US 20060153928Al
`
`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0153928 A1
`
` Kinoshita et a1. (43) Pub. Date: Jul. 13, 2006
`
`
`(54) AMINION—ORIGIN MEDICAL MATERIAL
`AND METHOD OF PREPARING THE SAME
`
`(30)
`
`Foreign Application Priority Data
`
`(76)
`
`Inventors: Shigeru Kinoshita, Osaka (JP);
`Takahiro Nakamura, Kyoto (JP)
`Correspondence Address:
`ARMSTRONG,
`TZ, QUINTOS, HANSON
`
`& BROOKS, LLP
`1725 K STREET. NW
`SUITE 1000
`WASHINGTON, DC 20006 (US)
`
`(2]) Appl. No.:
`
`10/546,774
`
`(22) PCT Filed:
`
`Feb. 17, 2004
`
`(86) PCT No.:
`
`PCT/JP04/01692
`
`Feb. 26, 2003 (JP) 2003049947
`
`Publication Classification
`
`(51)
`(52)
`
`Int Cl
`(2006.01)
`1321K“ 33/54
`.. .
`,
`.
`..............................................................
`
`424/582
`
`ABSTRACT
`(57)
`It is intended to provide an amnion-origin medical material
`which can be easily handled and fully sterilized and, more-
`over, favorably acts as a base material for forming a cell
`layer thereon. This material is prepared by: (i) removing the
`epithelial layer from the amnion while remaining at at lea set
`a part 01' the base membrane thereof; and (ii) drying it under
`such conditions that the remaining base membrane can
`sustain a structure allowing the adhesion and proliferation of
`cells thereon in using.
`
`MTF Ex. 1022, pg. 1
`
`

`

`Patent Application Publication
`
`Jul. 13, 2006 Sheet 1 0f 6
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`US 2006/0153928 A1
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`Fig.1
`
`
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`MTF Ex. 1022, pg. 2
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`

`

`Patent Application Publication Jul. 13, 2006 Sheet 2 of 6
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`US 2006/0153928 A]
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`Fig.2
`
`++|I
`
`+
`
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`HEBREBBb-E
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`c.5085
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`MTF EX. 1022, pg. 3
`
`
`

`

`Patent Application Publication Jul. 13, 2006 Sheet 3 of 6
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`US 2006/0153928 A1
`
`Fig.3
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`/[
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`7g gI\
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`:\\\V.\\\\\\\\\\\\V
`III/IIIIII,k§\""III"'II
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`III/[III],
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`\“““““‘\““““
`
`MTF Ex. 1022, pg. 4
`
`

`

`Patent Application Publication Jul. 13, 2006 Sheet 4 of 6
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`US 2006/0153928 A1
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`Fig.4
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`
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`MTF EX. 1022, pg. 5
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`

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`Patent Application Publication Jul. 13, 2006 Sheet 5 of 6
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`US 2006/0153928 A1
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`Fig.5
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`
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`MTF EX. 1022, pg. 6
`
`

`

`Patent Application Publication Jul. 13, 2006 Sheet 6 of 6
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`US 2006/0153928 A1
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`Fig.6
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`
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`MTF Ex. 1022, pg. 7
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`

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`US 2006/0153928 A1
`
`Jul. 13, 2006
`
`AMINION-ORIGIN MEDICAL MATERLAL AND
`METHOD OF PREPARING THE SAME
`
`TECHNICAL FIELD
`
`[0001] The present invention relates to a medical material
`derived from amniotic membrane and the use thereof. The
`medical material ofthe present invention can be used for, for
`example, an injury repairing material, an injury protective
`material, an injury curing material, an adhesion preventive
`material, an artificial tissue, and the like. Specifically, the
`medical material can be used for producing epithelium for
`transplantation (for example, corneal epithelium) or endot-
`helium for transplantation (for example, corneal endothe-
`lium).
`
`BACKGROUND ART
`
`[0002] Under circumstances of the development of tech-
`nology for cell cultivation and progress in medical technol—
`ogy, various trials for reconstructing (reproducing) tissues
`that cannot exhibit their original functions have been carried
`out with various tissues targeted. For example, in one trial,
`epidermis of the skin or corneal epithelium is reconstructed
`on an appropriate substrate in vitro, and the reconstructed
`tissue is used as tissue for transplantation. Such a substrate
`
`used for constructing tissue for transplantation requires
`
`
`
`properties of having a high a lnity (and low immunogenic-
`ity) with respect to a living body, and allowing the adhesion
`and proliferation of the targeted cells thereon. Hitherto, as a
`material having such properties, amniotic membrane has
`been widely used. Amniotic membrane is membrane that
`encloses a fetus together with amniotic fluid. It is thought
`that amniotic membrane is particularly preferred as a mate-
`rial for producing a graft because of its low immunogenicity.
`Amniotic membrane has a schematic structure in which an
`epithelial layer is formed on a stromal layer composed of
`collagen via a basement membrane. As mentioned above,
`amniotic membrane has low immunogenicity. However, in
`order to further reduce the immunogenicity and to construct
`tissue for transplantation by constructing a cell layer on the
`anmiotic membrane, it is necessary to prevent cells derived
`from amniotic membrane from being mixed. Therefore, it is
`thought to be preferable that an epithelial layer is removed
`in use
`(see
`Japanese Unexamined Publication No.
`H5—56987).
`DISCLOSURE OF THE INVENTION
`
`[0003] Amniotic membrane from whim an epithelial layer
`has been removed is prepared as follows. Firstly, collected
`anmiotic membrane is washed, unnecessary attached mate-
`rials are removed, and then an epithelial layer is denuded.
`The thus obtained anmiotic membrane that does not contain
`the epithelium is stored in refrigerating or freezing environ-
`ment before use except that it is used immediately after
`preparation. Therefore, a satisfactory thermal management
`before use is required and special attention should be paid in
`handling (in particular, at the time of delivery). Furthermore,
`since the material
`is transplanted into a living body, a
`sufficient sterilization process is required to be carried out
`before use. According to the investigation by the present
`inventors, however, when amniotic membrane that does not
`contain the epithelium in refrigerating or freezing environ-
`ment is subjected to treatment with a y beam or an electron
`beam, discoloration of a solution was observed, and the
`anmiotic membrane was not resistant to the necessary ster-
`ilization process.
`
`[0004] With the foregoing problems in mind, the present
`invention was made, and it
`is an object of the present
`invention to provide a medical material derived from anmi-
`otic membrane that can be handled easily, subjected to
`sufficient sterili7ation process, and favorably acts as a sub—
`strate on which a cell layer is constructed.
`
`inventors have paid attention to a
`[0005] The present
`basement membrane of amniotic membrane and thought it
`important that the basement membrane is allowed to remain
`and that the structure thereof is maintained in order to
`achieve a preferable medical material suitable as a substrate
`for constructing a cell layer. Furthermore, from the view-
`point of handling,
`the present inventors thought it most
`preferable that the medical material is in a dried state before
`use. Based on the above-mentioned thoughts, the present
`inventors attempted to construct a medical material capable
`of achieving the above-mentioned object by using anmiotic
`membrane as a starting material. Firstly, the epithelium is
`removed from the amniotic membrane with at least a part of
`the basement membrane left. Subsequently, water compo-
`nent is removed by vacuum lyophilization so as to obtain
`amniotic membrane in a dried state (amniotic membrane that
`does not contain the epithelium). ln order to examine the
`properties of the resultant anmiotic membrane that does not
`contain the epithelium, the present inventors attempted to
`construct a cell layer by using the amniotic membrane as a
`substrate. As a result,
`in a model system using corneal
`epithelial cells, favorable adhesion of cells to the amniotic
`membrane and proliferation, and further stratification were
`observed. When transplantation experiment was carried out
`by using the finally obtained cell layer (corneal epithelium-
`like sheet) as a graft, reliable survival of the graft to a living
`body and excellent effect of reconstructing the cornea was
`confirmed. The present invention was completed based on
`the above—mentioned findings and provides the following
`configurations.
`
`[l] A medical material derived from anmiotic
`:0006]
`membrane, comprising the following properties (1) to (3) of:
`:0007]
`(1) being in a dried state;
`:0008]
`(2) not having an epithelial layer; and
`:0009]
`(3) having a basement membrane that retains a
`structure allowing the adhesion and proliferation of cells
`thereon in use.
`
` :0012]
`
`[2] The medical material derived from amniotic
`:0010]
`membrane described in [1] further comprising the following
`property (4) that:
`:0011]
`(4) the medical material is contained in a container
`in a state that is not substantially in contact with oxygen.
`[3] The medical material derived from anmiotic
`membrane described in [l] or [2] wherein the state that is
`not substantially in contact with oxygen is a state in which
`the container has been evacuated or a state in which the gas
`in container has been replaced by nitrogen gas.
`[0013]
`[4] The medical material derived from amniotic
`membrane described in any of[l] to [3], wherein the cell is
`a conical epithelial cell, a corneal endothelial cell, or an oral
`mucosal epithelial cell.
`
`[5] A method of preparing a medical material
`[0014]
`derived from amniotic membrane, the method comprising
`the steps (i) and (ii):
`
`MTF EX. 1022, pg. 8
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`

`

`US 2006/0153928 A1
`
`Jul. 13, 2006
`
`layer from amniotic
`(i) removing an epithelial
`[0015]
`membrane with at least a part of a basement membrane left;
`and
`
`(C) plating the cell suspension on the medical
`:0032]
`material described in any of [l] to [4], and cultivating
`thereof.
`
`[15: The method of constructing a corneal endot-
`:0033]
`helium-like sheet described in [14], wherein the following
`step (13-1) is carried out after the step (B):
`
`(B- ) increasing a cell density of the cell suspen-
`:0034]
`sion by centrifugation.
`
`[16: The method of constructing a corneal endot—
`:0035]
`helium-like sheet described in [14] or [15], wherein, in the
`step (C), centrifugation is carried out after the cell suspen-
`sion is plated.
`
`[17: The method of constructing a corneal endot—
`:0036]
`helium-like sheet described in [14] or [15]. wherein the step
`C) comprise the following steps (C-l) to (C-5):
`
`(C— ) placing a container in a culture container, the
`:0037]
`container having a bottom surface including a membrane
`with a pore size allowing a culture solution to pass through;
`
`
`
`
`
`(ii) drying the amniotic membrane that does not
`[0016]
`contain the epithelium obtained in the step (i) under such
`conditions that the remaining basement membrane retains a
`structure allowing the adhesion and proliferation of cells
`thereon in use.
`
`[6] The method of preparing a medical material
`[0017]
`derived from amniotic membrane described in [5], wherein
`the step (ii) is carried out by lyophilization.
`
`[7] The method of preparing a medical material
`[0018]
`derived from amniotic membrane described in [5] or [6],
`further comprising the following step (iii):
`
`(iii) containing the dried material obtained in the
`[0019]
`step (ii) in a container in a state that is not substantially in
`contact with oxygen.
`
`[8] The method of preparing a medical material
`[0020]
`derived from amniotic membrane described in [7], wherein
`the state that is not substantially in contact with oxygen is a
`state in which the container has been evacuated or a state in
`which the gas in container has been replaced by nitrogen
`gas.
`
`[9] A method of constructing a corneal epithelium-
`[0021:
`like sheet, the method comprising the following step:
`
`cultivating corneal epithelial cells on the medical
`[0022:
`material described in any of [l] to [4].
`
`[10] A method of constructing a corneal epithe-
`[0023:
`lium-like sheet, the method comprising the following steps
`(a) and (b):
`
`a) cultivating corneal epithelial cells or cells having
`[0024:
`differentiation potency into corneal epithelial cell—like cells
`on the medical material described in any of [l] to [4]; and
`
`b) after cells were proliferated and stratified, bring—
`[0025:
`ing an outermost layer of the stratified cells into contact with
`air.
`
`
`
`[11] The method of constructing a corneal epithe-
`[0026:
`lium-like sheet described in [10], wherein the step (a) is
`carried out in he coexistence of supporter cells.
`
`[12] The method of constructing a corneal epithe—
`[0027:
`lium—like sheet described in [10], wherein the step (a) is
`carried out in the coexistence of supporter cells and in a state
`in which an isolation membrane having a pore size through
`which the supporter cells cannot pass is provided between
`the supporter cells and the medical material.
`
`[13] The method of constructing a corneal epithe-
`[0028]
`lium-like sheet according to any of [10] to [12], wherein the
`cell cultivated in the step (a) is an oral mucosa] epithelial
`cell.
`
`[14] A method of constructing a corneal endothe-
`[0029]
`lium—like sheet, the method comprising the following steps
`(A) to (C):
`
`(A) cultivating and proliferating collected corneal
`[0030]
`endothelial cells;
`
`(B) preparing a cell suspension by collecting the
`[0031]
`proliferated corneal endothelial cells; and
`
`
`
`(C-Z) placing the medical material described in any
`:0038]
`of [1] to [4] on the bottom surface of the container;
`
`:0039]
`material;
`
`:0040]
`
`:0041]
`
`(C3) plating the cell suspension on the medical
`
`(C—4) carrying out centrifugation; and
`
`(C-S) cultivating.
`
`in this specification,
`:0042] Unless otherwise specified,
`“corneal epithelial cell” is used as a term encompassing cells
`included in a corneal epithelial cell layer. That is to say, it
`also includes a corneal epithelial stem cell. Similarly, “cor-
`neal endothelial cell” is used as a term encompassing cells
`included in a corneal endothelial cell layer. That is to say, it
`also includes corneal endothelium stem cell. Furthermore, in
`this specification, “corneal epithelium-like sheet” is a term
`for meaning a cell layer that has similar features to those of
`the corneal epithelium and can be transplanted as a substi—
`tute for the corneal epithelitun. Similarly, a “corneal epithe-
`lium transplantation sheet” is used as a term for meaning a
`composition that has similar features to those of the corneal
`epithelium and can be transplanted for reconstructing the
`corneal epithelium. On the other hand, “corneal endothe-
`lium—like sheet” is a term for meaning a cell layer that has
`similar features to those of the corneal endothelium and can
`be transplanted as a substitute for the corneal endothelium.
`Similarly, a “corneal endothelium transplantation sheet” is
`used as a term for meaning a composition that has similar
`features to those of the corneal endothelium and can be
`transplanted for reconstructing the corneal endothelium.
`
`
`BRIEF DJSCRIPIION OF 1H,: DRAWINGS
`
`
`[0043] FIG. 1 shows scanning electron micrographs of
`images of amniotic membrane. A shows an image of the
`surface of anmiotic membrane in a normal state (a state in
`which the epithelium is kept intact); and B shows an image
`ofthe surface of the anmiotic membrane after the epithelium
`
`was denuded (0.02% :EDTA solution).
`In A, polygonal
`amniotic membrane epithelium is observed, whereas, in B,
`no anmiotic membrane epithelium is observed, which cori-
`firrns that the epithelium was completely denuded.
`
`MTF EX. 1022, pg. 9
`
`

`

`US 2006/0153928 A1
`
`Jul. 13, 2006
`
`[0044] FIG. 2 is a table summarizing the surface analysis
`of lyophili7ed amniotic membrane. ln the table, + denotes
`positive and — denotes negative. AM(—)FJ_)yray: dried amni-
`otic membrane obtained by the method according to
`Example 5, AM(+): sample of amniotic membrane subjected
`to only washing after collection, AM(—): sample of amniotic
`membrane from which the epithelium was removed but to
`which lyophilization was not carried out, AM(—)dispasc:
`sample of amniotic membrane from which the epithelium
`was removed by dispase treatment but to which drying
`process is not carried out, AM(—)FD: sample of amniotic
`membrane from which the epithelium was removed and to
`which lyophilization was carried out. but sterilization pro-
`cess was not carried out, AM(—)yray: a sample of amniotic
`membrane to which sterilization process was canded out
`without carrying out lyophilization after the epithelium was
`removed.
`
`[0045] FIG. 3 is a cross—sectional View schematically
`showing a state of an instrument, etc. when oral mucosa]
`epithelial cells are cultivated on amniotic membrane. In a
`culture dish 1, a culture insert 2 is disposed. On the bottom
`surface of the culture dish 1, a 3T3 cell layer 5 is formed.
`Furthermore, on the bottom surface of the culture insert 2.
`amniotic membrane 3 is placed and conical epithelial cells
`4 are cultivated on the amniotic membrane. Reference
`numeral 6 denotes a culture medium.
`
`[0046] FIG. 4 shows an HE (hematoxylin-eosin) stained
`image of a corneal epithelial cell layer formed on lyo—
`pliilized amniotic membrane.
`
`[0047] FIG. 5 shows a state of the ocular surface before
`operation (upper image), and a state of the ocular surface
`(fiuorescein—stained image) two days after transplantation of
`a corneal epithelium transplantation sheet (lower image). A
`portion stained with fiuorescein (arrow) is shown by hatch-
`ing. The ocular surface (transplanted surface) is not stained
`with fiuorescein, showing the survival of a graft onto the
`ocular surface. Furthermore,
`it is observed that the entire
`peripheral portion of the graft is stained with fiuorescein.
`
`[0048] FIG. 6 shows a state of the ocular surface 7 days
`after transplantation of a corneal epithelium transplantation
`sheet (upper view shows an image obtained by directly
`observing the ocular surface, and lower View shows an
`image stained with lluoresceiii). It is shown that the graft
`maintains transparency (upper View). A portion stained with
`fluorescein (arrow) is shown by hatching. It is shown that
`only a small portion is stained and that the graft remains on
`the ocular surface and further spreads toward the periphery
`as compared with two days after the transplantation,
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`[0049] The first aspect of the present invention relates to
`a medical material derived from amniotic membrane includ-
`ing the following features (1) to (3) of:
`
`[0050]
`
`(l) being in a dried state;
`
`[0051]
`
`(2) not having an epithelial layer; and
`
`(3) having a basement membrane that retains a
`[0052]
`structure allowing the adhesion and proliferation of cells
`thereon in use.
`
`
`
`Firstly, the medical material of the present inven-
`[0053]
`tion is characterized by being derived from amniotic inem—
`brane. Herein, “being derived from amniotic membrane”
`broadly means that the medical material is obtained by using
`the amniotic membrane as a starting material. From the
`Viewpoint of immunogenicity, it is preferable that the medial
`material of the present invention is derived from human
`amniotic membrane. Human amniotic membrane is a mem-
`brane covering the outermost layer of the uterus and the
`placenta, and has a structure in which a basement membrane
`and an epithelial layer are formed on stromal tissue that is
`rich in collagen.
`[0054] With the property (1), the medical material of the
`present invention can be handled easily. Specifically, the
`medical material can be stored even at room temperature
`(for example, from about 100 C. to about 350 C.). That is to
`say, the material is not required to be managed in freezing
`or refrigerating environment before use, thus facilitating the
`handling thereof (storing, delivery, e c.). However,
`the
`above-mentioned description does not mean that the medical
`material of the present invention cam10 be stored at lower
`temperatures. and it may be stored in freezing or refriger-
`ating environment if necessary.
`
`[0055] On the other hand, since the medical material is in
`a dried state. it can be subjected to an e ective sterilization
`process without afl'ecting the use thereof Furthermore, since
`the medical material is in a dried state, very little deterio—
`ration overtime occurs. As a result, the medical material can
`be maintained at high quality for a long time.
`[0056] The property (2) of the present invention contrib—
`utes to lowering the immunogenicity. That is to say, amniotic
`membrane is a material having low immunogenicity in
`nature, but when amniotic membrane is deprived of an
`epithelial layer completely, the immunogenicity ofthe amni-
`otic membrane is further reduced. On the other hand, the
`property of not having an epithelial layer is important in that
`the targeted cell layer is allowed to be favorably constructed
`on the medical material of the present invention. When a cell
`layer is intended to be constructed by plating certain cells on
`a substrate that is amniotic membrane with an epithelial
`layer left, the proliferation ofthe plated cells are inhibited by
`the epithelial cells of amniotic membrane. By using amni—
`otic membrane from which an epithelial layer is completely
`removed, such a proliferation inhibition due to amniotic
`membrane epithelial cell may not occur, Thus, with the
`property of not having the epithelium, the medical material
`of the present invention functions as a substrate suitable for
`constnicting a cell layer of interest.
`[0057] Since the medical material of the present invention
`has the property (3),
`it becomes a material that can be
`preferably used as a substrate for constructing a cell layer of
`interest. In amniotic membrane obtained from which the
`epithelium has been completely removed and which is
`air-dried, that is, air dried amniotic membrane consisting of
`only stromal layer, since a basement membrane component
`is generally decomposed, when cells are plated thereon, the
`cells do not adhere favorably. However, according to the
`medical material of the present
`invention,
`the basement
`membrane is allowed to remain and the structure is allowed
`to be highly retained. Therefore, the cells plated on the base
`membrane can be adhered well by an adhesion factor
`contained in the basement membrane. As a result, prolifera-
`tion and stratification of cells can be achieved.
`
`MTF Ex. 1022, pg. 10
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`

`

`US 2006/0153928 A1
`
`Jul. 13, 2006
`
`[0058] Note here that the term “in use” in property (3)
`means the time when the medical material of the present
`invention is soaked in an appropriate solution (sterilized
`water, physiological saline solution, phosphate buffer solu-
`tion, appropriate culture medium, and the like), and thereby
`the medical material is recovered from the dried state.
`
`[0059] The presence of the basement membrane can be
`confirmed by examining the presence of constituting com-
`ponents peculiar to the basement membrane, e.g., type IV
`collagen (otl, CL2 and (15), type VII collagen, laminine 5, and
`the like. 'lhat is to say, in the medical material of the present
`invention, when it is recovered, at least one, preferably a
`plurality of, and more preferably all of the above—mentioned
`components are detected. Note here that the detection of the
`components such as type VII collagen can be carried out by
`immunostaining procedure using antibodies specific to these
`components .
`
`[0060] Examples of cells that are allowed to proliferate on
`the medical material of the present invention may include
`epithelial cells (corneal epithelial cells, skin epithelial cells.
`oral mucosal epithelial cells, and the like), and endothelial
`cells (corneal endothelial cells, and the like). Note here that
`example of the oral mucosal epithelial cells include, for
`example, cells existing in the dental root part (oral crevicular
`mucosal epithelial cells), mucosal epithelial cells of labial
`part, mucosal epithelial cells of palate part, mucosal epithe-
`lial cells of buccal part, and the like.
`
`[0061] As mentioned above, medical material derived
`from amniotic membrane of the present invention is an
`extremely useful material for constructing a cell layer (tis—
`sue) that is intended to be used for transplantation.
`
`It is preferable that the medical material of the
`[0062]
`present invention is contained in a container in a state that
`is not substantially in contact with oxygen. In this embodi-
`ment, the medical material is not deteriorated by oxygen and
`can be maintained at high quality for a long time. The term
`“state that is not substantially in contact with oxygen” means
`a state in which oxygen is not substantially present in a
`container. Specifically, such a state can include, for example,
`a state in which the container is evacuated or a state in which
`nitrogen is filled (nitrogen—substituted) in a container. The
`“container” is not particularly limited as long as it is suitable
`for containing the medical material of the present invention.
`For example, a ba g or a tube made of plastic synthetic resin
`(a container may be formed by superimposing two sheets
`and sealing the peripheral portions thereof), or a bottle made
`of inorganic material such as glass may be used as a
`container of the present invention.
`
`[0063] The above-mentioned medical material of the
`present invention can be produced by the following method
`(a second aspect of the present invention).
`
`[0064] The second aspect of the present invention relates
`to a method of producing a medical material derived from
`amniotic membrane and the method includes the following
`steps (i) and (ii):
`
`layer from amniotic
`(i) removing an epithelial
`[0065]
`membrane with at least a part of a basement membrane left:
`and
`
`(ii) drying the amniotic membrane that does not
`[0066]
`contain the epithelium obtained in the step (i) under such
`
`conditions that the remaining basement membrane retains a
`structure allowing the adhesion and proliferation of cells
`thereon in use.
`
`In the step (i), an epithelial layer is removed. At this
`[0067]
`time, however, basement membrane is not removed together
`and at least a part of the basement membrane is allowed to
`remain. Such a process is carried out by denuding an
`epithelial layer with the use of a cell scraper, forceps, etc.
`Preferably, the adhesion between cells constituting the epi-
`thelia layer is loosen by using EDTA, proteolytic enzyme,
`and the like, in advance, and then the epithelial layer is
`denuded with the use of a cell scraper, etc. However, it is
`preferable that such a pre—process (process using EDTA,
`etc.) is carried out under conditions where the structure of
`the basement membrane involved in the adhesion of epithe-
`lial layer with respect to a stromal layer is not destroyed. For
`example, if process is carried out using dispase under the
`general conditions (for example, dispase is added so that the
`concentration becomes l.2 III and reaction is canied out at
`37° C. for one hour), not only an epithelial layer but also
`basement membrane are considerably damaged. When such
`a pre—processing is carried out, basement membrane holding
`the original
`function camiot be allowed to remain. An
`important point of the step (i) in the present invention is in
`that an epithelial
`layer is completely removed while a
`basement membrane is allowed to remain in a state in which
`at least a part of the basement membrane is allowed to
`remain with the original function thereof maintained. For
`example, when the pre—process is carried out will the use of
`EDTA (for example, EDTA is added so that the concentra-
`tion becomes 0.02% to 0.1% and reaction is carried out at 4°
`
`C. to 37° C. for one hour to four hours), the emect 011 the
`basement membrane is extremely small.
`
`
`
`[0068] Preferably, the amniotic membrane used in the step
`(i) is human anmiotic membrane. Human anmiotic mem-
`brane can be collected by, for example, htunan embryonic
`membrane, placenta, etc. obtained at the time of afterbirth at
`delivery. Specifically, the human amniotic membrane can be
`prepared by treating and purifying human embryonic mem-
`brane, placenta, and umbilical cord obtained right after
`delivery as a whole. The method of preparing human amni-
`otic membrane can employ a well-known method such as a
`method described in Japanese Patent Unexamined Publica—
`tion N o. HOS—56987, etc. That is to say, amniotic membrane
`can be prepared by detaching airmiotic membrane from the
`embryonic membrane obtained at delivery and removing the
`remaining tissue by a physical treatment such as ultrasonic
`cleansing and an enzyme treatment, and the like. The thus
`prepared human amniotic membrane can be cryopreserved
`innnediately before the step (i) is carried out. The human
`anmiotic membrane can be frozen in a liquid obtained by
`mixing equal volume ratio of DMEM (Dulbecco’s modified
`Eagle’s medium) and glycerol at, for example, —80° C. By
`the cryopreservation, not only the improvement in operation
`but also reduction of the antigenicity can be expected.
`
`In the step (ii) following the step (i), the amniotic
`[0069]
`membrane that does not contain the epithelium obtained in
`the step (i) is dried under such conditions that the remaining
`basement membrane retains a structure allowing the adhe-
`sion and proliferation of cells thereon in use. The drying
`method is not particularly limited as long as the method can
`be carried out under such conditions. However, lyophiliza-
`tion is preferably employed. In the lyophilization, in general,
`
`MTF Ex. 1022, pg. 11
`
`

`

`US 2006/0153928 A1
`
`Jul. 13, 2006
`
`in a low atmospheric pressure environment (vacurun) in
`which a boiling point is about —20° C. (l07 Pa, 0.8 Torr) to
`about —50° C. (4 Pa, 0.03 Torr), water content is removed by
`sublimation from a frozen and solidified sample (for
`example, a sample frozen at about —40° C.) According to
`the lyophilization, water can be removed also from the
`inside uniformly and high dryness can be achieved. There-
`fore, the material can be dried while highly maintaining the
`original function and form. Furthermore, the lyophilization
`has features: (1) less deterioration occurs during process; (2)
`sterilization can be achieved easily; (3) a dried material
`excellent in recovering property can be obtained; (4) a dried
`material excellent in preserving property can be obtained.
`and the like.
`
`[0070] The lyophilization can be carried out by a lyo—
`philization apparatus including a vacuum chamber, a cooling
`and heating device and an exhaust system (a cold trap and
`a vacuum pump). A number of lyophilization apparatuses
`are commercially available. In the step (ii) of the present
`invention, a lyophilization apparatus, which was arbitrarily
`selected from the above, can be used. Note here that the
`process conditions can be set based on the instruction for use
`attached to the apparatus to be used. At this time, size of
`samples subjected to drying process and dryness, etc. may
`be considered. The dryness may be set so that, for example,
`the water activity (AW) becomes less than 0.5.
`
`It is preferable that after the above-mentioned step
`[0071]
`(ii). the following step (iii) is carried out.
`
`(iii) containing the dried material obtained in the
`[0072]
`step (ii) in a container so that the material is not substantially
`in contact with oxygen.
`
`[0073] According to this step (iii), it is possible to obtain
`dried amniotic membrane that does not contain the epithe-
`lium that is packaged in a state that is substantially isolated
`from oxygen. Thus, a medical material derived from amni—
`otic membrane capable of being stored for a long time can
`be obtained.
`
`[0074] The step (iii) can be carried out, for example, by
`evacuating a container or by substituting nitrogen inside the
`container after the dried material is contained in an appro-
`priate container. Alternatively, it can be also carried out by
`enclosing deoxidant in a container. As the container, for
`example, a bag or a tube made of plastic synthetic resin (a
`container may be formed by superimposing two sheets and
`sealing the peripheral portions thereof), or a bottle made of
`inorganic material such as glass may be used as a container
`of the present invention.
`
`[0075] Another aspect of the present invention relates to a
`method of using the medical material derived from amniotic
`membrane provided in the first aspect of the present inven—
`tion. One embodiment is a method of constructing a corneal
`epithelium-like sheet. In this embodiment, on the medical
`material derived from amniotic membrane, comeal epithe—
`lial cells or cells having diflerentiation potency into comeal
`epithelial cell-like cells (hereinafter referred to as “cells for
`corneal epithelium“ collectively) are cultivated. The comeal
`epithelial cells can be obtained from an appropriate donor’s
`cornea. It is preferable to use a recipient’s own comeal
`epithelial cells if possible. It is advantageous because a
`corneal epithelium-like sheet that is free from immunologi-
`cal rejection in transplantation can be constructed, and thus
`
`transplantation that does not involve immunological rejec-
`tion can be carried out. When it is impossible or difficult to
`obtain a recipient’s own corneal epithelial cells, corneal
`epithelial cells of a person other than a recipient may be
`used. However, in this case, it is preferable to select a donor
`by considering immunological compatibility.
`
`[0076] The “cells having dillerentiation potency into cor—
`neal epithelial cell-like cells” mean cells capable of con-
`structing a corneal epithelium-like cell layer in the cultiva-
`tion in vitro or in the body after transplantation. Herein, the
`“comeal epithelium-li