EX. 1027EX. 1027
`
`Ex. 1027
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`Certified English-Language
`
`Certified English-LanguageCertified English-Language
`Translation of JP 2001-161353
`
`Translation of JP 2001-1613 53Translation of JP 2001-1613 53
`to Shimazaki at Ex. 1026
`
`
`
`to Shimazaki at EX. 1026to Shimazaki at EX. 1026
`
`

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`(19) Japan Patent Office (JP)
`
`(12) Japanese Unexamined Patent
`Application Publication (A)
`
`
`
`
`(11) Japanese Unexamined Patent
`Application Publication Number
`2001-161353
`(P2001-161353A)
`(43) Publication date June 19, 2001
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`5/00
`9/00
`
`
`590
`
`FI
`C 12 N
`A 61 F
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`
`
`Request for examination Not yet requested Number of claims 5 OL (Total of 8 pages)
`
`Subject codes (Reference)
`E 4B065
`
`Identification codes
`
`
`
`(51) Int. Cl.7
`C 12 N
`5/06
`A 61 F
`9/007
`
`
`(21) Application number
`(22) Date of application
`
`
`
`
`
`
`
`
`
`H11-349705
`December 9, 1999
`
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`(71) Applicant
`
`(72) Inventor
`
`(72) Inventor
`
`(74) Agent
`
`599173088
`Japan Ophthalmic Consultants Inc.
`5-26-7 Nishifuna, Funabashi-shi, Chiba-ken
`JUN SHIMAZAKI
`3-7-28-301 Shimomeguro, Meguro-ku, Tokyo
`KAZUO TSUBOTA
`5-26-7 Nishifuna, Funabashi-shi, Chiba-ken
`100099623
`Patent attorney Naokazu Okuyama
`
`
`
`F terms (reference)
`
`4B065 AA93X BB23 CA44
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`10: Cell sheet for transplantation
`
`16: Epithelial cells
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`14: Stem cell tissue
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`12: Amnion
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`
`
`(TITLE OF THE
`(54)
`for
`INVENTION) Cell sheet
`Transplantation and Method for Manufacturing the
`Same
`
`(57) (ABSTRACT)
`(PROBLEM) To provide a cell sheet for transplantation that
`provides a high epithelial reconstruction ratio and a method
`for its production.
`(MEANS FOR SOLVING) The cell sheet for transplantation 10
`comprises amnion 12, an epithelial stem cell tissue 14
`affixed to the amnion 12, and epithelial cells 16 that have
`proliferated from the stem cell tissue so as to cover the
`amnion surface. In addition, production of the cell sheet 10
`for transplantation involves fixing the amnion 12 to a dish
`plate, bonding the epithelial stem cell tissue 14 onto the
`amnion 12, and then carrying out culturing in this
`condition. As a result of this culturing, the epithelial cells
`16 proliferate from the stem cell tissue 14, and the surface
`of the amnion 12 is covered with the epithelial cells 16. As
`a result, a cell sheet for transplantation 10 is produced
`wherein the epithelial stem cell tissue 14 is affixed to the
`surface of the amnion 12, and the surface thereof is covered
`with epithelial cells 16 that have proliferated.
`
`
`MTF Ex. 1027, pg. 1
`
`

`
`(SCOPE OF PATENT CLAIMS)
`(CLAIM 1) A cell sheet for transplantation, comprising
`amnion with the sponge layer and epithelial layer having
`been removed;
`epithelial stem cell tissue that has been affixed to the
`surface of said amnion; and
`epithelial cells that have been proliferated in vitro from said
`stem cell tissue so as to cover the surface of said amnion.
`(CLAIM 2) The cell sheet for transplantation according to
`claim 1, characterized in that said epithelial cells are
`corneal epithelial cells or conjunctival epithelial cells.
`(CLAIM 3) A method for producing a cell sheet for
`transplantation, comprising:
`preparing amnion with the sponge layer and epithelial layer
`having been removed;
`affixing epithelial stem cell tissue to said amnion; and
`propagating said epithelial cells on said amnion.
`(CLAIM 4) The method for producing a cell sheet for
`transplantation according to claim 3, characterized in that
`said epithelial cells are corneal epithelial cells or
`conjunctival epithelial cells.
`(CLAIM 5) The method for producing a cell sheet for
`transplantation according to claim 3, characterized in that
`said stem cell tissue is corneal limbus cells.
`
`(DETAILED DESCRIPTION OF THE INVENTION)
`(0001)
`(TECHNICAL FIELD OF THE INVENTION) The present
`invention relates to a cell sheet for transplantation and a
`method for its production. In particular, the present
`invention relates to a cell sheet for transplantation in which
`corneal epithelial cells have been cultured and proliferated
`on amnion, and a method for its production.
`(0002)
`(PRIOR ART) The cornea is the outermost layer of the
`optical system that constitutes the eyeball. Being a tissue
`that is transparent and has no vasculature, it contributes to
`providing favorable eyesight through the formation of a
`smooth surface in conjunction with lachrymal fluid. In
`addition, the corneal epithelial cells are in continual contact
`with
`the external environment and
`thus also have
`preventative action in regard to protecting the eyeball from
`foreign objects such as microorganisms in the external
`environment and ultraviolet light or other types of sunlight.
`Specifically, the corneal epithelial cells play an extremely
`important
`role
`in preserving corneal
`transparency,
`protecting the eyeball as a whole, and maintaining
`homeostasis.
`(0003) For example, there are cases where the cornea
`experiences a loss of transparency due to clouding caused
`by keratitis, corneal ulcer, perforation, or other such
`disorders. Treatments involving corneal transplantation
`have been carried out in order to treat permanent loss of
`eyesight resulting from clouding of the cornea in this
`manner. This corneal transplantation involves removing
`the patient’s cornea
`that has
`lost
`transparency and
`transplanting a transparent cornea in its place. As a result
`of this transplantation, transparency is restored, and
`eyesight is once again returned.
`(0004) There are also disorders that cannot be treated
`simply by corneal transplantation in this manner, examples
`of which
`include Stevens-Johnson syndrome, ocular
`
`cicatricial pemphigoid, chemical injury, and heat injury.
`Normally, the corneal epithelial cells divide daily, with old
`cells separating away and new cells being regenerated from
`stem cell tissue. However, damage to the stem cell tissue
`that regenerates the cornea is seen in these disorders.
`(0005) The stem cell tissue that regenerates the corneal
`epithelium is referred to as “corneal limbus tissue.” This
`tissue is localized just at the interfacial region between the
`iris and the sclera and is in a special environment that is
`exposed to the external environment. For this reason, in the
`disorders described above, it is thought that the stem cell
`tissue itself is eradicated as a result of incurring some kind
`of injury. The defective regions resulting from the
`eradication of the stem cell tissue then become covered by
`the surrounding conjunctival epithelium, resulting in a loss
`of transparency and dramatic loss of vision.
`(0006) With these types of disorders, the corneal limbus is
`depleted, and so the transplanted cornea will not persist
`over a
`long period of
`time when simple corneal
`transplantation is performed. For this reason, in order to
`achieve lasting eye surface regeneration, it is necessary also
`to transplant the corneal limbus.
` One method for
`transplantation of the corneal limbus that has been
`developed is a transplantation method that employs amnion
`(Asahi Medical, September, 1999, pp. 62-65, N. Engl. J.
`Med. 340: 1697-1703, 1999).
`(0007) The amnion that is used in this transplantation
`method can be obtained from pregnant women who have
`undergone caesarean section. This amnion has a thick
`basement membrane and thus functions as a substrate for
`the proliferation and differentiation of corneal epithelial
`cells.
`
`In addition, amnion
`is nearly devoid of
`immunogenicity and thus also has the action of inhibiting
`
`inflammation and scarring. Amnion thus protects the
`corneal epithelium or stem cell tissue thereof that has
`proliferated on the amnion from rejection reactions and the
`like in the person receiving the transplant (recipient).
`(0008) With transplantation methods that employ amnion
`having these types of properties, as shown in Fig. 3, the
`corneal tissue that has experienced conjunctivalization or
`the like is first cut away, exposing the corneal stroma and
`sclera. The amnion 1 for eyeball surface regeneration is
`then attached onto the exposed corneal stroma and sclera.
`A central cornea (including epithelium, stroma, and
`endothelium) 2 is then cut out from the donated corneal
`tissue, the periphery of the corneal limbus tissue 3 is
`prepared, and the tissue is transplanted and sutured onto the
`exposed amnion and corneal stroma. The corneal limbus 3
`that has been transplanted in this manner differentiates and
`propagates with this amnion 1 as a substrate, while being
`protected by the amnion 1 that is not immunogenic. The
`corneal epithelium is thereby regenerated on the amnion 1.
`This method thus allows long-term maintenance and
`regeneration of the corneal epithelium.
`(0009)
`(PROBLEMS TO BE SOLVED BY THE INVENTION) However,
`with conventional transplantation methods that employ
`amnion, there are many cases in which epithelialization
`does not occur appropriately from the transplanted corneal
`limbus after surgery, and restoration of vision is not
`obtained.
`
`In
`addition,
`in
`long-term
`follow-up
`investigations of limbus transplantation in patients having
`
`MTF Ex. 1027, pg. 2
`
`

`
`experienced corneal chemical injury or patients suffering
`from Stevens-Johnson syndrome,
`the eyeball surface
`regeneration success ratio has been roughly 50%, with lack
`of epithelialization occurring in many of these 50% of
`cases in which success was not obtained.
`(0010) In the past, it was assumed that surgical treatment
`methods were not possible with these disorders, and
`although transplantation methods that employ amnion have
`allowed the transplantation success ratio to reach 50%,
`additional improvement in treatment outcome is desired.
`(0011) On the other hand, within the field of regenerative
`medicine,
`there has been ongoing development of
`transplantation sheets that are used for transplantation at
`sites of damage in which the entire layer of hypodermis and
`epithelium have been lost in human skin. For example,
`Japanese Unexamined Patent Application No. 10-277143
`describes a
`transplanting sheet and method for
`its
`production that is used for treating full-thickness skin
`wounds in human skin and the like.
`(0012) This transplantation sheet is produced by enclosing
`hypodermal tissue-derived fibroblasts in human fibrin
`sheet, with epidermis being affixed to the sheet surface.
`With this type of transplantation sheet, superior graft
`survival rates are exhibited in cases of transplantation into
`full-thickness wounds in which the hypodermis has been
`lost as well.
`(0013) The present invention was developed in light of the
`above problems, and an object of the invention is to
`provide a cell sheet for transplantation and a method for its
`production that achieves better treatment results in regard
`to epithelial cells such as corneal epithelial cells by
`employing amnion.
`(0014)
`(MEANS FOR SOLVING THE PROBLEMS) In order to attain the
`objectives described above,
`the present
`invention
`is
`characterized in that epithelial stem cell tissue is affixed
`onto amnion from which the sponge layer and epithelial
`layer have been removed, and the epithelial cells are
`regenerated in vitro from the stem cell tissue so as to cover
`the amnion surface.
`(0015) In accordance with the present invention as
`described above, epithelial cells are propagated from stem
`cell tissue that has been affixed in advance to amnion in a
`cell sheet for transplantation so as to cover the amnion
`surface, thereby improving the epithelial regeneration ratio
`relative to transplantation methods using conventional
`amnion and achieving better treatment outcomes.
`(0016) In addition, with the cell sheet for transplantation,
`because stem cell tissue has been affixed in advance on
`amnion, the transplantation time is shortened relative to
`conventional cases in which the amnion and corneal limbus
`are separately transplanted onto the eyeball surface.
`(0017) Depending on the condition in which the stem cell
`tissue has been obtained, the tissue may include epithelial
`cells at the periphery of the stem cell tissue in addition to
`stem cell tissue. For this reason, the epithelial cells that
`have been produced as a result of propagation by culturing
`the stem cell tissue on amnion may also include epithelial
`cells from the donor in addition to epithelial cells that have
`propagated from the stem cell tissue.
`(0018)
`
`(EMBODIMENTS OF THE INVENTION) Preferred embodiments
`of the present invention are described below with reference
`to the drawings.
`for
`sheet
`cell
`embodiment) The
`(0019)
`(First
`transplantation of the first embodiment is shown in Fig. 1.
`(0020) As shown schematically in Fig. 1, the cell sheet for
`transplantation 10 comprises amnion 12, epithelial stem
`cell tissue 14 that has been affixed to the amnion 12, and
`epithelial cells 16 that have been propagated from the stem
`cell tissue so as to cover the amnion surface.
`(0021) The amnion 12 is the membrane that directly coves
`the fetus on the innermost side of the amniotic sac of
`vertebrates. The amnion 12 has almost no immunogenicity
`and also has an anti-inflammatory action. Although the
`amnion 12 has almost no immunogenicity in this manner,
`in order to more reliably prevent elicitation of an immune
`response subsequent to transplanting with the cell sheet for
`transplantation into humans, it is preferable to use amnion
`that is derived from the same animal species, i.e., human.
`(0022) When this amnion is derived from the same species,
`specifically, if the amnion is derived from humans when
`transplanting
`into humans,
`then the material
`is not
`restricted to humans that are related by blood, such as
`parents or children, and material that is derived from any
`human may be used.
`(0023) Human-derived amnion can be obtained, for
`example, from pregnant females who have undergone
`caesarean section. This amnion typically has a sponge
`layer, a compact layer, a basement membrane layer, and an
`epithelial layer, in sequence from the bottom. However,
`with the “amnion” used in the cell sheet for transplantation,
`the sponge layer and epithelial layer are not necessary, and
`so material may be used that has been treated to remove
`these layers.
`(0024) The epithelial stem cell tissue 14 that is affixed to
`the amnion 12 is cell tissue that has the potential for
`regenerating epithelial cells. The term “epithelial cells”
`used herein includes, for example, corneal epithelial cells
`and conjunctival epithelial cells.
`(0025) Examples of epithelial stem cell tissue 14 include
`corneal limbus tissue which is the stem cell tissue for
`corneal epithelial cells.
` If
`the epithelial cells are
`conjunctival epithelial cells, the stem cell tissue 14 is stem
`cell tissue for conjunctival epithelial cells which present is
`at the conjunctival border, or the like. The corneal limbus
`cells referred to above are localized at the boundary of the
`sclera and the iris of the eyeball and are exposed at the
`eyeball surface. These cells may be obtained from corneal
`donors and the like.
`(0026) The epithelial stem cell tissue 14 may be pure stem
`cell tissue or may include surrounding epithelial cells,
`fibroblasts, and vascular endothelial cells.
`(0027) The epithelial stem cell tissue 14 may be obtained
`from a source other than the recipient patient, and the donor
`may be a parent or sibling of the recipient, or a person that
`has no blood relationship to the recipient.
`(0028) With epithelial stem cell tissue 14 that is obtained
`from an unrelated person, it is preferable to use material
`that is derived from a donor that has a matching HLA type
`in order to avoid the potential for immune-based rejection
`reactions. However, when epithelial cell tissue cannot be
`obtained from a donor with a matching HLA type, the
`
`MTF Ex. 1027, pg. 3
`
`

`
`material may be obtained from a donor that does not have a
`matching HLA type.
`(0029) In addition, in order to prevent infection resulting
`from transplantation, the donated tissue may be tissue that
`has been confirmed in advance to pose no threat of
`infection.
`(0030) In addition, the epithelial stem cell tissue 14
`preferably is in a favorable state and can generate epithelial
`cells. The size of the epithelial stem cell tissue 14 will be
`different depending on the condition of the cells. For
`example, when the condition of the epithelial stem cell
`tissue 14 is favorable, the size may be about 1 mm2, for
`example, for an amnion size of 2 cm2.
`(0031) In addition, the epithelial stem cell tissue 14 may be
`a single tissue sheet or multiple tissue sheets. These
`epithelial stem cell tissues 14, when carried on the amnion,
`are preferably placed on the amnion by arranging them in a
`configuration in which they are intrinsically disposed in the
`body.
`(0032) Fig. 1 shows a preferred state of disposition for case
`in which corneal limbus tissue is used for the epithelial
`stem cell tissue 14. Specifically, corneal limbus tissue is
`normally localized at the boundary between the sclera and
`the iris, and so a case is shown in which the epithelial stem
`cell tissue 14 is disposed at locations corresponding to the
`boundary thereof.
`(0033) The epithelial cells 16 that have been made to
`proliferate on the amnion 12 include cells that have been
`made to proliferate by in vitro culturing of the epithelial
`stem cell tissue 14 that is affixed to the amnion 12, and, for
`a situation in which surrounding epithelial cells are bound
`to the donated stem cell tissue, donor epithelial cells in
`addition to the epithelial cells that have proliferated from
`the stem cell tissue.
`(0034) In addition, the epithelial cells 16 that have
`proliferated on the amnion are preferably in a favorable
`condition at the time of transplanting. For example, cells
`are preferred that are in their growth stage whereby a first-
`order straight line that rises to the right is exhibited upon
`drawing a growth curve.
`(0035) As stated above, the cell sheet for transplantation 10
`that is covered with the epithelial cells 16 and that has
`epithelial stem cell tissue provided on the surface of
`amnion 12 is transplanted at a lesion in which there has
`been eradication or damage of the epithelial cells and the
`stem cell
`tissue
`thereof.
` With
`the cell sheet for
`transplantation 10, by propagating the epithelial cells 16
`from the epithelial stem cell tissue 14 in advance so that the
`surface of the amnion 12 is covered, it is possible to
`improve
`the epithelial
`tissue
`regeneration
`ratio
`in
`comparison to methods in which the epithelial cells are
`propagated after transplanting at the lesion, as has been
`done in the past.
`(0036) (Second embodiment) In this embodiment, the
`method for producing the cell sheet for transplantation 10 is
`described.
`(0037) (1) Preparation of the amnion
`
`The steps for preparing the amnion are shown in
`Fig. 2.
`(0038) As shown in Fig. 2, when transplanting into a
`human, the amnion that will serve as the substrate for the
`cell sheet for transplantation is obtained by harvesting the
`
`amnion from a pregnant female that has undergone
`caesarean section (S200).
`(0039) If the amnion will not be used immediately upon
`harvesting (S201), it can be stored (S202). The storage
`method can involve treatment for the storage, followed by
`immersion in storage solution and storage at -80°C.
`(0040) Treatment of the amnion for storage can involve, for
`example, preparing physiological saline containing DMSO
`(dimethyl sulfoxide) at concentrations of, for example, 4.2,
`8.5, and 15.0%, then immersing the amnion in the solutions
`for about 30 min each, sequentially starting from the lower
`DMSO concentration.
`the
`(0041) The amnion storage solution may be
`physiological saline containing 15% DMSO referred to
`above.
`(0042) The amnion that has been recovered from storage or
`the amnion that has been harvested as described above is
`subjected to a pretreatment in order to facilitate separation
`of the unnecessary epithelial layer and sponge layer that
`constitute the amnion (S203). This pretreatment method
`has no particular restrictions, but may involve treatment
`with 10% aqueous ammonia, for example.
`(0043) The amnion that has been subjected to the
`pretreatment is then cut to a suitable size as necessary
`(S204). In cases where the eyeball surface is to be
`regenerated, the cut size is about 2 cm2, for example, which
`allows the exposed eyeball surface to be well covered.
`However, the size may be determined in accordance with
`the surface area of the transplantation site.
`(0044) The amnion that has been cut is introduced into dish
`plates for cell culture (e.g., 35 mm diameter). The amnion
`that has been introduced into a plate has had the sponge
`layer and epithelial layer stripped, and so only the compact
`layer and the basement membrane remain (S205). The
`amnion is then fixed to the plate bottom with the basement
`membrane oriented upwards (S206).
`(0045) Fixing of the amnion to the plate has no particular
`restrictions, provided that the method does not significantly
`damage the amnion. However, a method may be used, for
`example, that involves a combination of freezing and
`drying as described below.
`(0046) Specifically, amnion with the compact layer and
`basement membrane remaining is completely frozen at -
`80°C and is then returned to room temperature, whereupon
`the water content in the plate is removed by suctioning.
`The amnion is then preferably arranged with the wrinkles
`spread out and the epithelial side facing upwards, and the
`amnion surface is then completely dried in an aseptic state.
`(0047) After drying, the material may be used as-is, or may
`be frozen again at -80°C. When frozen, the amnion is
`returned to room temperature prior to use, and the amnion
`can then be used after the water content affixed to the dish
`plate has been removed.
`(0048) (2) Culturing and propagation of the epithelial cells
`on the amnion
`
`The epithelial stem cell tissue that has been
`harvested from the donor is affixed to the amnion, and
`epithelial cells are cultured and made to propagate.
`(0049) Specifically, the stem cell tissue that is used herein
`are preferably in good condition, and, for example, the stem
`cells that have been obtained from the donor in good
`condition are preferably used immediately after donation.
`
`MTF Ex. 1027, pg. 4
`
`

`
`Upon obtaining this stem cell tissue in good condition, the
`tissue is affixed to the surface of the amnion that has been
`fixed to the aforementioned plate, medium is introduced in
`an amount whereby the stem cell tissue does not float, and
`culturing is initiated. However, after affixing the stem cell
`tissue to the amnion, it is preferable to increase the amount
`of culture medium to an amount whereby the stem cell
`tissue or the epithelial cells that have started propagating
`are well infused with medium.
`(0050) The size of the stem cell tissue that has been affixed
`to the amnion is preferably on the large side. For example,
`with corneal limbus tissue, it is preferable to use about 1
`mm2 for 2 cm2 of amnion. However, the size can be
`increased or decreased depending on the condition of the
`cell tissue.
`(0051) The medium that is used for culturing of the stem
`cell tissue has no particular restrictions, provided that it is a
`medium whereby the epithelial cells can be suitably
`propagated from the stem cell tissue. For example, when
`initiating culturing of the stem cells, SHEM medium or the
`like may be used.
` In addition, after initiation of
`propagation of the epithelial cells, basal medium for
`epithelial cell culturing such as medium 165 may be used.
`The compositions of SHEM medium and medium 165 are
`described below.
`(0052) Serum required for cell culture may be added to
`these media. Fetal calf serum (FCS) or the like may be
`used for this serum, but it is preferable to use serum from
`the recipient or serum from the donor of the stem cell
`tissue.
`(0053) When FCS or the like is used, it is preferable to use
`the material after centrifuging the commercial product and
`passing it through a filter. In addition, when using human
`serum, it is preferable to use material after heat treatment
`(e.g., about 3 min at 56°C) or after passage through a 0.22
`μm filter or the like to remove cells or the like such as
`leukocytes that can elicit an immune-based rejection
`response.
`(0054) In addition, the added amount of serum can be
`adjusted to a high concentration of, for example, about 15%
`at the time of initiation of culturing of the stem cell tissue.
`On the other hand, a comparatively low concentration of
`about 3%, for example, may be used after initiation of
`propagation of the epithelial cells. The time at which the
`media is switched over may be the stage at which
`propagation of
`epithelial
`cells
`is microscopically
`confirmed, or the stage at which acidification due to cell
`growth is indicated by an indicator when a pH indicator or
`the like has been added to the medium.
`(0055) Culturing can be carried out in an environment that
`is desirable for cell propagation. For example, a CO2
`incubator set to 37°C or the like may be used. In addition,
`the culturing time may be as short a time as necessary for
`the surface of the amnion to be covered and as long as it
`takes for vigorous propagation of epithelial cells. For
`example, it is preferable for the time to be within the range
`of the growth stage during which a first-order straight line
`results from plotting cell growth on a graph.
`(0056) For example, an approximate time period may be
`used that is required for achieving confluence, meaning that
`the bottom surface of the culture plate is completely
`covered with epithelial cells that have propagated. More
`
`specifically, when corneal limbus tissue in good condition
`(1 mm2) is cultured on amnion (2 cm2) that has been fixed
`to a dish plate (diameter 3.5 cm), a culturing period of
`about 10 to 14 days can be used.
`(0057) (3) Recovery of cell sheet for transplantation
`
`As a result of culturing in the dish plate, the stem
`cell tissue adheres to the surface of the amnion that
`functions as a substrate, and a cell sheet for transplantation
`is produced in which epithelial cells that have propagated
`from the stem cell tissue have spread out so as to cover the
`amnion surface. The cell sheet for transplantation that is
`thereby produced is recovered by peeling the amnion from
`the dish plate. The cell sheet for transplantation that has
`been recovered is then transplanted at the lesion site of the
`recipient soon after recovery.
`(0058) (Third embodiment) In this embodiment, a kit for
`producing a cell sheet for transplantation will be described.
`(0059) The cell sheet for transplantation can be produced
`by
`the series of methods presented
`in
`the second
`embodiment. However, in order to facilitate production, a
`kit may also be offered that includes the culture solutions,
`reagents, and the like that are required for producing the
`cell sheet for transplantation, as well as a manual that
`describes the production methods.
`(0060) For example, this kit can include media required for
`culturing the amnion and stem cell tissue that has been
`fixed to a plate. In addition, as necessary, the kit may
`include amnion or the like. By offering a kit that includes
`amnion, media, and the like in this manner, it is possible to
`produce a cell sheet for transplantation comparatively
`easily in accordance with the production methods described
`above, provided that the stem cell tissue and the like is
`prepared.
`(0061)
`(EXAMPLES) The present invention is described in detail
`below using examples, but the present invention is not
`restricted to the examples.
`(0062) (Example 1) Preparation of amnion
`
`Amnion is prepared for use as the substrate for
`the cell sheet for transplantation. Amnion is harvested
`from a pregnant mother having undergone caesarean
`section or the like. When the harvested amnion is to be
`stored, treatment solution for storage is prepared. The
`treatment solution is prepared as 4.2, 8.5, and 15.0%
`solutions that are produced by adding DMSO at a final
`concentration of 4.2, 8.5, and 15.0% to sterile physiological
`saline or sterilized PBS (-). The prepared solutions are
`introduced into respective containers.
`(0063) Pretreatment of the harvested amnion is carried out
`by immersing the amnion for 30 min in each of the
`solutions,
`sequentially,
`starting
`from
`the
`lowest
`concentration, i.e., the 4.2% solution, the 8.5% solution,
`then the 15.0% solution. After treatment, the amnion
`epithelium is removed in cases where the amnion is to be
`used immediately. Otherwise, when storing, the amnion is
`introduced into a storage container containing the 15%
`solution and is stored at -80°C.
`(0064) 15% solution is poured into a dish plate for cell
`culture (e.g., 100 mm diameter), and the amnion is then
`transferred therein. The amnion is cut to the size needed
`for use, and the material is then transferred into individual
`dish plates (e.g., 35 mm diameter). The harvested amnion
`
`MTF Ex. 1027, pg. 5
`
`

`
`has a sponge layer, compact layer, basement membrane,
`and epithelium in sequence from the bottom layer, but the
`sponge layer which is the bottom-most layer of them is
`stripped using a pincers. After stripping the sponge layer,
`the amnion is immersed in 10% aqueous ammonia and is
`left for 30 min at room temperature.
`(0065) Next, the top-most epithelium layer is stripped from
`the amnion using a scraper. After stripping the epithelium,
`the 10% aqueous ammonia is then removed by aspiration.
`Sterilized PBS is then introduced into the dish plate to wash
`the amnion.
`(0066) The washed amnion is then used as substrate for
`propagating and culturing the epithelial cells. To this end,
`the amnion is applied to the bottom of a dish plate and
`fixed. The application and fixing are carried out so that the
`amnion will not float when the culture solution is
`subsequently added.
`(0067) The amnion is then frozen and dried in order to affix
`the amnion to the bottom of the plate. Specifically, with
`methods in which two repetitions of freezing are carried
`out, the amnion that has been washed with PBS is first
`placed at -80°C and is completely frozen.
`(0068) Next, the amnion that has been frozen is returned to
`room temperature on the day prior to culturing of the
`epithelial cells, and the water content in the plate is
`aspirated with an aspirator. After removing the water
`content with the aspirator, the amnion is placed with the
`upper layer facing upwards, and the wrinkles in the amnion
`are eliminated by spreading it with a forceps. In this state,
`the dish plate is placed in an incubator with the lid off, and
`the material is dried for one hour or longer using a fan.
`After confirming that the upper layer surface of the amnion
`is completely dry, the material is again frozen at -80°C and
`is returned to room temperature one hour prior to use. The
`material is then used after removing the water content that
`has affixed to the dish plate.
`(0069) When the amnion is to be fixed to the bottom of the
`plate, the second freezing step can be omitted.
`
`<SHEM medium>
`Component
`Dulbecco’s modified Eagles’ medium
`containing HEPES (D-MEM/F12)
`NaHCO3
`Insulin (human recombinant, expressed
`in E. coli)
`Human epithelial cell growth factor
`(human-EGF)
`Cholera toxin
`Dimethyl sulfoxide (DMSO)
`Antibiotic
`(e.g.,
`benzylpenicillin,
`streptomycin)
`
`165 medium
`Component
`165 medium
`for human corneal
`(Basal medium
`epithelial cell culture)
`Insulin
`Human-EGF
`
`Source
`Gibco BRL
`
`Wako Pure Chemical
`SIGMA
`
`Gibco BRL
`
`Gibco BRL
`SIGMA
`
`
`Source
`KURABO
`
`SIGMA
`Gibco BRL
`
`for
`(Example 2) Production of cell sheet
`(0070)
`transplantation using corneal limbus tissue obtained from
`the recipient’s parent, sibling, or the like
` The corneal limbus tissue is harvested from the donor,
`and serum is separated. The separated serum is heat-treated
`for 30 min at 56°C to inactivate it. The serum is then
`added
`to SHEM medium,
`thereby preparing SHEM
`medium containing 15% serum. Some of this prepared
`medium is introduced in advance into a vessel, and the
`corneal limbus tissue from the donor is harvested. The
`harvested corneal limbus tissue is then immediately
`introduced into the container containing the medium.
`(0071) The corneal limbus tissue is placed on the
`aforementioned amnion that has been fixed to the dish
`plate, and SHEM medium containi

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