripheral retina sufficiently separated from the macula is removed. Epithelial cells are separated from the collected retinal pigment epithelium by trypsin treatment or manual method. The separated epithelial cells (RPE cells) are cultured in an appropriate culture medium in which RPE cells can grow as necessary. As the culture medium, for example, a medium obtained by adding serum, antibiotics (amphotericin, gentamicin, penicillin, streptomycin, etc.) to a commercially available DMEM medium can be used.

 (b-2) Passage culture, cell suspension preparation

 RPE cell passages and cell suspensions can be prepared using the same procedures and conditions as for IPE.

 [0029] Step (1) and step (2) described above are performed independently. Therefore, the power to say that any step can be performed first (or both at the same time). Considering the nature of the material (ie, collagen, cells) handled in each step, the preparation of the collagen gel thin film is usually preceded. And implement.

[0030] (3) Step of seeding and culturing cells In this step, the cells (IPE cells or RPE cells) prepared by the above procedure are seeded on the collagen gel thin film prepared by the above procedure. Specifically, for example, a culture solution is added to a dried collagen gel prepared in a culture vessel and incubated (for example, 37 ° C, about 5 to 20 minutes). The cell suspension is seeded on the vitrified collagen gel thin film thus obtained. At this time, it is preferable to adjust the number of cells to be seeded so that a cell layer having a desired cell density is formed in the finally prepared cultured cell sheet. Specifically, for example, about 100 to about 5000 cells, preferably about 500 to 1000 cells are seeded per 1 mm ² so that a cell layer with a cell density of about 1000 to about 10,000 cells Zmm ² is formed. To do. The culture can be performed under the same conditions as the primary culture described above. The culture time varies depending on the state of the cells used, but is, for example, 1 day to 60 days.

[0031] The cell culture here is preferably performed in the absence of heterologous animal cells. In the present invention, “in the absence of heterologous animal cells” means that animal cells that are heterologous to the cells are not used as conditions for culturing IPE cells or RPE cells. Specifically, when human cells are used as IP E cells or RPE cells, the conditions are such that cells of animal species other than humans, such as mice and rats, do not exist (coexist) in the culture medium. By culturing under such conditions, there is no possibility that components derived from different species (including heterologous cells themselves) are mixed into the cell components of the transplant material (ie, cultured cell sheet) finally obtained. The primary culture (and subculture as necessary) is also preferably performed in the absence of heterologous animal cells.

 [0032] Rather than seeding the collected IPE cells or RPE cells once cultured and grown and then seeding on the collagen gel thin film, it may be seeded directly on the collagen gel thin film and cultured. That is, the culture process (primary culture and subculture of IPE cells or RPE cells) performed before seeding on the collagen gel thin film may be omitted. In this way, the culturing operation can be simplified, and the cell layer can be constructed by a substantially one-step culturing operation.

 [0033] (4) Step of collecting cultured cell sheet

By culturing by the above method, a cultured cell in which a cell layer composed of IPE-derived cells (when IPE cells are used) or RPE-derived cells (when RPE cells are used) is formed on a collagen gel thin film. A sheet is obtained. Contact between the culture vessel and the collagen gel film The cultured cell sheet can be collected by releasing the attachment. For example, the collagen gel thin film is peeled off using tweezers or a knife. Here, when a cultured cell sheet is produced using a collagen gel thin film with a support, the collage gel thin film can be peeled off relatively easily in the culture container by grasping the support with tweezers. In addition, the support can maintain the shape of the collagen gel thin film and the cultured cell sheet at the time of peeling. Thus, if a collagen gel thin film with a support is used, a cultured cell sheet in good condition can be collected by an easy operation.

 [0034] (Provided form of cultured cell sheet)

 The cultured cell sheet of the present invention is provided, for example, in a state of being stored in a glass or plastic container and immersed in a cell culture medium or a storage solution such as a UW solution.

 [0035] (Applicable cases of cultured cell sheet)

 The cultured cell sheet prepared by the above method is a transplant material for the treatment of diseases that require RPE reconstruction, such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, myopic choroidal atrophy, and retinal pigment tumors. It can be used as an alternative to the pigment epithelium.

 [0036] (Method of transplanting cultured cell sheet)

 As an example of a method for transplanting cultured cell sheets, the method for transplanting to age-related macular degeneration (AMD) patients is described below.

 First, the presence of choroidal neovascularization (CNV) is confirmed prior to treatment. This is usually done by observing changes in bleeding and eluate around the macula, fluoresceinangiography (FAG) or mdocyamne green angiography (for Ij-G noriki lj 2 · d.

 3 ports are prepared and the vitreous body is excised with a vitreous cutter, and is weak against the retina near the CNV that has been confirmed by a scanning laser ophthalmoscope (S LO) examination before surgery After solidification of diathermy, make a small hole and grasp CNV with an insulator to remove it. After excision of CNV, the subretinal space is washed with a cell transplant needle to form a shallow retinal detachment state. The cultured cell sheet is transplanted into the space thus formed.

 Example

 [0037] 1. Preparation of vitrified collagen gel thin film

Thickness prepared according to the method described in WO 2005/014774 pamphlet A vitrified collagen gel thin film having a thickness of about 20 μm was obtained from Asahi Techno Glass Co., Ltd. (Chiba, Japan). The outline of the method for preparing the collagen gel thin film is described below.

 First, a circular support as shown in FIG. 1 was prepared by cutting a nylon membrane (Amersham # RPN1782B), sterilized, and placed in a hydrophobic polystyrene 24-wall plate. A cell culture solution 0.5% type 1 collagen aqueous solution (Ushi type I collagen, Nitta Gelatin Co., Ltd.) was placed in a sterile conical tube cooled on ice and mixed uniformly. The collagen mixture was placed in a 24-well plate containing the above support, and then gelled by maintaining it in a humidified incubator at 37 ° C in the presence of 5% CO / 95% air for 2 hours. This final concentration 0.25% collagen gel was vitrified by aseptically drying for 2 days in a clean bench under conditions of 10 ° C and 40% humidity with the lid removed. By adding 3 ml of PBS to this, the dried vitrified collagen gel was rehydrated. Rinse several times with 3 ml of PBS. Furthermore, this collagen gel thin film was aseptically dried for 2 days in a clean bench under conditions of 10 ° C and 40% humidity with the lid removed. Thereafter, it was kept aseptically stored at room temperature. In the following experiments, a dried collagen gel that had been stored for 40 days or longer was used. In addition, when stored and maintained for 40 days or more, the collagen gel thin film obtained by returning the dried collagen gel to a wet state has very high transparency and strength (for details, see International Publication No. 2005Z014774 pamphlet). In addition, almost no swelling is observed when moving from a dry state to a wet state.

2. Cultivation using animal cells Preparation of IPE sheet (1)

 A cultured IPE sheet using porcine IPE cells was prepared by the following procedure.

2- 1. Tissue collection

After euthanizing pigs slaughtered for food purposes, the eyeballs were removed from the corpses. The eyeball was immersed in about 20 ml of intraocular perfusate containing penicillin and streptomycin within 10 hours, and the following treatment was performed within 1 hour. The outer part of the cornea was cut about 5 mm with scissors and separated into an anterior ocular tissue containing the cornea and a posterior ocular tissue containing the retina. An eye tissue force was removed using cesi. The iris pigmented tissue could be obtained by thoroughly washing with an intraocular perfusate except for the jelly-like substance contained therein. Subsequent processing is When there was no description, it carried out paying attention to aseptic operation in the clean bench.

 [0039] 2- 2. Isolation of IPE cells

 2- Transfer the iris pigmented tissue collected in 1 to a 60 mm dish containing 6 ml of BSS (Santen Pharmaceutical) and then move to a 60 mm dish containing 6 ml of BSS containing lOmM EDTA and leave it still at 37 ° Incubated for 10 minutes. Remove dish from incubator, IPE culture solution (F12 medium (Kohjin bio) 450 ml, FCS (ICN) 50 ml, amphotericin (Wako) 1.25 mg, gentamicin (Gibco) 250 z 1, bFGF (Promega) 5 μg) 2 ml After the addition, epithelial cells were carefully detached from the tissue using a scraper. The iris pigmented epithelial cells are black, and the area of the iris tissue from which the cells have been detached becomes pale and skin-colored. Therefore, cell detachment was continued until the black area could not be visually confirmed from the iris tissue. After removing the cell detachment iris tissue, the solution was aspirated with a pipette and poured into a 15 ml centrifuge tube, and 2 ml of the IPE culture solution was poured into the dish and again poured into a 15 ml centrifuge tube to wash the dish. After another washing operation, centrifugation was performed at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, 5 ml of IPE culture solution was added and the pellet was resuspended, followed by centrifugation at 4 ° C and 3000 rpm for 2 minutes. The cell pellet was recovered by removing the supernatant.

 [0040] 2- 3. Culture of IPE cells

 Unless otherwise stated, IPE medium was used for IPE cell culture. 2-4 To the tube containing the pellet obtained in 2 above, 4 ml of IPE medium was added to suspend the pellet, and the whole solution was added to a 6-well plate (IWAKI) coated with type I collagen. One extra IPE cell is added to one well. Cell culture is incubator with 5% CO and 37 ° C

 2

Went in. The medium was not changed after the culture, and the plate was left to stand. This is the period when IPE cells adhere to the plate, and the cells were observed daily. It takes about 7-10 days for the cells to adhere to the plate. Thereafter, the IPE medium was changed every 3 days. The medium is exchanged according to the following procedure. First, the IPE culture solution was removed using an lml pipette, and the PBS was removed by washing the lml cache plate, and then washed. When culturing IPE cells, black granules are seen in the medium. This granule is removed by the washing operation. The PBS washing operation is repeated until no granules are observed in the collected PBS. Normally, the washing operation is performed 5 times at the first medium change, The medium was changed twice. Thereafter, the IPE culture solution was returned to the lml cache incubator and the culture was continued. The cells became confluent after 2 weeks of culture, and the number of cells reached about 5-7 × 10 ⁵ cells / well.

[0041] 2-4. Passage of IPE cells

 Experiments were conducted using wells that had reached confluence by culturing for 3 weeks. The IPE culture solution was removed using a 1 ml pipette, 0.05% trypsin / EDTA solution (Invitrogen) was added to each well and placed in a 37 ° C incubator. After an appropriate period of time, the plate was observed under a stereomicroscope while shaking, and the treatment was continued until almost all cells were floating. Usually, trypsin reaction time was 15 minutes. Into the well, lml of the IPE culture solution was poured into a 15ml centrifuge tube using a pipette. 1 ml of IPE culture solution was put into the well and poured into a 15 ml centrifuge tube again to wash the well. After another washing operation, centrifugation was performed at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, 16 ml of IPE culture solution was added to suspend the pellet, and 4 ml of each was added to a 6-well plate coated with type I collagen, and transferred to an incubator with 5% CO and 37 ° C. The cells were allowed to stand until they were attached to the plate, and this period was usually 2-3 days. After the cells adhered, the medium was changed once every 3 days in the same manner as in 2-3.

[0042] 2- 5. Preservation of IPE cells

Experiments were carried out using cells that had reached confluence after culturing for 3 weeks by the method described in 2-3. Cells were collected in a 15 ml tube in the same manner as in 2_4. Then, 5 ml of bun bunker (Nippon Gentex) was added and the pellet was resuspended and centrifuged at 4 ° C and 3000 rpm for 2 minutes. Remove the supernatant and collect the cell pellet. Add 10 ml of bunker to suspend the pellet. Add 1 ml of the bunker to a 2 ml cryogenic tube and place it in a deep freezer at 80 ° C in advance. Then, it was transferred into an insulated container and frozen at _80 ° C. One day later, the tube was transferred to a liquid nitrogen container and left for one week. One week later, the tube was transferred to a high-temperature bath preliminarily kept at 37 ° C and thawed immediately. The solution in the tube was transferred to a 15 ml tube, 4 ml of IPE medium was added, and centrifuged at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, add 1 ml of IPE culture solution, suspend, and inoculate the cells on a 24-well plate coated with type I collagen and incubate under conditions of 5% CO and 37 ° C. The culture was carried out. Observation was performed one week later, and cells adhering to the plate were observed.

[0043] 2-6. Construction of cell layer

A pellet containing 5 X 10 ⁴ IPE cells was prepared according to 2-4, and 2 ml of IPE culture solution was added and suspended to obtain a cell suspension. On the other hand, the IPE culture solution was placed in a 24-well plate prepared in step 1 to which a dry collagen gel with a thickness of 20 μm was attached, and incubated at 37 ° C for 15 minutes. The dried body was rehydrated. After adding 1 ml of the above cell suspension onto the wet collagen gel thin film (about 20 μm thick) obtained as a result, the plate was transferred to an incubator with 5% CO and 37 ° C, and cultured. did. After culturing, the IPE medium was changed every 3 days using the same method as in 2-3. About 2 to 3 days after the start of culture, the cells adhered to the collagen gel thin film and reached confluence about 2 weeks later (Fig. 2).

[0044] 3. Cultivation using animal cells Production of RPE sheet (1)

 A cultured RPE sheet using porcine RPE cells was prepared by the following procedure.

 3-1. Tissue collection

 After euthanizing pigs slaughtered for food purposes, the eyeballs were removed from the corpses. The eyeball was immersed in about 20 ml of intraocular perfusate containing penicillin and streptomycin within 10 hours, and the following treatment was performed within 1 hour. The outer part of the cornea was cut about 5 mm with scissors and separated into anterior ocular tissue containing the cornea and posterior ocular tissue containing the retina. Tissue strength was obtained by removing the vitreous body and retina using cesi, and then removing the scleral retinal pigment membrane and the choroid present on the sclera side. Retinal pigment tissue could be obtained by thorough washing with intraocular perfusate. Subsequent processing was performed with care in aseptic operation in a clean bench unless otherwise stated.

[0045] 3-2. Isolation of RPE cells

Transfer the retinal pigment tissue collected in 3-1 to a 60 mm dish containing 6 ml of BSS (Santen Pharmaceutical) and then move to a 60 mm dish containing 6 ml of BSS containing lOmM EDTA and leave it at 37 ° Incubated for 10 minutes. The dish is removed from the incubator and RPE culture solution (DMEM medium (Kohjin bio) 450 ml, FCS (ICN) 50 ml, amphotericin (Wako) 1.25 mg, After adding 2 ml of ntamycin (Gibco) 250 μl), epithelial cells were carefully detached from the tissue using a scraper. The retinal pigment epithelial cells are black, and the area of the retina pigment tissue from which the cells have been detached becomes white. Therefore, cell detachment was continued until the black area from the retinal pigment tissue could not be visually confirmed. After removing the cell detachment retinal pigment tissue, the solution was aspirated with a pipette and poured into a 15 ml centrifuge tube, and 2 ml of RPE culture solution was poured into the dish and again poured into a 15 ml centrifuge tube to wash the dish. After another washing operation, centrifugation was performed at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, 1 ml of 0.05% trypsin / EDTA solution was added and re-suspended, and left in a 37 ° C incubator for 2 minutes. Centrifugation of 2 ml of RPE medium at 3000 卬 m for 2 minutes was performed. After removing the supernatant, 5 ml of RPE medium was added and the pellet was resuspended and centrifuged at 4 ° C and 3000 rpm for 2 minutes. The cell pellet was recovered by removing the supernatant.

[0046] 3-3. Culture of RPE cells

RPE medium was used for RPE cell culture unless otherwise stated. 3- In the tube containing the pellet obtained in 2 above, 4 ml of RPE medium was added to suspend the pellet, and the entire solution was added to a 6-well plate (IWAKI) coated with type I collagen. This means that one eye has RPE cells per eye. Cell culture was performed in an incubator with 5% CO and 37 ° C. The medium was not changed after the culture, and the plate was left to stand. This was the time when RPE cells adhered to the plate, and the cells were observed daily. It takes about 7-10 days for the cells to adhere to the plate. Thereafter, the RPE medium was changed every 3 days. The medium was exchanged by the same procedure as 2-3. Cells became confluent after 3 weeks of culture and the cell count reached approximately 3-5 x 10 ⁵ cells / well.

[0047] 3-4. Passage of RPE cells

 Experiments were carried out using wells with cells that reached confluence by culturing for 4 weeks. Cells were passaged using the same method as in 2-4.

[0048] 3-5. Preservation of RPE cells

RPE cells were preserved in the same manner as 2-5. Cryopreservation One week after RPE culture Attempting to cultivate in lysis, cells observed to adhere to the plate one week after the start of culture. It was.

 [0049] 3 -6. Construction of cell layer

 A cultured RPE sheet was prepared in the same manner as in 2- 6. About 2 to 3 days after the start of sheet preparation, the cells adhered to the collagen sheet and reached confluence after about 3 weeks (Fig. 3).

 [0050] 4. Cell sheet evaluation

 (a) Presence or absence of phagocytic ability (1)

The phagocytic ability of the cultured IPE sheet was tested using latex beads. In the test, the cultured IPE sheet obtained in 2-6. Was used. First, 1.0 × 10 ⁹ uncoated latex beads (polyscience) having an average diameter of 1 μm were placed in an IPE culture solution in which an IPE sheet was immersed. The bead is 1.0 X 10 ⁹ pieces / lml / 1 sheet. A 24-well plate containing a cultured IPE sheet was placed in a 37 ° C incubator and allowed to stand. After 6 hours, the latex bead-containing IPE medium was removed. After adding 1 ml of PBS and shaking the plate, washing was performed by removing PBS. This washing operation was performed until latex beads were no longer observed in the removed PBS, and was usually performed five times. By carrying out the washing operation, free beads that have not been taken up into the cells are removed from the culture medium. After completion of the washing operation, PBS was removed, lml of 100% acetone was added to the well, and the mixture was allowed to stand for 15 minutes. By this operation, the cells are fixed. Acetone was removed, and the well was washed by adding 1 ml of PBS. The washing operation was performed twice. The culture IPE sheet was carefully peeled from the well using cesi, and the culture IPE sheet was placed on a slide glass so that the cell surface was up. 30 μl of mounting medium (Vector) was added to the cultured IPE sheet, sealed with a cover glass, and then observed under a microscope. When beads are taken up into cells, small granules are observed, so that the phagocytic ability can be evaluated by observing the presence or absence of granules compared to the cultured koji sheet without taking up beads. As a result, it was confirmed that granules were observed only in the cultured koji sheet to which beads were added, and that the cultured koji sheet had phagocytic ability (Fig. 4).

 The phagocytic ability of the cultured RPE sheet obtained in 3-6 was also evaluated using the same procedure. Granules were observed only in the cultured RPE sheet with beads added, confirming the phagocytic ability (Fig. 5).

[0051] (b) Presence of phagocytic ability (2) It was verified whether or not the cultured IPE sheet has phagocytic ability to the optic nerve outer segment. First, an outer optic nerve segment was obtained using a butterfly eyeball. The eyeballs used were porcine tissues slaughtered for food in Ikeda Foods Co., Ltd. A portion of the porcine eyeball 5 mm outside the corneal ring was cut into a circle, and the eyeball was separated into the anterior and posterior segments. Thereafter, the eye part was cut into two parts with scissors, and the vitreous body was removed using cesi to obtain a retina. After thorough washing with BSS, it was transferred to a 60 Φ dish containing 6 ml of BSS and the tissue on the choroid side was rubbed with a scraper. After removing the retinal tissue, the pellet was obtained by centrifuging at 4 ° C and 6000 mm for 3 minutes and removing the supernatant. This pellet contains mainly the optic nerve outer segment. The pellet was suspended by adding 500 μl of pH 8.0 PBS containing 100 mM NaCl, and 1 mg of fluorescent dye SNAEFL succinimidino ester dissolved in 100 μl of anhydrous dimethylformamide was collected. Fluorescent labels were introduced by standing at room temperature in a dark place for 30 minutes. After centrifuging at 6000 μm for 3 minutes to remove the supernatant, lml of PBS was added to suspend and centrifuged, followed by washing. After three washing operations, the suspension was resuspended in 200 μΐ PBS.

 Add the optic nerve outer segment labeled in the above procedure to the culture obtained in 2-6. 50 μΐ per well, and incubate for 3 hours under conditions of 5% CO and 37 ° C.

 2

did. After removing the medium, lml of DMEM / F12 was added and the plate was shaken, and then washing was performed by removing DMEM / F12. This washing operation was usually performed 5 times. The optic nerve segment that was not taken up by the cells is removed during the washing process. The cell sheet was observed with a fluorescence microscope to observe phagocytosis of the optic nerve outer segment to the cells. After washing, lml of 100% acetone was added to the well and left for 15 minutes. By this operation, the cells are fixed. Acetone was removed, and 1 ml of PBS was added to the well for washing. The washing operation was performed twice. The culture IPE sheet was carefully peeled off from the tool using sushi, and the culture IPE sheet was placed on a slide glass so that the cell surface was up. 30 μL of mounting medium (Vector) was placed on the sheet, sealed with a cover glass, and then observed under a fluorescence microscope. Since fluorescence is observed when the optic nerve outer segment is taken up into the cell, phagocytic ability can be evaluated by observing the presence or absence of fluorescence in comparison with an acupuncture sheet without optic nerve outer segment added. As a result, fluorescence was observed only in the cultured ΙΡΕ sheet with the optic nerve outer segment added, confirming that the cultured ΙΡΕ sheet has phagocytic ability. The phagocytic ability of the cultured RPE sheet obtained in 3-6. Was also evaluated in the same manner. As a result, fluorescence was observed only in the cultured RPE sheet with the nerve outer segment covered, and the phagocytic ability was confirmed.

 [0052] 5. Cultivation using animal cells Production of IPE sheet (2)

 Preparation of cultured IPE sheets using monkey IPE cells was attempted. The operating procedure was the same as when porcine IPE cells were used. Monkey IPE cells grew slightly slower than porcine IPE cells, and took 2-3 weeks to reach confluency during culture, but cultured IPE sheets equivalent to those using porcine IPE cells Was able to be produced. From this result, it was confirmed that the above-described production method was excellent in versatility that was not effective only when porcine IPE cells were used.

 [0053] 6. Cultivation using animal cells Production of RPE sheet (2)

 An attempt was made to prepare cultured REP sheets using monkey RPE cells. The operating procedure was the same as when porcine IPE cells were used. Although monkey RPE cells differed from porcine RPE cells in that the cell growth was slightly slower, it took 3 to 4 weeks to reach confluence, but when cultured with porcine RPE cells, the same culture RPE sheet was used. Was able to be produced. From this result, it was confirmed that the above production method is not effective only when porcine RPE cells are used, and is excellent in versatility.

 [0054] 7. Preparation of cultured IPE sheet using human autologous cells

 7- 1. Tissue collection

 An incision is made from the patient's informed consent through the upper corneal region under local anesthesia, and the upper iris is removed surgically, and the iris pigmented tissue is removed approximately 2 X 2 mm and the tissue is removed. The excised tissue should be immersed in about 2 ml of intraocular perfusate and the following treatment should be performed within 1 hour. If there is no other description, perform the subsequent processing with care for aseptic operation in a clean bench.

[0055] 7- 2. Isolation of IPE cells

7- Move the iris pigment tissue collected in 1. to a 60 mm dish containing 6 ml of BSS (Santen Pharmaceutical), and then move to a 60 mm dish containing 6 ml of BSS containing lOmM EDTA. Incubate for 10 minutes. Take out the dish from the incubator, IPE culture solution (F12 medium (Kohjin bio) 450 ml, FCS (ICN) 50 ml, amphotericin (Wako) 1.25 mg, Genta 2 ml of mycin (Gibco) 250 z 1, bFGF (Promega) 5 μg) is added, and then the epithelial cells are carefully detached from the tissue using a scraper. The iris pigment epithelial cells are black, and the area of the iris tissue from which the cells have been detached becomes a light skin color. Therefore, detachment of the cells is continued until the black area cannot be visually confirmed from the iris tissue. After removing the cell detachment iris tissue, aspirate the solution with a pipette and inject it into a 15 ml centrifuge tube. Add 2 ml of IPE culture solution to the dish, and again inject into the 15 ml centrifuge tube to wash the dish. After another washing, centrifuge at 4 ° C and 3000rpm for 2 minutes. After removing the supernatant, add 5 ml of IPE culture solution, resuspend the pellet, and centrifuge at 4 ° C and 3000 rpm for 2 minutes. Collect the cell pellet by removing the supernatant.

[0056] 7- 3. Culture of IPE cells

 Use IPE medium unless otherwise specified for IPE cell culture. 7— Add lml of IPE medium to the tube containing the pellet obtained in step 2 to suspend the pellet, and add the whole solution to a 24 well plate (IWAKI) coated with type I collagen. One tissue worth of IPE cells is added to one tool. Cell culture is performed in an incubator with 5% CO and 37 ° C. After the culture, the medium is not changed, and the plate is left still. This is the period when IP E cells adhere to the plate, and the cells are observed daily. It takes about 7-10 days for the cells to adhere to the plate. After that, change the IPE medium every 3 days. The medium is exchanged according to the following procedure. First, remove the IPE broth using an lml pipette, add lml of PBS, shake the plate gently, remove the PBS, and wash. When culturing IPE cells, black granules are seen in the medium. The force that this granule is removed by the washing operation Repeat the PBS washing operation until no granules are observed from the recovered PBS. Usually, the washing operation is performed 5 times for the first medium change and twice for the subsequent medium change. Then, add lml of IPE medium and return to the incubator to continue the culture. Cells become confluent about 2-3 weeks after the start of culture.

[0057] 7-4. Passage of IPE cells

Experiment with a well with cells that have reached confluence in a 24 well plate. Remove the IPE culture solution using an lml pipette, add 0.05 ml of 0.05% trypsin / EDTA solution (Invitrogen), and leave it in a 37 ° C incubator. After a certain amount of time has passed While observing under a stereomicroscope while shaking the rate, continue the treatment until almost all cells float. Usually, trypsin reaction time is 15 minutes. Add lml of IPE culture solution to the well and inject into a 15ml centrifuge tube using a pipette. Add lml of IPE culture solution to the well and pour it again into a 15ml centrifuge tube to wash the well. Repeat the washing procedure, and then centrifuge at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, add 4 ml of IPE broth to suspend the pellet, add 1 ml of each well to a 24-well plate coated with type I collagen, and transfer to an incubator with 5% CO at 37 ° C. . The cells are allowed to stand until they adhere to the plate, and this period is usually about 2 to 3 days. After the cells have adhered, the medium is changed once every 3 days in the same way as in 7-3.

[0058] 7-5. Preservation of IPE cells

 Incubate for 3 weeks using the method described in 7-3 and then use the cells that have reached confluent cells. Collect cells in a 15 ml tube in the same manner as in 7-4. Then, add 5 ml of bun bunker (Nihon Genenetics), resuspend the pellet, and centrifuge at 4 ° C and 3000 mm for 2 minutes. Remove the supernatant and collect the cell pellet. Add lml of bun bunker to the pellet, add it to the 2ml cryogenic tube, place it in a deep freezer at 80 ° C, and transfer it to the insulated container. Freeze at –80 ° C. After one day, transfer the tube to a liquid nitrogen container and leave it for a week. After one week, transfer the tube to a high-temperature bath pre-incubated at 37 ° C and immediately thaw. Transfer the solution in the tube to a 15 ml tube, and centrifuge the IPE culture solution at 4 ml, 4 ° C, 3000 rpm for 2 minutes. After removing the supernatant, lml of IPE culture solution was added and suspended, and the cells were seeded on a 24-well plate coated with type I collagen, and 5% CO, 3

Transfer to an incubator at 7 ° C and culture. Observe one week later and observe the cells attached to the plate.

[0059] 7-6. Construction of cell layer

Prepare a pellet containing the specified number of IPE cells according to 7-4. Add 2 ml of IPE broth and suspend to make a cell suspension. On the other hand, the IPE culture solution was placed in a 24-well plate prepared in step 1 to which a dry collagen gel with a thickness of 20 μm was attached, and incubated at 37 ° C for 15 minutes. Rehydrate the dried body. As a result, the cell suspension is placed on the wet collagen gel thin film (about 20 μm thick). After adding lml, transfer the plate to an incubator with 5% CO and 37 ° C, and incubate

. After incubation, replace the IPE medium every 3 days using the same method as described in 7-3. About 2 to 3 days after the start of the culture, the cells adhere to the collagen gel thin film and reach confluence after about 2 weeks of culture.

[0060] 8. Preparation of cultured RPE sheet using human autologous cells

 8-1. Tissue collection

 A scleral incision is performed under local anesthesia for patients with informed consent. A vitrectomy is performed, and about 2 X 2 mm of healthy RPE is taken from the subretinal area sufficiently far from the macula. The excised tissue should be immersed in about 2 ml of intraocular perfusate, and the following treatment should be performed within 1 hour. If there is no other description, perform the subsequent processing with care for aseptic operation in a clean bench.

 [0061] 8-2. Isolation of RPE cells

 Move the retinal pigment tissue collected in 8-1 to a 60 mm dish containing 6 ml of BSS (Santen Pharmaceutical) and then move to a 60 mm dish containing 6 mM BED containing 6 mM EDTA and leave it at 37 ° Incubate for 10 minutes. Remove the dish from the incubator, add 2 ml of RPE culture solution (DMEM medium (Kohjin bio) 450 ml, FCS (ICN) 50 ml, amphotericin (Wako) 1.25 mg, gentamicin (Gibco) 250 μ1), and then use a scraper. Carefully exfoliate and separate epithelial cells. The retinal pigment epithelial cells are black, and the area of the retina pigment tissue from which the cells have been detached becomes white. Therefore, detachment of the cells is continued until no black area can be visually confirmed from the retinal pigment tissue. After removing the cell detachment retinal pigment tissue, aspirate the solution with a pipette and inject it into a 15 ml centrifuge tube. Add 2 ml of RPE culture solution to the dish and inject again into the 15 ml centrifuge tube to wash the dish. Repeat the washing procedure, and then centrifuge at 4 ° C and 3000 mm for 2 minutes. After removing the supernatant, add 1 ml of 0.05% trypsin / EDTA solution, re-suspend, and leave in a 37 ° C incubator for 2 minutes. Add 2 ml of RPE medium and centrifuge at 3000 rpm for 2 minutes. After removing the supernatant, add 5 ml of RPE medium and resuspend the pellet, and centrifuge at 4 ° C and 3000 µm for 2 minutes. Collect the pellets of the cells by removing the supernatant.

[0062] 8-3. RPE cell culture Use RPE medium unless otherwise stated for RPE cell culture. 8- Add lml RPE medium to the tube containing the pellet obtained in step 2 to suspend the pellet, and add the whole solution to a 24-well plate (IWAKI) coated with type I collagen. This means that one tissue of RPE cells has been saved per tool. Cell culture is performed in an incubator with 5% CO and 37 ° C. After the culture, the medium is not changed, and the plate is left still. This is the time when RPE cells adhere to the plate, and the cells are observed daily. It takes about 7-10 days for the cells to adhere to the plate. After that, change the RPE medium every 3 days. Replace the medium in the same way as in 7-3. Cells become confluent about 3-4 weeks after the start of culture.

[0063] 8 -4. Passage of RPE cells

 Passage cells in the same way as in 7-4.

[0064] 8-5. Preservation of RPE cells

 7- Store RPE cells in the same way as in 5.

[0065] 8 -6. Construction of cell layer

 Prepare a culture RPE sheet in the same way as in 7-6.

[0066] 9. Preparation of cultured IPE sheets using human allogeneic cells

 Allogeneic IPE can be collected using an eyeball obtained from an eye bank. After removing the whole eyeball from the corpse, place it in an ocular storage solution such as optisol as soon as possible. Corneal limbal force About 5 mm outside is cut with scissors to separate the anterior ocular tissue including the cornea and the posterior ocular tissue including the retina. Anterior tissue force using cesi Remove the iris tissue except the cornea and lens. Iris pigment tissue can be obtained by thoroughly washing with BSS except for the jelly-like substance contained. Using the iris pigment tissue obtained in this way, prepare a cultured IPE sheet in the same manner as in 7-2 to 7_6.

[0067] 10. Cultivation using human allogeneic cells Production of RPE sheet

Allogeneic RPE can be collected using an eyeball obtained from an eye bank. After removing the whole eyeball from the cadaver, place it in an ophthalmic preservation solution such as optisol as soon as possible. Corneal limbal force About 5 mm outside is cut with scissors to separate the anterior ocular tissue including the cornea and the posterior ocular tissue including the retina. Remove the posterior ocular tissue force from the vitreous and retina using cesi, then retina pigmented membrane from the sclera And the choroid present on the sclera side is peeled to obtain a retinal pigment tissue. Retinal pigment tissue can be obtained by thorough washing with BSS. Using the retinal pigment tissue thus obtained, a cultured RPE sheet is prepared in the same manner as in 8_2 to 8_6.

 [0068] 11. Preparation of cultured cell sheet by one-step culture method

 (a) Culture IPE sheet

 Obtain isolated IPE cells by the methods described in 7-1. And 7-2. Add the culture solution to this to prepare the cell suspension. Cell suspension is seeded on the vitrified collagen gel thin film prepared by the method described in 1., and then cultured in the same manner as in 7-6. As described above, the collected IPE cells are directly cultured on a collagen gel thin film to obtain a cultured IPE sheet.

 (b) Culture RPE sheet

 Obtain isolated RPE cells by the methods described in 8-1. and 8-2. Add the culture solution to this to prepare the cell suspension. Cell suspension is seeded on the vitrified collagen gel thin film prepared by the method described in 1., and then cultured in the same manner as in 8-6. As described above, the collected RPE cells are directly cultured on a collagen gel thin film to obtain a cultured RPE sheet.

 [0069] 12. Transplantation of cultured cell sheet under the retina

 Transplantation of cultured IPE sheet or cultured RPE sheet can be carried out by the following procedure. First, after placing 2% methylcellulose on the recipient cornea, a contact lens for vitreous surgery is placed and a scleral incision is made at 11 o'clock. After vitrectomy, insert a 36G needle from the scleral incision to make a retinal incision at the lesion site. Artificial retinal detachment is made by injecting a small amount of intraocular perfusate from the retinal incision into the subretinal region. Spread the scleral incision and insert a cultured cell sheet (cultured IPE sheet or cultured RPE sheet) under the retina. After transplanting the cultured cell sheet, laser photocoagulation is performed on the retinal incision, and the lower eye is replaced with air or expandable gas. After transplanting, keep the prone position until air or gas is absorbed.

 [0070] 13. Examination of culture conditions and culture method

13-1. Dish coating for IPE primary culture In 2-3, the cells were cultured using a plasma-treated non-coated dish, a type IV collagen dish, a laminin coated dish, a fibronectin coated dish, or a matrigel coated dish instead of the type I collagen coated dish. One week after the start of primary culture, each dish was observed, and the optimal carrier type for culture was examined using the number of adherent cells as an index. As a result, under the conditions using type IV collagen dish or fibronectin coated dish, the number of adherent cells was almost the same as when using type I collagen coated dish. On the other hand, a slight decrease in the number of adherent cells was observed under the conditions using the plasma-treated non-coated dish or laminin-coated dish, and no cell proliferation was observed under the conditions using the matrigel-coated dish. Based on the above results, it was considered that the best dish for the primary culture of IPE cells was the coating of type I collagen on the surface in terms of cost and effectiveness. On the other hand, it was found that a plasma-treated non-coated dish was also able to withstand practical use although the cell growth rate was slightly inferior.

 [0071] 13-2. Serum concentration during primary cell culture

 The relationship between FCS concentration in the culture medium during primary culture and cell viability (cell proliferation rate) was examined. In 2−3 and 3−3, culture is performed under the condition that the concentration of FCS in the culture solution is reduced from 10% to 5, 3, 1, or 0%. The existence was observed. When adherent cells were present, the number of adherent cells was compared with the FCS 10% condition. As a result, in the culture of IPE cells, the number of adherent cells was the same as the condition of FCS 10% when FCS was 5% or more, and the number of adherent cells was slightly reduced under the condition of FCS 3%, F CS 1% or less Adherent cells were not observed under the conditions. In RPE cell culture, the number of adherent cells was the same as FCS 10% when FCS was 3% or higher, slightly lower when FCS was 1%, and adherent cells were observed when FCS was 0%. Was not. From the above results, it can be said that the serum concentration in the culture medium during primary culture can be lowered to at least 3% in the case of IPE cells and to at least 1% in the case of RPE cells. By lowering the serum concentration, it is possible to reduce the culture cost, and in addition, it is possible to reduce the risk of contamination with foreign proteins and viruses contained in the serum.

 [0072] 13-3. Serum-free culture (at the time of primary cell culture)

We examined whether primary culture could be performed in serum-free culture. 2- 3. and 3— 3. In each case, immediately after cell isolation, incubate under conditions that suspend in HSM medium (2-3) and epilarif (3-3), observe the dish one week after the start of culture, and observe adherent cells. In either case, cell adhesion to the dish was not observed. Next, after culturing for 1 week by the usual method (2-3 or 3-3) using IPE culture medium or RPE culture medium, the culture medium is cultured under the conditions for changing to HSM medium or Epilife. We observed whether it would become confluent. As a result, good cell proliferation was observed for both IPE cells and RPE cells, reaching confluence after about 3 weeks for IPE cells and about 4 weeks for RPE cells. From the above results, it was revealed that the serum-free culture method can be adopted as the primary culture method for both IPE cells and RPE cells.

13 -4. Substrate type and thickness

 The type and thickness of the substrate were examined. First, in 2-6 and 3-6, a cultured cell sheet was prepared under the condition that the origin of collagen as a component of the substrate (collagen gel thin film) was swine, and the morphology of the cell layer was observed. Specifically, it was observed whether the cells constituting the cell layer had a honeycomb shape. As a result, it was found that even when a substrate made of porcine collagen was used, a culture sheet having a cell layer equivalent to that obtained when a substrate having collagen power derived from ushi was used. In other words, it was clarified that even a substrate prepared using collagen derived from other animal species as a raw material, not only from ushi, is useful for producing cultured cell sheets.

 Next, the thickness of the substrate was changed from 20 μm to 10 am, and an attempt was made to produce a cultured cell sheet. In addition, both a substrate made of ushi collagen and a substrate made of porcine collagen were examined. As a result, regardless of the origin of collagen, even when the thickness of the substrate was 10 μπι, a cultured cell sheet equivalent to the thickness of 2 μμπι could be produced. Moreover, the obtained cultured cell sheet had sufficient strength and form retention required for the transplant material.

It is desirable that the thickness of the cultured cell sheet, which has less physical space under the macular part retina to which the cultured cell sheet is transplanted, is 20 zm or less in total. From the above study, it became clear that a 10 μm-thick substrate could be used, and in fact, a cultured cell sheet with a thickness of 20 μm or less was successfully constructed. Industrial applicability

 [0074] The cultured cell sheet of the present invention can be used as a transplant material for various diseases that require transplantation of retinal pigment epithelium (RPE). The cultured cell sheet of the present invention includes a cell layer having a structure similar to that of living body RPE.

 On the other hand, the cultured cell sheet of the present invention is formed by culturing and proliferating collected IPE cells or RPE cells in vitro. Therefore, it is possible to produce transplant material based on a small number of IPE cells or RPE cells, and even patients with a reduced number of these cells can make transplant material using their own cells. . This means that a network transplantation that does not cause rejection can be realized.

 [0075] Currently, reconstruction of tissues using undifferentiated cells typified by ES cells has been attempted for various tissues. However, in the current situation where it is desired to use autologous cells where possible, the use of ES cells is not realistic and there are many points to be overcome. The cultured cell sheet of the present invention can be constructed using autologous cells. If autologous cells are used, it can be said that a cell sheet that exhibits high engraftment and has an excellent therapeutic effect can be constructed.

 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

 The contents of papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

Claims

Claims A cultured cell comprising a vitrified collagen gel thin film having a thickness of about 20 μm or less and an iris pigment epithelial cell or retinal pigment epithelial cell-derived cell layer formed thereon. Sheet. 2. The cultured cell sheet according to claim 1, wherein the collagen gel thin film has a thickness of about 5 μm to about 10 μm. The cultured cell sheet according to claim 1 or 2, wherein the collagen gel thin film comprises type I collagen. The cultured cell sheet according to any one of claims 1 to 3, wherein a support for maintaining the shape of the collagen gel thin film is attached. The cultured cell sheet according to any one of claims 1 to 4, comprising one or more characteristics selected from the group consisting of the following (1) to (8):  (1) the cell layer has a single layer structure; (2) the cell density of the cell layer is about 1000-10000 cells / mm ² ;  (3) The planar view shape of the cells constituting the cell layer is a polygonal shape;  (4) The cells are arranged almost uniformly and regularly in the cell layer;  (5) Tight junctions are observed between cells in the cell layer;  (6) the cell layer has phagocytic ability;  (7) melanin deposition is observed in the cell layer;  (8) Expression of a retinal pigment epithelial cell marker is observed in the cell layer.  6. The cultured cell sheet according to claim 5, comprising all of the features (1) to (8).  A method for producing a cultured cell sheet comprising the following steps:  (1) providing a vitrified collagen gel thin film having a thickness of about 20 / m or less; (2) preparing iris pigment epithelial cells or retinal pigment epithelial cells;  (3) seeding and culturing the cells on the collagen gel thin film;  (4) A step of recovering the collagen gel thin film and the cell layer formed thereon after culturing. 8. The composition of claim 7, wherein the collagen gel thin film has a thickness of about 5 μm to about 10 μm. Manufacturing method.  [9] The method according to claim 7 or 8, wherein the collagen gel thin film comprises type I collagen.  [10] The production method according to any one of claims 7 to 9, wherein the collagen gel thin film is provided with a support for maintaining the shape thereof. [11] A substrate composed of a vitrified collagen gel thin film having a thickness of about 10 μm or less.