`
`Ex. 1024
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`Certified English-Language
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`Certified English-LanguageCertified English-Language
`Translation of
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`Translation ofTranslation of
`WO 2007/013331 to
`Kinoshita et al. at Ex. 1023
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`WO 2007/013331 toWO 2007/013331 to
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`Kinoshita et al. at EX. 1023Kinoshita et al. at EX. 1023
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`Specification
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`Sheet-Like Composition
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`Technical Field
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`[0001]
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`The present invention relates to a sheet-like composition using an amnion, and a method for
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`producing the same. The sheet-like composition in the present invention can be used as a culture
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`substrate for producing artificial tissue (such as corneal epithelium), as a graft material for
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`reconstructing ocular surfaces and skin, and as an adhesion-preventing material.
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`Background Art
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`[0002]
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`Amnion has excellent properties as a graft material, such as very good biocompatibility and
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`flexibility, and has been used in the reconstruction of corneal epithelium and other tissues (see,
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`for example, Patent Documents 1-4). There are two general uses for amnion. It can be applied
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`directly to the affected area to reconstruct tissue, and can be used as a culture substrate for a cell
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`culture. The flexibility of amnion is an important characteristic in both of these applications.
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`Because it is very flexible, amnion covers the affected area without leaving any gaps, and is able
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`to adhere well to the affected area and become engrafted, resulting in a favorable therapeutic
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`effect. When amnion is used as the culture substrate in a cell culture, its flexibility promotes
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`good cell proliferation and proper organization (differentiation).
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`[0003]
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`Patent Document 1: Japanese Patent Publication No.5-56987A
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`MTF Ex. 1024, pg. 1
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`Patent Document 2: International Publication No.03/043542A1
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`Patent Document 3: International Publication No.03/092762A1
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`Patent Document 4: International Publication No.2004/078225A1
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`Disclosure of the Invention
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`Problem Solved by the Invention
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`[0004]
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`Amnion covers the outermost layer of the uterus and placenta in mammals, and is obtained
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`during delivery. Since intact amnion is not always available, there is demand in clinical
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`applications for amnion which can be stored for a long period of time and which is easy to
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`handle. Therefore, harvested amnion has been stored in a dry state to improve storage and
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`handling properties. While drying preserves storage stability, it also denatures the constituent
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`proteins. This greatly reduces the flexibility of the amnion when restored to a moist state.
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`Because of the reduced flexibility, the amnion may not cover the affected area without leaving
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`any gaps, and may not adhere well to the affected area and become engrafted. Also, amnion that
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`has been dried has a lower cell proliferation rate, and impedes stratification and proper
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`organization (differentiation). This may be due to the significantly reduced flatness of the
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`amnion surface in addition to the reduction in flexibility. It is an object of the present invention
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`to solve this problem by providing a sheet-like composition comprising amnion which has
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`superior storage and handling properties, as well as very good flexibility during use.
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`Means of Solving the Problem
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`[0005]
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`MTF Ex. 1024, pg. 2
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`In order to achieve this object, the present inventors modified amnion to improve its flexibility.
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`As a result, they discovered that the flexibility of amnion treated with trehalose, a disaccharide,
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`could be restored when returned to a moist state after drying. In other words, it became clear that
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`treatment with trehalose is effective at improving the flexibility of amnion. It was also
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`discovered that treatment with trehalose had the surprising effect of improving the transparency
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`of amnion. Therefore, treatment of amnion with trehalose is extremely advantageous when using
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`an amnion in any application requiring a high degree of transparency (such as in corneal
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`reconstruction). In addition, treatment with trehalose increased the tensile strength of amnion and
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`resulted in an amnion that is tougher than intact amnion. It has also become clear that treatment
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`with trehalose is effective at improving storage and handling properties, and shape retention of
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`an amnion after transplantation. It was also found that the biocompatibility of an amnion treated
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`with trehalose is as good as that of intact amnion, and that treatment with trehalose does not
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`adversely affect biocompatibility. After obtaining these findings, the effect of trehalose on the
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`functions of amnion as a cell culture substrate was examined. More specifically, corneal
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`epithelial cells were cultured on amnion treated with trehalose, and cell proliferation and cell
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`stratification on the substrate were determined. The results revealed favorable cell proliferation
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`and stratification of from 5 to 7 layers, indicating a significant improvement compared to
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`stratification (1 or 2 layers) on amnion that had not been treated with trehalose. This
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`demonstrated that treatment with trehalose is also effective on amnion used as a cell culture
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`substrate. Sheets of amnion with cell layers formed on top were transplanted on the ocular
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`surfaces of animals to determine the reconstructive effect. As a result, very good adhesion and
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`engrafting was discovered. Defect-free ocular surfaces could be reconstructed, and a high degree
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`of transparency was retained. The present invention provides the following sheet-like
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`composition based primarily on these findings.
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`
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`[1] A sheet-like composition comprising an amnion having trehalose added thereto.
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`[2] The sheet-like composition according to [1], wherein the amnion is in a frozen or dry state.
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`[3] The sheet-like composition according to [2], wherein the amnion is in a freeze-dried state.
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`MTF Ex. 1024, pg. 3
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`[4] The sheet-like composition according to [1] through [3], wherein the amnion is an amnion
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`having the epithelial cell layer removed therefrom.
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`[5] The sheet-like composition according to [1] through [4], wherein the amnion includes basal
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`membrane components Collagen IV, Collagen VII, and Laminin 5 detected at a strength
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`comparable to those of untreated amnion.
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`[6] The sheet-like composition according to [1] through [5], wherein the amnion is a human
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`amnion.
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`[7] The sheet-like composition according to [1] through [6], wherein a cell layer of biologically
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`derived cells is formed on the amnion.
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`[8] The sheet-like composition according to [7], wherein the biologically derived cells are
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`stratified in the cell layer.
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`[9] The sheet-like composition according to [7], wherein the biologically derived cells are
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`derived from the corneal epithelium, conjunctival epithelium, cutaneous epidermis, follicle
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`epithelium, oral mucosal epithelium, iris pigment epithelium, retinal pigment epithelium,
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`respiratory tract mucosal epithelium, or intestinal mucosa.
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`[10] The sheet-like composition according to [7], wherein the cell layer is composed of cells
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`stratified in approximately five to seven layers, and has properties similar to those of the corneal
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`epithelium.
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`[11] The sheet-like composition according to any one of [1] through [6], wherein the sheet-like
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`composition is used as an adhesion-preventing material or reconstructive material for organ
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`surface tissue damage caused by surgical stress.
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`[12] The sheet-like composition according to any one of [1] through [11], wherein an adhesive
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`component is attached to the amnion on the chorion side.
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`MTF Ex. 1024, pg. 4
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`[13] The sheet-like composition according to [12], wherein the adhesive component comprises
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`fibrinogen and thrombin.
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`[14] The sheet-like composition according to [12], wherein the adhesive component comprises
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`fibrinogen, thrombin and aprotinin.
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`[15] The sheet-like composition according to any one of [1] through [14], wherein the chorion
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`side of the amnion is coated with a bioabsorbable material.
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`[0006] In another aspect of the present invention, the following transplant method is provided.
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`[16] A transplant method using as a graft material the sheet-like composition according to any
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`one of [1] through [15].
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`[0007] In another aspect of the present invention, the following production method is provided.
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`[17] A method for producing a sheet-like composition, the method comprising the steps of:
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`(a) preparing amnion; and
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`(b) adding trehalose to the amnion.
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`[18] The method according to [17] further comprising the step of:
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`(c) freezing or drying the amnion after step (b).
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`[19] The method according to [18] further comprising the step of:
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`(d) sterilizing the amnion after step (c).
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`[20] The method according to any one of [17] through [19], wherein step (a) comprises the step
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`of:
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`(a1) removing the epithelium from the amnion.
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`MTF Ex. 1024, pg. 5
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`[21] The method according to [20], wherein step (a1) comprises the steps of:
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`(1) preparing an amnion separated from living tissue;
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`(2) freezing and thawing the amnion;
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`(3) treating the amnion with trypsin after the freezing and thawing;
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`(4) and washing the amnion after treatment with trypsin.
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`[22] The method according to [21], wherein the freezing temperature is from -20°C to -80°C, and
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`the thawing temperature is from 4°C to 50°C in the freezing and thawing process.
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`[23] The method according to [21] or [22], wherein the freezing and thawing process is repeated
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`two or more times.
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`[24] The method according to any one of [20] through [23], wherein the trypsin treatment is
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`performed using a trypsin solution having a trypsin concentration of from 0.01% (w/v) to 0.05%
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`(w/v).
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`[25] The method according to [24], wherein the trypsin solution comprising from 0.1 mM to 0.6
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`mM of a chelator selected from a group including EDTA, NTA, DTPA, HEDTA, GLDA, and
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`any combination thereof.
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`[26] The method according to any one of [20] through [25], wherein the trypsin treatment is
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`performed under conditions enabling the trypsin solution to come into contact with only the
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`epithelium side of the amnion.
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`[27] The method according to any one of [20] through [26], wherein the following step is
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`performed after step (b):
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`(A) forming a cell layer of biologically derived cells on the amnion.
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`Brief Description of the Drawings
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`[0008]
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`MTF Ex. 1024, pg. 6
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`[FIG. 1]
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`FIG. 1 is a table summarizing the applications of the sheet-like composition as a tissue-
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`reconstructing material (the primary application site, an example of application method, the
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`preferred form of amnion, and the primary purpose).
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`[FIG. 2]
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`FIG. 2 is diagram used to explain the methods used to secure the amnion. In FIG. 2 (a), the
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`amnion is interposed between a pair of frames. In FIG. 2 (b), the amnion is interposed between a
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`frame and a plate.
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`[FIG. 3]
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`FIG. 3 is a graph showing the result of a physical property evaluation (thickness) of trehalose-
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`treated/freeze-dried amnions.
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`[FIG. 4]
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`FIG. 4 is a graph showing the result of a physical property evaluation (clarity) of trehalose-
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`treated/freeze-dried amnions.
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`[FIG. 5]
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`FIG. 5 is a graph showing the result of a physical property evaluation (tensile strength) of
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`trehalose-treated/freeze-dried amnions.
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`[FIG. 6]
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`MTF Ex. 1024, pg. 7
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`FIG. 6 is a graph showing the result of a physical property evaluation (flexibility) of trehalose-
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`treated/freeze-dried amnions.
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`[FIG. 7]
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`FIG. 7 is a pair of photographs showing the results of the biocompatibility of a trehalose-
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`treated/freeze-dried amnion. The trehalose-treated/freeze-dried amnion was transplanted between
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`corneal stroma layers in a rabbit, and the condition of the ocular surface was monitored. The
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`photograph on the left shows the condition of the ocular surface immediately after the transplant,
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`and the photograph on the right shows the condition of the ocular surface one month after the
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`transplant.
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`[FIG. 8]
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`FIG. 8 is a pair of photographs showing the results of the biocompatibility of a trehalose-
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`treated/freeze-dried amnion. The trehalose-treated/freeze-dried amnion was transplanted between
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`corneal stroma layers in a rabbit, and a portion of the cornea including the transplant was excised
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`and subjected to HE staining one month after transplantation.
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`[FIG. 9]
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`FIG. 9 is a cross-sectional view schematically showing the tools used to culture corneal epithelial
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`cells on a trehalose-treated/freeze-dried amnion. A culture insert 12 was placed in a culture dish
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`11, and 3T3 cell layer 15 was formed on the bottom surface of the culture dish 11. Also, an
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`amnion 13 was placed on the bottom surface of the culture insert 12, and the corneal epithelial
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`cells 14 were cultured on top. In the figure, 16 denotes the culture medium.
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`[FIG. 10]
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`FIG. 10 is a pair of photographs (HE staining images) showing the cell layer formed on the
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`trehalose-treated/freeze-dried amnion. For the sake of comparison, a cell layer formed on an
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`MTF Ex. 1024, pg. 8
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`amnion which had been treated and freeze-dried without being treated with trehalose
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`(untreated/freeze-dried amnion) is also shown.
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`[FIG. 11]
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`FIG. 11 is a series of photographs showing HE staining images and immuno-staining images of a
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`sheet (cultured corneal epithelial sheet) in which a cell layer was formed on the trehalose-
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`treated/freeze-dried amnion. The signals of each antibody in the immuno-staining images are
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`green. The cell nuclei are indicated in red. Corneal keratin 3 is expressed (+), but keratinized
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`cutaneous keratin 10 (-) and corneal keratin 13 are not expressed (-).
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`[FIG. 12]
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`FIG. 12 is a series of photographs showing the reconstructive effect of a cultured corneal
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`epithelial sheet made with a trehalose-treated/freeze-dried amnion. The condition of the ocular
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`surface (top) and its fluorescein staining image (bottom) are shown two and fourteen days after
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`transplantation of the cultured corneal epithelial sheet.
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`[FIG. 13]
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`FIG. 13 is a series of photographs showing the HE staining images (top left) and immuno-
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`staining images (top right and bottom) of the cultured corneal epithelial sheets with the various
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`keratins two weeks after transplantation. Here, ** denotes trehalose-treated/freeze-dried amnions
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`(TH-AM).
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`[FIG. 14]
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`FIG. 14 is a series of photographs showing the results of immuno-staining the trehalose-
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`treated/freeze-dried amnions using an antibody directed against a basal membrane-specific
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`component and an antibody directed against a stratum compactum-specific component. The
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`immuno-staining images (C) of the trehalose-treated/freeze-dried amnion are shown with those
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`MTF Ex. 1024, pg. 9
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`of untreated amnions (A) and of freeze-dried amnion without trehalose-treatment (B) for the sake
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`of comparison. 1: Staining image of Collagen I; 2: staining images of Collagen III; 3: staining
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`images of Collagen IV; 4: staining images of Collagen V; 5: staining images of Collagen VII; 6:
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`staining images of Laminin 5; and 7: staining images of fibronectin.
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`[FIG. 15]
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`FIG. 15 is a flow chart showing the procedure for removing an amniotic epithelium.
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`[FIG. 16]
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`FIG. 16 is a pair of figures showing the methods used to perform the trypsin treatment. In FIG.
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`16 (a), an amnion secured to a frame is immersed in a trypsin solution with the epithelial side
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`facing downward. In FIG. 16 (b), a trypsin solution is poured over the frame so that the trypsin
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`can act upon the epithelial side of the amnion.
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`[FIG. 17]
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`FIG. 17 is a series of photographs showing HE staining images of trypsin-treated, untreated (with
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`epithelium), and manually-treated amnions.
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`[FIG. 18]
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`FIG. 18 is a series of photographs showing the immuno-staining images of the trypsin-treated
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`amnion.
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`[FIG. 19]
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`FIG. 19 is a series of photographs showing the immuno-staining images of the trypsin-treated
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`amnion.
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`MTF Ex. 1024, pg. 10
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`[FIG. 20]
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`FIG. 20 is a series of photographs showing the immuno-staining images of the untreated amnion
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`(with epithelium).
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`[FIG. 21]
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`FIG. 21 is a series of photographs showing the immuno-staining images of the manually treated
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`amnion.
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`[FIG. 22]
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`FIG. 22 is a table summarizing the results of the HE staining and immuno-staining experiments.
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`Key to the Drawings
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`[0009]
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`1, 2, 3: frame
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`4: plate
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`5: trypsin solution
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`10: amnion
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`11: culture dish
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`12: culture insert
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`13: amnion
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`14: corneal epithelial cells
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`15: 3T3 cell layer
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`16: culture medium
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`Best Mode of Carrying Out the Invention
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`MTF Ex. 1024, pg. 11
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`[0010]
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`The first aspect of the present invention relates to a sheet-like composition. The main component
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`of the sheet-like composition of the present invention is an amnion. The clarity and toughness of
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`amnion can form a sheet-like composition with improved clarity and strength. The very good
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`biocompatibility and low immunogenicity of amnion impart better biocompatibility and low
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`immunogenicity of the resulting sheet-like composition. Use of amnion can also be expected to
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`impart various actions, such as anti-inflammatory action, suppression of scar formation, and
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`inhibition of angiogenesis. Use of amnion is also preferable with respect to cell layer formation,
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`if the cell layer is contained in the sheet-like composition of the present invention. More
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`specifically, as described below in greater detail, the use of amnion promotes adhesion, cell
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`proliferation, and cell layer formation when the sheet-like composition includes a cell layer
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`formed by seeding and culturing certain cells on an amnion serving as the substrate (support),
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`because amnion has properties facilitating good adhesion and cell proliferation.
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`[0011]
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`(Origin of Amnion)
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`Amnion is a membrane covering the outermost layer of the uterus and placenta in mammals, and
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`is composed of a basal membrane and epithelial layer formed on collagen-rich parenchymal
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`tissue. Amnion from a human, monkey, chimpanzee, pig, horse or cow can be used. Among
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`these, human amnion is preferred from the standpoint of safety, low immunogenicity and virus
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`infection probability.
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`[0012]
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`(Addition of Trehalose)
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`The sheet-like composition of the present invention uses an amnion to which trehalose has been
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`added. As a result of extensive research, the present inventors discovered that the addition of
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`MTF Ex. 1024, pg. 12
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`trehalose improves the flexibility of the amnion, especially when the amnion has been freeze-
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`dried. In addition, as will be explained in examples below, the present inventors discovered that
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`an amnion to which trehalose has been added serves as a very good substrate for culturing cells.
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`The sheet-like composition of the present invention created on the basis of these discoveries has
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`very good flexibility, and promotes cell proliferation and stratification when used as a substrate
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`for culturing cells. When the matrix proteins in an amnion are weakened, the strength of the
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`amnion is reduced, making the amnion more susceptible to damage. Weakened amnion can no
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`longer firmly retain moisture, and become brittle and less resilient. The trehalose added to the
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`amnion is believed to act on sites where the binding of matrix proteins has become loose,
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`reinforcing the binding between proteins, normalizing the retention of moisture inside the
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`amnion, and imparting the moisture retention, integrity and flexibility intrinsic to amnion. The
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`addition of trehalose also keeps the matrix proteins in the amnion from becoming soft during the
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`freeze-drying process, and which effectively protects them from swelling and becoming
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`weakened in water. Trehalose (substance name, generic name) is a compound represented by α-
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`D-glucopyranosyl (1,1)-α-D-glucopyranoside. Trehalose can be added to an amnion by treating
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`the amnion with a trehalose solution. A method for adding trehalose is described below in greater
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`detail. One embodiment of the present invention consists essentially of an amnion to which
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`trehalose has been added.
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`[0013]
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`(State of the Amnion)
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`
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`In one embodiment of the present invention, amnion with the epithelial cell layer removed is
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`used. An amnion with the epithelial cell layer removed is much safer since it does not cause
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`immunological rejection or other problems caused by epithelial cells. Because cell adhesion and
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`proliferation that takes place on an amnion with the epithelium removed yields superior results, a
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`high-quality cell sheet can be created in a shorter period of time, thereby providing an advantage
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`in the manufacture of the sheet-like composition. The removal of the epithelial layer on the
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`amnion can be determined by confirming the absence of cells from the amniotic epithelial layer
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`on the sheet-like composition of the present invention. Amnion with the epithelial layer retained
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`MTF Ex. 1024, pg. 13
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`can also be used in the creation of the sheet-like composition of the present invention. Retention
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`of the epithelial layer on the amnion allows for thorough sterilization using gamma-ray treatment,
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`thereby improving the safety of the sheet-like composition.
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`[0014]
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`(Use of Reconstituted Amnion)
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` A
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` reconstituted amnion can be used to construct the sheet-like composition according to the
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`invention. More specifically, an amnion can be broken down using a homogenizer, ultrasound
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`treatment, or enzymatic treatment, and then reconstituted in membrane form. The breakdown
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`treatment is preferably performed using a homogenizer because it can be expected to retain a
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`structure with a fine basal membrane. Treatment using a homogenizer can be performed under
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`(rotation speed) conditions from 3,000 rpm to 50,000 rpm, preferably from 10,000 rpm to 40,000
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`rpm, and more preferably at approximately 30,000 rpm.
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`[0015]
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`(Thickness)
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`
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`The use of an amnion in the sheet-like composition of the present invention enables an extremely
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`thin sheet to be obtained. The sheet-like composition of the present invention can be prepared to
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`a thickness from 10 μm to 500 μm. Such a thin sheet allows for a greater number of uses. An
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`amnion with a portion of the stratum compactum on the chorionic side removed (for example,
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`from 10 μm to 30 μm) can be used to create the sheet-like composition of the present invention.
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`It may be coated with bioabsorbable material to obtain a thickness, for example, from 100 μm to
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`500 μm.
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`[0016]
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`(Use of Adhesive Component)
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`MTF Ex. 1024, pg. 14
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`In one embodiment of the present invention, fibrinogen and thrombin (hereinafter, w referred to
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`as the “adhesive components”) is attached to the surface of amnion. When a sheet-like
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`composition of the present invention is transplanted, the fibrinogen is specifically hydrolyzed by
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`the thrombin to form fibrin, and the fibrin is then polymerized to form a stable fibrin clot which
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`imparts an adhesive effect. Because it is highly adhesive, the sheet-like composition, after being
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`attached to the affected area, can attain sufficient adhesion without suturing, thereby simplifying
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`the surgical procedure. In the present specification, an amnion with an attached epithelium and
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`adhesive components is referred to as an “amnion with attached adhesive components and
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`epithelium” and amnion without an attached epithelium but with attached adhesive components
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`is referred to as an “amnion with attached adhesive components but no epithelium”.
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`[0017]
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`Fibrinogen and thrombin are attached on either side or on both sides of the amnion depending on
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`the intended application of the sheet-like composition of the present invention. When attached to
`
`one side, the chorionic side of the amnion (that is, the side opposite the epithelium) receives the
`
`attached fibrinogen and thrombin regardless of the presence or absence of the epithelium.
`
`Therefore, when used, the sheet-like composition is transplanted to the application site with the
`
`epithelium facing outward. A sheet-like composition used as an anti-adhesive can receive the
`
`adhesive components on either side of the amnion. However, a sheet-like composition implanted
`
`in vivo as a bioadhesive should receive adhesive components on both sides of the amnion.
`
`
`
`[0018]
`
`
`
`As mentioned below, the sheet-like composition of the present embodiment is prepared in an
`
`appropriate state (for example, in a dry state or in a moist state) using a step in which fibrinogen
`
`and thrombin are attached to the surface of amnion in accordance with the intended use.
`
`Therefore, fibrin should be produced by some of the fibrinogen before the sheet-like composition
`
`is used in accordance with the state during the process and/or the final state. Therefore, a sheet-
`
`MTF Ex. 1024, pg. 15
`
`
`
`like composition of the present invention may include fibrin or fibrin clots generated for this
`
`reason.
`
`
`
`[0019]
`
`
`
`There are no particular restrictions on the origin of the fibrinogen and thrombin. The fibrinogen
`
`and thrombin can be prepared using the blood of, for example, humans, monkeys, chimpanzees,
`
`cattle, horses, sheep, or pigs, as the starting material. Fibrinogen and thrombin obtained as a
`
`recombinant with cultured cells (for example, CHO or COS cells) may be used. Use of
`
`fibrinogen and thrombin derived from humans (especially, human-derived recombinants) is
`
`preferred. This is advantageous from the standpoint of safety and immunogenicity. Use of a
`
`recombinant is preferred from the standpoint of stability, quality and infection resistance. It is
`
`especially preferable to use fibrinogen and thrombin derived from the blood of the patient
`
`(recipient) receiving the transplanted sheet-like composition of the present invention. This is
`
`especially advantageous because immunological rejection is less likely to occur. The fibrinogen
`
`and thrombin do not have to have the same origin. For example, a combination of fibrinogen
`
`derived from human blood and thrombin derived from bovine blood may be used.
`
`
`
`[0020]
`
`
`
`There are no particular restrictions on the amount of fibrinogen and thrombin attached. For
`
`example, the attached amount of fibrinogen can be set in a range from 0.1 mg to 50 mg per 1 cm2
`
`of amnion. Similarly, the attached amount of thrombin can be set in a range from 0.5 μmg to 10
`
`mg per 1 cm2 of amnion. Adhesive force is the primary consideration when determining the
`
`amount of fibrinogen and thrombin to be attached. In other words, the amounts of these
`
`components are determined so as to obtain the necessary adhesive force. When the amount of
`
`attached fibrinogen and thrombin is too large, an immune reaction or angiogenesis may occur,
`
`although this often depends on the origin of fibrinogen that is used. Here, when a sheet-like
`
`composition is used to reconstruct an ocular surface (and angiogenesis caused by these
`
`components may occur after transplantation), the amount attached should be reduced to
`
`minimize the possibility of angiogenesis. By establishing the attached amounts of these
`
`MTF Ex. 1024, pg. 16
`
`
`
`components as low as possible, post-transplantation angiogenesis can be suppressed and a
`
`superior therapeutic effect can be expected. In experiments conducted by the present inventors,
`
`excellent adhesive force was observed with respect to the ocular surface when the attached
`
`amount of fibrinogen is greater than 0.5 mg per 1 cm2 of amnion. At the same time, excellent
`
`adhesive force was observed with respect to the ocular surface when the attached amount of
`
`thrombin is from 1 μg to 1 mg per 1 cm2 of amnion. Based upon these findings, the attached
`
`amount of fibrinogen is preferably from 0.5 mg to 20 mg per 1 cm2 of amnion, more preferably
`
`from 0.5 mg to 10 mg per 1 cm2 of amnion, and even more preferably from 0.5 mg to 6 mg
`
`(more specifically, 0.5 mg, 1 mg, or 2 mg) per 1 cm2 of amnion. Similarly, the amount of
`
`attached thrombin is preferably from 1 μg to 1 mg per 1 cm2 of amnion, more preferably from 5
`
`μg to 200 μg per 1 cm2 of amnion, and even more preferably from 10 μg to 100 μg (more
`
`specifically, 10 μg, 20 μg, or 30 μg) per 1 cm2 of amnion.
`
`
`
`[0021]
`
`
`
`In one embodiment of the present invention, aprotinin is attached to the surface of amnion in
`
`addition to fibrinogen and thrombin. Aprotinin inhibits fibrin clots from being dissolved plasmin
`
`due to the effect of thrombin. Therefore, by including aprotinin, the breakdown of fibrin clots
`
`can be suppressed. As a result, adhesive force can be maintained or reinforced. There are no
`
`particular restrictions on the origin of the aprotinin. The aprotinin can be derived from the
`
`pancreas of a cow, horse, sheep, pig, monkey, or chimpanzee. Recombinant aprotinin obtained
`
`using cultured cells (for example, CHO or COS cells) may also be used. Use of a recombinant is
`
`preferred from the standpoint of stability, quality, and infection prevention. When aprotinin is
`
`used, there are no particular restrictions on the amount attached. For example, the amount of
`
`aprotinin attached can be set in a range from 0.1 KIU to 200 KIU per 1 cm2 of amnion. The
`
`present inventors also investigated how a change in the amount of attached aprotinin affected the
`
`adhesive force during the investigation of the amount of attached fibrinogen. As a result, they
`
`discovered that, even when the amount of aprotinin is set in a range from 1 KIU to 2 KIU, there
`
`was sufficient adhesive force with respect to an ocular surface. Based on this discovery, the
`
`range of the amount of attached aprotinin is set preferably from 1 KIU to 100 KIU per 1 cm2 of
`
`amnion, more preferably from 1 KIU to 20 KIU per 1 cm2 of amnion, and even more preferably
`
`MTF Ex. 1024, pg. 17
`
`
`
`from 1 KIU to 10 KIU (for example, 1 KIU, 2 KIU, or 3 KIU) per 1 cm2 of amnion. When the
`
`amount of the aprotinin is too high, manufacturing costs increase and side effects caused by the
`
`immunogenicity, etc. of the aprotinin may also increase. When the amount of aprotinin is too low,
`
`the effect of aprotinin in suppressing the deposition of fibrin clots may not be sufficiently
`
`imparted. The fibrin clots may be used as an adhesive for some purposes, usually in combination
`
`with aprotinin. As a result of an investigation by the present inventors, it was discovered that,
`
`even when aprotinin is not used in the sheet-like composition of the present invention, sufficient
`
`adhesive force can be obtained with respect to living tissue. When aprotinin is not used, the
`
`configuration can be simplified, and lower manufacturing costs can be achieved. In addition, side
`
`effects caused by the immunogenicity etc. of aprotinin can be disregarded.
`
`
`
`[0022]
`
`
`
`(Reinforcement Using Bioabsorbable Materials)
`
`
`
`The chorionic side of the amnion can be coated with a bioabsorbable material in order to
`
`reinforce the sheet-like composition of the present invention. The bioabsorbable materials used
`
`for this purpose can be any material that is broken down and absorbed before the amnion.
`
`Examples of such bioabsorbable materials include Polygractin 910, gelatins, collagens, and
`
`polylactic acid. There are no particular restrictions on the shape of the bioabsorbable material
`
`used for reinforcement purposes. The bioabsorbable material may be formed into a mesh or a
`
`sheet used to cover the chorionic side of the amnion in order to reinforce the amnion. During the
`
`reinforcement process, the amnion may be either in a moistened state or dry state, although the
`
`final state of the amnion is preferably dry from the standpoint of superior handling and storage
`
`properties. Amnion which has been reinforced in this manner is referred to herein as “hybrid
`
`amnion”.
`
`
`
`[0023]
`
`
`
`(Cell Layer)
`
`
`
`MTF Ex. 1024, pg. 18
`
`
`
`In one embodiment of the present invention, the sheet-like composition includes a cell layer on
`
`top of the amnion. If adhesive components are used in this composition, the adhesive
`
`components (such as fibrinogen) are attached to the side of the amnion on which the cell layer
`
`was not formed. In the present embodiment, an amnion from which the epithelium has been
`
`removed is generally used. The cell layer is formed on the side where the epithelium has been
`
`removed. This cell layer is formed from cells of biolo