U.S. patent application number 15/647742 was filed with the patent office on 2017-10-26 for method for producing the laminate of a sheet-shaped cell culture and fibrin gels.
This patent application is currently assigned to TERUMO KABUSHIKI KAISHA. The applicant listed for this patent is OSAKA UNIVERSITY, TERUMO KABUSHIKI KAISHA. Invention is credited to Shigeru MIYAGAWA, Fumiya OOHASHI, Atsuhiro SAITO, Tadashi SAMESHIMA, Yoshiki SAWA, Ryouhei TAKEUCHI.
Application Number | 20170304504 15/647742 |
Document ID | / |
Family ID | 55401291 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170304504 |
Kind Code |
A1 |
OOHASHI; Fumiya ; et
al. |
October 26, 2017 |
METHOD FOR PRODUCING THE LAMINATE OF A SHEET-SHAPED CELL CULTURE
AND FIBRIN GELS
Abstract
A method for treating a disease associated with tissue
abnormality in a subject. The method includes administering an
amount of a pharmaceutical composition to a subject in need of
treating a disease associated with tissue abnormality, the
pharmaceutical composition comprising a laminate comprising a
fibrin gel and a cell culture. The laminate is produced by a
method, which includes forming a fibrin gel layer on an upper
surface of the cell culture by a reaction between fibrinogen and
thrombin, wherein: the cell culture is prepared on a culture dish
and the laminate of the fibrin gel and the cell culture are
transferred onto a subject's heart. The laminate further includes a
reinforcing portion made of a fibrin gel, the reinforcing portion
formed by laminating the fibrin gel on the fibrin gel layer of the
laminate of the cell culture and the fibrin gel.
Inventors: |
OOHASHI; Fumiya;
(Ashigarakami-gun, JP) ; TAKEUCHI; Ryouhei;
(Ashigarakami-gun, JP) ; SAMESHIMA; Tadashi;
(Ashigarakami-gun, JP) ; MIYAGAWA; Shigeru;
(Osaka, JP) ; SAWA; Yoshiki; (Osaka, JP) ;
SAITO; Atsuhiro; (Osaka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TERUMO KABUSHIKI KAISHA
OSAKA UNIVERSITY |
Tokyo
Osaka |
|
JP
JP |
|
|
Assignee: |
TERUMO KABUSHIKI KAISHA
Tokyo
JP
OSAKA UNIVERSITY
Osaka
JP
|
Family ID: |
55401291 |
Appl. No.: |
15/647742 |
Filed: |
July 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14844259 |
Sep 3, 2015 |
9737636 |
|
|
15647742 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/38 20130101;
A61L 27/52 20130101; A61L 27/58 20130101; A61L 2430/20 20130101;
A61P 43/00 20180101; A61L 27/225 20130101 |
International
Class: |
A61L 27/58 20060101
A61L027/58; A61L 27/22 20060101 A61L027/22; A61L 27/38 20060101
A61L027/38; A61L 27/52 20060101 A61L027/52 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 2014 |
JP |
2014-179150 |
Claims
1. A method for treating a disease associated with tissue
abnormality in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; and the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart, wherein the laminate further comprises: a
reinforcing portion made of a fibrin gel, the reinforcing portion
formed by laminating the fibrin gel on the fibrin gel layer of the
laminate of the cell culture and the fibrin gel.
2. The method according to claim 1, wherein the reinforcing portion
is made on an end of the laminate.
3. The method according to claim 1, further comprising: after
forming the fibrin gel layer, trimming excess fibrin gel.
4. The method according to claim 1, comprising: suturing the
laminate of the fibrin gel and the cell culture to the tissue
abnormality in the subject.
5. The method according to claim 1, comprising: a ratio of
fibrinogen to thrombin adhering to the laminate of about 8:5 to
about 8:75.
6. A method for treating a disease associated with tissue
abnormality in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart; and suturing the laminate of the fibrin gel
and the cell culture to the tissue abnormality in the subject.
7. The method according to claim 6, wherein the laminate further
comprises: a reinforcing portion made of a fibrin gel, the
reinforcing portion formed by laminating the fibrin gel on the
fibrin gel layer of the laminate of the cell culture and the fibrin
gel.
8. The method according to claim 7, wherein the reinforcing portion
is made on an end of the laminate.
9. The method according to claim 6, further comprising: after
forming the fibrin gel layer, trimming excess fibrin gel.
10. The method according to claim 6, comprising: suturing the
reinforcing portion of the laminate of the fibrin gel and the cell
culture to the tissue abnormality in the subject.
11. The method according to claim 6, comprising: a ratio of
fibrinogen to thrombin adhering to the laminate of about 8:5 to
about 8:75.
12. A method for treating a disease associated with tissue
abnormality in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; and the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart; and placing a side on which the fibrin gel
is not formed of the laminate of the fibrin gel and the cell
culture on a surface of a subject's heart.
13. The method according to claim 12, wherein the side on which the
fibrin gel is not formed of the laminate had been attached to a
surface of culture dish.
14. The method according to claim 12, wherein the laminate further
comprises: a reinforcing portion made of a fibrin gel, the
reinforcing portion formed by laminating the fibrin gel on the
fibrin gel layer of the laminate of the cell culture and the fibrin
gel.
15. The method according to claim 14, wherein the reinforcing
portion is made on an end of the laminate.
16. The method according to claim 12, further comprising: after
forming the fibrin gel layer, trimming excess fibrin gel.
17. The method according to claim 15, comprising: suturing the
reinforcing portion of the laminate of the fibrin gel and the cell
culture to the tissue abnormality in the subject.
18. The method according to claim 12, comprising: a ratio of
fibrinogen to thrombin adhering to the laminate of about 8:5 to
about 8:75.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/844,259 filed on Sep. 3, 2015, which claims
priority to Japanese Application No. 2014-179150 filed on Sep. 3,
2014, the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present disclosure generally relates to a method for
producing a laminate of a sheet-shaped cell culture and a fibrin
gel, a laminate produced by the method, a pharmaceutical
composition containing the laminate, and a method for treating a
disease using the laminate.
BACKGROUND DISCUSSION
[0003] In recent years, for the repair of damaged tissues, attempts
have been made to transplant various cells. For example, for the
repair of cardiac muscle tissue damaged due to an ischemic heart
disease such as angina pectoris, or myocardial infarction, attempts
have been made to use fetal cardiomyocytes, skeletal myoblasts,
mesenchymal stem cells, cardiac stem cells, and ES cells (See,
Haraguchi et al., Stem Cells Transl Med., 2012 February; 1 (2):
136-41).
[0004] As part of such attempts, a cell structure formed using a
scaffold and a sheet-shaped cell culture obtained by forming cells
into a sheet have been developed. (See JP-T-2007-528755).
[0005] Applications of sheet-shaped cell cultures to treatment have
been studied focusing on regenerative medicine, including the use
of a cultured epidermal sheet for skin damage due to a burn, the
use of a sheet-shaped corneal epithelial cell culture for corneal
damage, and the use of a sheet-shaped oral mucosa cell culture for
the endoscopic excision of esophageal cancer.
[0006] Sheet-shaped cell cultures can be highly useful in
regenerative medicine. However, they can be brittle as they are,
and wrinkling, earing, and the like often occur during isolation
from a culture medium or in the following operations. Thus, it can
be extremely difficult to perform operations like transfer,
storage, and implantation. In order to solve such problems, a
method in which a fibrinogen liquid and a thrombin liquid are
simultaneously sprayed onto a sheet-shaped cell culture to form a
support layer containing fibrin on the sheet-shaped cell culture,
thereby producing a laminate of a sheet-shaped cell culture and
fibrin, has been attempted (See, JP-A-2011-172925).
[0007] With respect to the laminate of a sheet-shaped cell culture
and fibrin described in JP-A-2011-172925, it has been found that
when the laminate is isolated from a culture medium, there may be
the case where the laminate tears, the case where isolation is not
possible, or the case where the support layer comes off the
sheet-shaped cell culture, making it impossible to obtain a
complete laminate, for example. It has also been found that when
the support layer is thickened to solve such problems, the cell
function can be inhibited.
SUMMARY
[0008] Accordingly, a sheet-shaped cell culture laminate is
disclosed that does not have the problems disclosed above, has
excellent operability, and is suitable for implantation. In
addition, a method for producing the same, a pharmaceutical
composition containing the laminate, and a method for treating a
disease using the laminate are disclosed.
[0009] In accordance with an exemplary embodiment, for the
formation of a fibrin gel layer on a sheet-shaped cell culture,
when a liquid containing fibrinogen is first dripped onto a
sheet-shaped cell culture, and then a liquid containing thrombin is
sprayed, a fibrin gel layer formed by a reaction between fibrinogen
and thrombin can firmly adhere to the sheet-shaped cell culture;
and as a result, when the obtained laminate of a fibrin gel and a
sheet-shaped cell culture is isolated from a container, the fibrin
gel layer does not come off the sheet-shaped cell culture.
[0010] In accordance with an exemplary embodiment, a method is
disclosed for producing a laminate of a fibrin gel and a
sheet-shaped cell culture, including a step of dripping a liquid
containing fibrinogen onto an upper surface of a sheet-shaped cell
culture, a step of spraying a liquid containing thrombin onto the
surface, and a step of forming a fibrin gel layer on the surface by
a reaction between fibrinogen and thrombin.
[0011] In accordance with an exemplary embodiment of the method as
disclosed, further including, after the step of forming a fibrin
gel layer, a step of washing the laminate.
[0012] In accordance with an exemplary embodiment of the method as
disclosed, further including, after the step of forming a fibrin
gel layer, a step of trimming excess fibrin gel.
[0013] In accordance with an exemplary embodiment, a laminate of a
fibrin gel and a sheet-shaped cell culture produced by the methods
as disclosed herein.
[0014] In accordance with an exemplary embodiment, a pharmaceutical
composition containing the laminate as disclosed herein.
[0015] In accordance with an exemplary embodiment, the
pharmaceutical composition disclosed herein, for treating a disease
associated with tissue abnormality.
[0016] In accordance with an exemplary embodiment, a method is
disclosed for treating a disease associated with tissue abnormality
in a subject, including administering an effective amount of the
laminate according or the pharmaceutical composition according to a
subject in need of treating a disease associated with tissue
abnormality.
[0017] In accordance with an exemplary embodiment, a laminate
produced by the method of the disclosure can be relatively strong,
having excellent operability, can be relatively easy to apply to
the affected area, and also does not make a significant operational
difference depending on the skill of the user. Therefore, diseases
can be reliably treated, and the spread and expand of regenerative
medicine using the laminate can be expected. In addition, the
laminate of the disclosure can be made of a biocompatible
component, and thus can also be directly implanted at an
implantation site, followed by the degradation of the fibrin gel in
vivo, which can help eliminate the need of a further surgery or the
need of using a special jig for the implantation of a sheet-shaped
cell culture, for example, making sheet-shaped cell cultures much
more convenient.
[0018] In accordance with an exemplary embodiment, a method is
disclosed for treating a disease associated with tissue abnormality
in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; and the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart, wherein the laminate further comprises: a
reinforcing portion made of a fibrin gel, the reinforcing portion
formed by laminating the fibrin gel on the fibrin gel layer of the
laminate of the cell culture and the fibrin gel.
[0019] In accordance with another exemplary embodiment, a method is
disclosed for treating a disease associated with tissue abnormality
in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart; and suturing the laminate of the fibrin gel
and the cell culture to the tissue abnormality in the subject.
[0020] In accordance with a further exemplary embodiment, a method
is disclosed for treating a disease associated with tissue
abnormality in a subject, comprising: administering an amount of a
pharmaceutical composition to a subject in need of treating a
disease associated with tissue abnormality, the pharmaceutical
composition comprising a laminate comprising a fibrin gel and a
cell culture, and wherein the laminate is produced by a method
comprising: forming a fibrin gel layer on an upper surface of the
cell culture by a reaction between fibrinogen and thrombin,
wherein: the cell culture is prepared on a culture dish; and the
laminate of the fibrin gel and the cell culture are transferred
onto a subject's heart; and placing a side on which the fibrin gel
is not formed of the laminate of the fibrin gel and the cell
culture on a surface of a subject's heart.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a photograph showing a sheet-shaped cell culture
in a culture dish in accordance with an exemplary embodiment.
[0022] FIG. 2 is a photograph showing the operation of dripping a
fibrinogen liquid onto a sheet-shaped cell culture.
[0023] FIG. 3 is a photograph showing the operation of spraying a
thrombin liquid onto a sheet-shaped cell culture.
[0024] FIG. 4 is a photograph showing the operation of trimming a
fibrin gel solidified other than on a sheet-shaped cell
culture.
[0025] FIG. 5 is a photograph showing a sheet-shaped cell culture
after simultaneously spraying a fibrinogen liquid and a thrombin
liquid.
[0026] FIG. 6 is a photograph showing the failure of isolation of a
laminate of a sheet-shaped cell culture and a fibrin gel obtained
by simultaneously spraying a fibrinogen liquid and a thrombin
liquid.
[0027] FIG. 7 is a photograph of a laminate of the disclosure
placed on an intestinal spatula.
[0028] FIG. 8 is a photograph showing a laminate having a
reinforcing portion of the disclosure in accordance with an
exemplary embodiment, and wherein the black arrow indicates the
reinforcing portion, and it can be seen that a suture is passing
through the center of the reinforcing portion.
[0029] FIG. 9 is a photograph showing the operation of hanging a
suture on a laminate of the disclosure placed on an intestinal
spatula.
[0030] FIG. 10 is a photograph showing the operation of
transferring a laminate of the disclosure from an intestinal
spatula to an implantation site.
[0031] FIG. 11 is a photograph showing the operation of
transferring a laminate of the disclosure from an intestinal
spatula to an implantation site.
[0032] FIG. 12 is a photograph showing the detachment of a suture
from a laminate of the disclosure transferred to an implantation
site.
[0033] FIG. 13 is a photograph showing the resuturing of a laminate
of the disclosure to an implantation site.
[0034] FIG. 14 is a photograph showing the operation of
transferring a laminate of the disclosure to an intestinal
spatula.
[0035] FIG. 15 is a photograph showing the operation of hanging a
suture on a marginal part of a laminate of the disclosure.
[0036] FIG. 16 is a photograph showing the operation of hanging a
suture on a marginal part of a laminate of the disclosure.
[0037] FIG. 17 is a photograph showing the operation of hanging a
suture on a marginal part of a laminate of the disclosure.
[0038] FIG. 18 is a photograph showing a laminate of the disclosure
that has been transferred to the side of a bottle.
DETAILED DESCRIPTION
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. All patents, applications, and other
publications (including information available from the Internet)
referred to herein are incorporated herein by reference in its
entirety.
[0040] A method is disclosed for producing a laminate of a fibrin
gel and a sheet-shaped cell culture, including a step of dripping a
liquid containing fibrinogen onto an upper surface of a
sheet-shaped cell culture, a step of spraying a liquid containing
thrombin onto the surface, and a step of forming a fibrin gel layer
on the surface by a reaction between fibrinogen and thrombin.
[0041] In the disclosure, "sheet-shaped cell culture" means cells
connected to each other to form a sheet. Cells may be connected to
each other directly (including connection through cell factors,
such as adhesion molecules) and/or through an intervening
substance. The intervening substance is not particularly limited as
long as it is a substance that is capable of at least physically
(mechanically) connecting cells, and may be an extracellular
matrix, for example. Preferred examples of intervening substances
include those derived from cells, for example, particularly those
derived from cells forming the cell culture. Cells are at least
physically (mechanically) connected, and may further be
functionally connected, such as chemically or electrically, for
example. The sheet-shaped cell culture may be composed of one cell
layer (monolayer) or may also be composed of two or more cell
layers (laminated (multilayer), for example, two layers, three
layers, four layers, five layers, and six layers).
[0042] In accordance with an exemplary embodiment, it can be
preferable that the sheet-shaped cell culture does not contain a
scaffold (support). Scaffolds are sometimes used in this technical
field to maintain the physical unity of a sheet-shaped cell culture
by attaching cells to the surface and/or inside thereof. For
example, a film made of polyvinylidene difluoride (PVDF) is known.
However, the sheet-shaped cell culture in the disclosure may be
capable of maintaining the physical unity even without such a
scaffold. In addition, it can be preferable that the sheet-shaped
cell culture is made only of a substance derived from cells forming
the cell culture and does not contain other substances.
[0043] Cells forming the sheet-shaped cell culture are not
particularly limited as long as they are capable of forming a
sheet-shaped cell culture, and examples thereof include adherent
cells (adhesive cells). Examples of adherent cells can include
adhesive somatic cells (for example, cardiomyocytes, fibroblasts,
epithelial cells, endothelial cells, hepatocytes, pancreatic cells,
renal cells, adrenal cells, periodontal ligament cells, gingival
cells, periosteal cells, skin cells, synoviocytes, and
chondrocytes) and stem cells (for example, tissue stem cells such
as myoblasts, cardiac stem cells, multipotential stem cells such as
embryonic stem cells, iPS (induced pluripotent stem) cells, and
mesenchymal stem cells). Somatic cells may be differentiated from
stem cells, particularly iPS cells. Non-limiting examples of cells
forming the sheet-shaped cell culture include myoblasts (for
example, skeletal myoblasts), mesenchymal stem cells (for example,
those derived from bone marrow, fat tissue, peripheral blood, skin,
hair root, muscle tissue, endometrium, placenta, and umbilical cord
blood), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic
stem cells, iPS cells, synoviocytes, chondrocytes, epithelial cells
(for example, oral mucosa epithelial cells, retinal pigment
epithelial cells, nasal mucosa epithelial cells), endothelial cells
(for example, vascular endothelial cells), hepatocytes (for
example, hepatic parenchymal cells), pancreatic cells (for example,
islet cells), renal cells, adrenal cells, periodontal ligament
cells, gingival cells, periosteal cells, and skin cells.
[0044] In accordance with an exemplary embodiment, cells forming
the sheet-shaped cell culture may be derived from any organism that
allows for treatment with the sheet-shaped cell culture. Such
organisms are not limited, and examples thereof include humans,
nonhuman primates, dogs, cats, pigs, horses, goats, sheep, rodent
animals (for example, mice, rats, hamsters, and guinea pigs), and
rabbits. In addition, the cells forming the sheet-shaped cell
culture may be one kind of cells, and it is possible to use two or
more kinds of cells. In a preferred embodiment of the disclosure,
in the case two or more kinds of cells are used to form a
sheet-shaped cell culture, the proportion (purity) of the
predominant cells can be, for example, 60% or more, preferably 70%
or more, and more preferably 75% or more, at the time of the
completion of the production of the sheet-shaped cell culture.
[0045] The cells forming the sheet-shaped cell culture may be cells
derived from a different species or cells derived from the same
species. Here, "cells derived from a different species" means that
in the case where the sheet-shaped cell culture is used for
implantation, the cells are derived from an organism of a different
species from the recipient. For example, in the case where the
recipient is a human, the cells derived from a different species
are cells derived from a monkey or a pig, for example. In addition,
"cells derived from the same species" means that the cells are
derived from an organism of the same species as the recipient. For
example, in the case where the recipient is a human, the cells
derived from the same species are human cells. Cells derived from
the same species can include self-derived cells (also referred to
as self-cells or autologous cells), for example, cells derived from
the recipient, and non-self-derived cells of the same species
(referred to as heterologous cells). Self-derived cells do not
cause a rejection reaction when implanted, and thus are preferable
in the disclosure. However, cells derived from a different species
and non-self-derived cells of the same species can be used. In the
case where cells derived from a different species or
non-self-derived cells of the same species are used,
immunosuppressive treatment may be required in order to suppress
the rejection reaction. Incidentally, as used herein, cells other
than self-derived cells, for example, cells derived from a
different species and non-self-derived cells of the same species,
may be collectively referred to as non-self-derived cells. In an
exemplary embodiment of the disclosure, the cells can be autologous
cells or heterologous cells. In an exemplary embodiment of the
disclosure, the cells can be autologous cells. In another exemplary
embodiment mode of the disclosure, the cells can be heterologous
cells.
[0046] The sheet-shaped cell culture can be produced by known
methods (see, for example, JP-T-2007-528755, JP-A-2011-172925,
JP-A-2010-081829, JP-A-2011-110368). A method for producing a
sheet-shaped cell culture can include, but is not limited to, a
step of seeding cells on a culture medium, a step of forming the
seeded cells into a sheet, and a step of isolating the sheet-shaped
cell culture from the culture medium. Before the step of seeding
cells on a culture medium, a step of freezing cells and a step of
thawing cells may also be performed. Further, after the step of
thawing cells, a step of washing cells may also be performed. These
steps may each be performed by any known technique suitable for the
production of a sheet-shaped cell culture. The production method of
the disclosure may further include a step of producing a
sheet-shaped cell culture. In that case, the step of producing a
sheet-shaped cell culture may include one or more of the above
sub-steps.
[0047] The liquid containing fibrinogen (hereinafter sometimes
referred to as fibrinogen liquid) is not particularly limited as
long as it is capable of reacting with thrombin to form a fibrin
gel. Examples thereof include liquids containing fibrinogen at
concentrations of, for example, 1 mg/mL to 500 mg/mL, 5 mg/mL to
400 mg/mL, 10 mg/mL to 250 mg/mL, 20 mg/mL to 150 mg/mL, 40 mg/mL
to 100 mg/mL, and 50 mg/mL to 90 mg/mL. The solvent of the
fibrinogen liquid is typically water. The fibrinogen liquid may
also contain, in addition to fibrinogen, other components such as
Factor XIII, aprotinin, serum albumin, glycine, L-arginine
hydrochloride, L-isoleucine, monosodium L-glutamate, D-mannitol,
sodium citrate hydrate, and sodium chloride. Fibrinogen liquids are
commercially available, but may also be produced based on a known
technique. Commercially available fibrinogen liquids are not
limited, and examples thereof include a solution of the content of
Vial 1 (a lyophilized powder of fibrinogen) of tissue adhesive
BOLHEAL.RTM. (manufactured by Teijin Pharma Ltd.) dissolved in the
content of Vial 2 (a fibrinogen solution) and a solution of the
content of Vial 1 (a fibrinogen powder) of tissue adhesive
Beriplast.RTM. Combi-Set (manufactured by CSL Bering) dissolved in
the content of Vial 2 (an aprotinin solution).
[0048] The dripping of the fibrinogen liquid may be performed using
known techniques, such as a syringe or a pipet. As a syringe, for
example, a needle-less syringe having a volume of 0.5 mL to 5 mL, a
needle-equipped syringe (for example, a syringe equipped with a
needle of 18 G to 27 G), the two-component mixing set of the
preparation set attached to tissue adhesive BOLHEAL.RTM. (equipped
with an application nozzle about 1 mm in inner diameter,
manufactured by Nipro Corporation), or the two-component mixing set
of the preparation set of Beriplast (equipped with an application
nozzle, manufactured by Nipro Corporation) can be used. The
dripping amount of the fibrinogen liquid is not particularly
limited as long as the upper surface of the sheet-shaped cell
culture can be covered, and may be, for example, about 6
.mu.L/cm.sup.2 to about 70 .mu.L/cm.sup.2, about 9 .mu.L/cm.sup.2
to about 50 .mu.L/cm.sup.2, about 12 .mu.L/cm.sup.2 to about 45
.mu.L/cm.sup.2, about 15 .mu.L/cm.sup.2 to about 40 .mu.L/cm.sup.2,
or about 18 .mu.L/cm.sup.2 to about 32 .mu.L/cm.sup.2. Non-limiting
examples of fibrinogen liquid dripping amounts can include, for
example, relative to a 45-mm-diameter sheet-shaped cell culture,
about 100 .mu.L to about 1000 .mu.L, about 150 .mu.L to about 800
.mu.L, about 200 .mu.L to about 700 .mu.L, about 250 .mu.L to about
600 .mu.L, and about 300 .mu.L to about 500 .mu.L. The particle
size of droplets of the fibrinogen liquid during dripping is not
particularly limited, and may be, for example, within a range such
that the diameter of droplets adhering to the sheet-shaped cell
culture after dripping will be, for example, about 0.2 cm to about
2.0 cm. In addition, the weight of the droplets is not limited, and
may be, for example, within a range of about 10 mg to about 100 mg,
about 15 mg to about 50 mg, or about 20 mg to about 30 mg. The
particle size and weight of droplets may be suitably adjusted by
selecting whether the syringe is equipped with a needle, the gauge
of the applied needle, or the shape of the needle tip.
[0049] The liquid containing thrombin (hereinafter sometimes
referred to as thrombin liquid) is not particularly limited as long
as it is capable of reacting with fibrinogen to form a fibrin gel.
Examples thereof include liquids containing thrombin at
concentrations of, for example, 1 unit/mL to 10000 units/mL, 10
units/mL to 5000 units/mL, 25 units/mL to 2500 units/mL, 50
units/mL to 1000 units/mL, 100 units/mL to 500 units/mL, and 250
units/mL to 300 units/mL. The solvent of the thrombin liquid is
typically water. The thrombin liquid may also contain, in addition
to thrombin, other components such as sodium citrate hydrate, and
sodium chloride. Thrombin liquids are commercially available, but
may also be produced based on a known technique. Commercially
available thrombin liquids are not limited, and examples thereof
include a solution of the content of Vial 3 (a lyophilized powder
of thrombin) of tissue adhesive BOLHEAL.RTM. (manufactured by
Teijin Pharma Ltd.) dissolved in the content of Vial 4 (a thrombin
solution) and a solution of the content of Vial 3 (a thrombin
powder) of tissue adhesive Beriplast.RTM. Combi-Set (manufactured
by CSL Bering) dissolved in the content of Vial 4 (an calcium
chloride solution).
[0050] The spraying of the thrombin liquid may be performed using
known techniques, such as a spray. Non-limiting examples of such
sprays include BOLHEAL.RTM. spray set (manufactured by Akita
Sumitomo Bakelite Co., Ltd) and the two-component mixing set
attached to tissue adhesive Beriplast.RTM. Combi-Set (manufactured
by CSL Bering) equipped with a spray chip. The spraying amount of
the thrombin liquid is not particularly limited as long as the
upper surface of the sheet-shaped cell culture can be covered, and
may be, as an estimated adhesion amount to the sheet-shaped cell
culture, for example, about 3 .mu.L/cm.sup.2 to about 70
.mu.L/cm.sup.2, about 5 .mu.L/cm.sup.2 to about 50 .mu.L/cm.sup.2,
about 6 .mu.L/cm.sup.2 to about 45 .mu.L/cm.sup.2, about 12
.mu.L/cm.sup.2 to about 40 .mu.L/cm.sup.2, or about 18
.mu.L/cm.sup.2 to about 32 .mu.L/cm.sup.2. Non-limiting examples of
thrombin liquid spraying amounts include, as estimated adhesion
amounts to a 45-mm-diameter sheet-shaped cell culture, for example,
about 50 .mu.L to about 1000 .mu.L, about 80 .mu.L to about 800
.mu.L, about 100 .mu.L to about 700 .mu.L, about 200 .mu.L to about
600 .mu.L, and about 300 .mu.L to about 500 .mu.L. Incidentally,
the estimated adhesion amount to a sheet-shaped cell culture can be
calculated as follows: a predetermined amount of thrombin liquid is
sprayed employing a spray, a height, a spray pressure, and a spray
angle that are to be used for actual spraying, and the weight of
the liquid adhering to a predetermined region (for example, within
a 45-mm-diameter circle) is measured and divided by the density of
the thrombin liquid (0.999973 g/cm.sup.3).
[0051] In accordance with an exemplary embodiment, spraying
conditions can be determined that will result in the desired
estimated adhesion amount of the thrombin liquid based on the
descriptions herein without requiring undue experimentation. For
example, in Example 1 below, it is described that estimated
adhesion amounts corresponding to spraying amounts of, for example,
300 .mu.L, 500 .mu.L, 600 .mu.L, and 900 .mu.L are 100 .mu.L, 180
.mu.L, 300 .mu.L, and 450 .mu.L, respectively. Accordingly, by the
least squares method, an approximate curve is obtained as follows:
estimated adhesion amount (.mu.L)=spraying amount
(.mu.L).times.0.6-88 (.mu.L). Based on this, a spraying amount that
results in the desired estimated adhesion amount can be determined.
The spraying of the thrombin liquid is not particularly limited as
long as the thrombin liquid can adhere to the upper surface,
preferably uniformly all over the upper surface, of a sheet-shaped
cell culture. For example, spraying can performed from a height of,
for example, about 2 cm to about 15 cm at a spray angle of about
15.degree. to about 150.degree. at a pressure of about 0.005 MPa to
about 0.1 MPa.
[0052] The ratio between the fibrinogen liquid and thrombin liquid
applied to the sheet-shaped cell culture is not particularly
limited as long as the operation of implanting the obtained
laminate is not excessively inhibited. For example, as the volume
ratio between the dripping amount of the fibrinogen liquid and the
estimated adhesion amount of the thrombin liquid, it may be, for
example, about 5:1 to about 1:3, about 4:1 to about 1:2, about 3:1
to about 1:1.5, about 2.5:1 to about 1:1, about 2:1 to about 1:1,
or about 1.5:1 to about 1:1, and may particularly be about 1:1. In
addition, as the ratio (mg: unit) between fibrinogen and thrombin
adhering to the laminate, it may be, for example, about 8:5 to
about 8:75, about 32:25 to about 4:25, about 24:25 to about 16:75,
about 4:5 to about 8:25, about 16:25 to about 8:25, about 12:25 to
about 8:25, and may particularly be about 8:25.
[0053] By adjusting the concentrations and amounts of the
fibrinogen liquid and the thrombin liquid, the thickness of the
resulting laminate and its properties (for example, flexibility and
adhesiveness) can be changed. For example, by increasing the amount
of the fibrinogen liquid, the thickness of the laminate can be
increased. As the volume ratio between the dripping amount of the
fibrinogen liquid (80 mg/mL) and the estimated adhesion amount of
the thrombin liquid (250 units/mL) approaches 1:1, and as the ratio
(mg:unit) between fibrinogen and thrombin adhering to the laminate
approaches 8:25, the flexibility and adhesiveness of the laminate
increase, resulting in improved operability.
[0054] The step of forming a fibrin gel layer is not limited, and
may be performed, for example, by allowing the sheet-shaped cell
culture to stand for a given period of time after spraying the
thrombin liquid. The standing time is not limited, and may be, for
example, about 1 minute to about 60 minutes, about 2 minutes to
about 30 minutes, about 3 minutes to about 20 minutes, or about 4
minutes to about 10 minutes. More specifically, it may be, for
example, about 1 minute, about 2 minutes, about 3 minutes, about 4
minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8
minutes, about 9 minutes, or about 10 minutes, and may particularly
be about 5 minutes.
[0055] After the step of forming a fibrin gel layer, a step of
washing the laminate may be performed. Washing may be performed by
adding a predetermined amount of washing liquid to the container
containing the laminate, and then discarding the same. Examples of
washing liquids include, but are not limited to, water,
physiological saline, various buffers (for example, PBS, and HBSS),
and various liquid media (for example, DMEM, MEM, F12, DME,
RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, and 199), L15,
SkBM, RITC80-7, and DMEM/F12). The washing step can be performed
once or more. In addition, the washing liquid added to the
container may be discarded immediately, or may also be allowed to
stand for a predetermined period of time (for example, without
being limited thereto, for example, about 1 minute to about 60
minutes, about 5 minutes to about 30 minutes, or about 10 minutes
to about 20 minutes, and particularly about 15 minutes) and then
discarded.
[0056] After the step of forming a fibrin gel layer (in the case
where a step of washing a laminate is included, after the washing
step), as necessary, a step of trimming excess fibrin gel may be
performed. Trimming may be performed using any instrument that can
be used for cutting a gel-like substance. Examples of such
instruments include, but are not limited to, scalpels, scissors,
and surgical knives. The fibrin gel may be trimmed along the
outline of the sheet-shaped cell culture, or may also be trimmed
such that the fibrin gel protrudes around the sheet-shaped cell
culture, for example, such that the margin of the laminate is made
only of a fibrin gel.
[0057] The step of dripping a liquid containing fibrinogen, step of
spraying a liquid containing thrombin, step of forming a fibrin gel
layer, step of washing a laminate, and/or step of trimming excess
fibrin gel mentioned above may be performed on a sheet-shaped cell
culture still adhering to the culture medium, or may also be
performed on a sheet-shaped cell culture that has been isolated
from the culture medium.
[0058] In accordance with an exemplary embodiment of the production
method of the disclosure, all the steps are performed in vitro. In
another exemplary embodiment, the production method of the
disclosure is not limited to steps performed in vivo, and may
include, for example, a step of collecting cells or a cell source
tissue from a subject. In one exemplary embodiment of the
production method of the disclosure, all the steps are performed
under aseptic conditions. In an exemplary embodiment, the
production method of the disclosure is performed such that the
laminate finally obtained will be substantially aseptic. In an
exemplary embodiment, the production method of the disclosure is
performed such that the laminate finally obtained will be
aseptic.
[0059] Another aspect of the disclosure relates to a laminate of a
sheet-shaped cell culture and a fibrin gel produced by the
production method of the disclosure. As compared with, for example,
a laminate produced by simultaneously spraying a fibrinogen liquid
and a thrombin liquid onto a sheet-shaped cell culture as described
in JP-A-2011-172925, in the laminate of the disclosure, the
adhesion of the fibrin gel layer to the sheet-shaped cell culture
is stronger, and the fibrin gel layer does not come off the
sheet-shaped cell culture during operation. In accordance with an
exemplary embodiment, it is believed that in the laminate of the
disclosure, before reacting with thrombin to form fibrin,
fibrinogen permeates inside the sheet-shaped cell culture (for
example, intercellular space), whereby a fibrin gel is
three-dimensionally bound to the sheet-shaped cell culture and
anchored, while the area of contact between the fibrin gel and the
sheet-shaped cell culture is increased, resulting in firm adhesion
between the two. In contrast, with respect to the laminate
described in JP-A-2011-172925, it is believed that fibrinogen and
thrombin simultaneously come into contact with a sheet-shaped cell
culture and immediately react to form a fibrin gel; as a result,
the fibrin gel is unlikely to form such a three-dimensional bond
with the sheet-shaped cell culture. Therefore, it is believed that
the laminate of the disclosure is different from the laminate
described in JP-A-2011-172925 both in quality and structure.
[0060] The strength of the laminate is not limited. For example, as
measured by the method described in Example 2, it may be about
0.010 N or more, about 0.015 N or more, about 0.020 N or more,
about 0.025 N or more, about 0.030 N or more, about 0.035 N or
more, about 0.040 N or more, or about 0.045 N or more, and may also
be within a range of about 0.010 N to about 0.200 N, about 0.015 N
to about 0.100 N, or about 0.020 N to about 0.50 N. In addition,
the strength of the laminate may be, as compared with a
sheet-shaped cell culture that is the same as one contained in the
laminate except that a fibrin gel is not laminated, for example,
about 1.5 times or more, about 2 times or more, about 3 times or
more, about 4 times or more, about 5 times or more, about 6 times
or more, about 7 times or more, about 8 times or more, about 9
times or more, or about 10 times or more the strength of such a
sheet-shaped cell culture, for example, and may also be within a
range of about 1.5 times to about 20 times, about 2 times to about
15 times, or about 2.5 times to about 10 times, for example.
[0061] In addition, the margin of the laminate may be made of the
fibrin gel and the sheet-shaped cell culture laminated together, or
may also be made of the fibrin gel alone or the sheet-shaped cell
culture alone.
[0062] Another aspect of the disclosure relates to a laminate of a
sheet-shaped cell culture and a fibrin gel, which has a reinforcing
portion made of a fibrin gel (hereinafter sometimes referred to as
reinforced laminate). The reinforced laminate of the disclosure has
at least one reinforcing portion formed by further laminating a
fibrin gel on the fibrin gel layer of the laminate of a
sheet-shaped cell culture and a fibrin gel. By forming the
reinforcing portion, the strength of a necessary part can be
increased without increasing the entire thickness of the laminate.
The reinforcing portion can be used as a suture insertion portion,
a portion to be held with tweezers, for example. The reinforcing
portion is formed by dripping a fibrinogen liquid and a thrombin
liquid onto a fibrin gel layer. Various thicknesses and shapes can
be made by adjusting the dripping amount, the dripping position, or
the dripping pattern. For example, when a fibrinogen liquid (80
mg/mL) and a thrombin liquid (250 units/mL) are dripped each in an
amount of 50 .mu.L, a reinforcing portion having a base area of
about 1 cm.sup.2.times.a height of about 1 mm to 2 mm can be
obtained, while when they are dripped each in an amount of 100
.mu.L, a reinforcing portion having a base area of about 1
cm.sup.2.times.a height of about 2 mm to 3 mm can be obtained.
[0063] The ratio between the fibrinogen liquid and thrombin liquid
dripped is not particularly limited as long as a reinforcing
portion having desired strength can be obtained. For example, as
the volume ratio between the fibrinogen liquid and thrombin liquid
dripped, it may be about 5:1 to about 1:3, about 4:1 to about 1:2,
about 3:1 to about 1:1.5, about 2.5:1 to about 1:1, about 2:1 to
about 1:1, or about 1.5:1 to about 1:1, and may particularly be
about 1:1. In addition, as the ratio (mg:unit) between fibrinogen
and thrombin contained in the reinforcing portion, for example, it
may be about 8:5 to about 8:75, about 32:25 to about 4:25, about
24:25 to about 16:75, about 4:5 to about 8:25, about 16:25 to about
8:25, or about 12:25 to about 8:25, and may particularly be about
8:25.
[0064] The strength of the reinforcing portion is not limited. For
example, as measured by the method described in Example 2, it may
be about 0.04 N or more, about 0.05 N or more, about 0.06 N or
more, about 0.07 N or more, about 0.08 N or more, about 0.09 N or
more, about 0.10 N or more, about 0.12 N or more, or about 0.15 N
or more, and may also be within a range of about 0.04 N to about
0.50 N, about 0.05 N to about 0.40 N, about 0.06 N to about 0.30 N,
or about 0.07 N to about 0.25 N. In addition, the strength of the
reinforcing portion may be about 1.5 times or more, about 2 times
or more, about 3 times or more, about 4 times or more, about 5
times or more, about 6 times or more, about 7 times or more, about
8 times or more, about 9 times or more, or about 10 times or more
the strength of a non-reinforcing portion, for example, and may
also be within a range of about 1.5 times to about 25 times, about
2 times to about 20 times, about 3 times to about 15 times, or
about 4 times to about 10 times, for example.
[0065] Another aspect of the disclosure relates to a method for
producing a laminate of a sheet-shaped cell culture and a fibrin
gel having a reinforcing portion made of a fibrin gel, the method
can include a step of dripping a liquid containing fibrinogen onto
an upper surface of a sheet-shaped cell culture, a step of spraying
a liquid containing thrombin onto the surface, a step of forming a
fibrin gel layer on the surface by a reaction between fibrinogen
and thrombin, a step of dripping a liquid containing thrombin and a
liquid containing fibrinogen onto the fibrin gel layer in such a
manner that the two liquids are mixed on the fibrin gel layer, and
a step of forming a reinforcing portion made of a fibrin gel on the
fibrin gel layer by a reaction between fibrinogen and thrombin.
[0066] The step of dripping a liquid containing fibrinogen onto an
upper surface of a sheet-shaped cell culture, the step of spraying
a liquid containing thrombin onto the surface, the step of forming
a fibrin gel layer on the surface by a reaction between fibrinogen
and thrombin, the liquid containing thrombin, and the liquid
containing fibrin are as described above with respect to the method
for producing a laminate of a sheet-shaped cell culture and a
fibrin gel. The method for producing a reinforced laminate may
include a step of forming a laminate of a sheet-shaped cell culture
and a fibrin gel by the method for producing a laminate of a
sheet-shaped cell culture and a fibrin gel mentioned above, a step
of dripping a liquid containing thrombin and a liquid containing
fibrinogen onto the fibrin gel layer in such a manner that the two
liquids are mixed on the fibrin gel layer, and a step of forming a
reinforcing portion made of a fibrin gel on the fibrin gel layer by
a reaction between fibrinogen and thrombin. Therefore, the method
for producing a reinforced laminate may also include the various
steps described above with respect to the method for producing a
laminate, such as a step of washing the laminate after the step of
forming a fibrin gel layer, a step of trimming excess fibrin gel
after the step of forming a fibrin gel layer (in the case where a
step of washing a laminate is included, after the washing step),
further a step of producing a sheet-shaped cell culture.
[0067] The dripping of the thrombin liquid and the fibrinogen
liquid onto the fibrin gel layer may be performed in any order or
may also be simultaneous. For example, the step of dripping a
thrombin liquid and a fibrinogen liquid onto a fibrin gel layer is
not limited, and encompasses (1) a step in which a thrombin liquid
is dripped onto a fibrin gel layer, and then a fibrinogen liquid is
dripped onto the dripped thrombin liquid, (2) a step in which a
fibrinogen liquid is dripped onto a fibrin gel layer, and then a
thrombin liquid is dripped onto the dripped fibrinogen liquid, and
(3) a step in which a thrombin liquid and a fibrinogen liquid are
simultaneously dripped onto a fibrin gel layer, for example.
[0068] The dripping of the thrombin liquid and the fibrinogen
liquid onto a fibrin gel layer may be performed using known
techniques, such as a syringe or a pipet. As a syringe, for
example, a needle-less syringe having a volume of 0.5 mL to 5 mL, a
needle-equipped syringe (for example, a syringe equipped with a
needle of 18 G to 27 G), the two-component mixing set of the
preparation set attached to tissue adhesive BOLHEAL.RTM. (equipped
with an application nozzle about 1 mm in inner diameter,
manufactured by Nipro Corporation), or the two-component mixing set
of the preparation set of Beriplast (equipped with an application
nozzle, manufactured by Nipro Corporation) can be used. The
particle size of droplets of the thrombin liquid or fibrinogen
liquid during dripping is not particularly limited, and may be, for
example, within a range such that the diameter of droplets adhering
to the sheet-shaped cell culture after dripping will be about 0.2
cm to about 2.0 cm. In addition, the weight of droplets of the
thrombin liquid or fibrinogen liquid during dripping is not
limited, and may be, for example, within a range of, for example,
about 10 mg to about 100 mg, about 15 mg to about 50 mg, or about
20 mg to about 30 mg. The particle size and weight of droplets may
be suitably adjusted by selecting whether the syringe is equipped
with a needle, the gauge of the applied needle, or the shape of the
needle tip. The dripping amounts of the thrombin liquid and the
fibrinogen liquid and the ratio between the thrombin liquid and
fibrinogen liquid dripped are as described above with respect to
the reinforced laminate.
[0069] The step of forming a reinforcing portion made of a fibrin
gel on a fibrin gel layer is not limited, and may be performed, for
example, by allowing a laminate to stand for a given period of time
after dripping the thrombin liquid and the fibrinogen liquid. The
standing time is not limited, and may be, for example, about 1
minute to about 60 minutes, about 2 minutes to about 30 minutes,
about 3 minutes to about 20 minutes, or about 4 minutes to about 10
minutes. More specifically, it may be, for example, about 1 minute,
about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes,
about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes,
or about 10 minutes, and may particularly be about 5 minutes.
[0070] After the step of forming a reinforcing portion made of a
fibrin gel, a step of washing the reinforced laminate may be
performed. Washing may be performed by adding a predetermined
amount of washing liquid to the container containing the reinforced
laminate, and then discarding the same. Examples of washing liquids
include, but are not limited to, water, physiological saline,
various buffers (for example, PBS, and HBSS), and various liquid
media (for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102,
104, 107, 120, 131, 153, and 199), L15, SkBM, RITC80-7, and
DMEM/F12). The washing step can be performed once or more. In
addition, the washing liquid added to the container may be
discarded immediately, or may also be allowed to stand for a
predetermined period of time (for example, without being limited
thereto, for example, about 1 minute to about 60 minutes, about 5
minutes to about 30 minutes, or about 10 minutes to about 20
minutes, and particularly about 15 minutes) and then discarded.
[0071] The laminate of the disclosure (including the reinforced
laminate; the same hereinafter) is useful for the treatment of
various diseases associated with tissue abnormality. Thus, in one
exemplary embodiment, the laminate of the disclosure is for use in
the treatment of a disease associated with tissue abnormality. The
laminate of the disclosure is configured such that a biocompatible
fibrin gel layer is laminated on one side of a sheet-shaped cell
culture. Thus, the other side of the sheet-shaped cell culture is
intact, and the fibrin gel is eventually degraded in vivo and
disappears. Therefore, the laminate can be applied to tissues and
diseases that can be treated with a sheet-shaped cell culture.
Treatment target tissues are not limited, and examples thereof
include cardiac muscle, cornea, retina, esophagus, skin, joints,
cartilage, liver, pancreas, gums, kidney, thyroid gland, skeletal
muscle, and middle ear. In addition, treatment target diseases are
not limited, and examples thereof cardiac diseases (for example,
myocardial damage (myocardial infarction, and cardiac injury),
cardiomyopathy (ischemic cardiomyopathy, dilated cardiomyopathy,
and dilated phase of hypertrophic cardiomyopathy)), corneal
diseases (for example, corneal epithelial stem cell deficiency,
corneal damage (thermal/chemical erosion), corneal ulcer, corneal
opacity, corneal perforation, corneal scar, Stevens-Johnson
syndrome, and ocular pemphigoid), retinal diseases (for example,
pigmentary retinopathy, and age-related macular degeneration),
esophageal diseases (for example, the prevention of esophageal
inflammation or stricture after esophageal surgery (removal of
esophageal cancer), skin diseases (for example, skin damage
(traumatic injury, thermal burn)), joint diseases (for example,
arthritis deformans), cartilage diseases (for example, cartilage
damage), hepatic diseases (for example, chronic hepatic diseases),
pancreatic diseases (for example, diabetes), dental diseases (for
example, periodontosis), renal diseases (for example, renal
insufficiency, renal anemia, and renal osteodystrophy), thyroid
diseases (for example, hypothyroidism), muscular diseases (for
example, muscle damage, and myositis), and middle ear diseases (for
example, otitis media).
[0072] The usefulness of a sheet-shaped cell culture for these
diseases is described in, for example, JP-T-2007-528755, Haraguchi
et al., Stem Cells Transl Med., 2012 February; 1 (2): 136-41,
Arauchi et al., Tissue Eng Part A., 2009 December; 15 (12): 3943-9,
Ito et al., Tissue Eng., 2005 March-April; 11 (3-4): 489-96, Yaji
et al., Biomaterials., 2009 February; 30 (5): 797-803, Yaguchi et
al., Acta Otolaryngol., 2007 October; 127 (10): 1038-44, Watanabe
et al., Transplantation., 2011 Apr. 15; 91 (7): 700-6, Shimizu et
al., Biomaterials., 2009 October; 30 (30): 5943-9, Ebihara et al.,
Biomaterials., 2012 May; 33 (15): 3846-51, and Takagi et al., World
J Gastroenterol., 2012 Oct. 7; 18 (37): 5145-50.
[0073] The laminate of the disclosure can be applied to a treatment
target tissue and used to restore and regenerate the same, and can
also be implanted at a site other than the treatment target tissue
(for example, subcutaneous tissue) as a source of physiologically
active substances, such as hormones (for example, Arauchi et al.,
Tissue Eng Part A., 2009 December; 15 (12): 3943-9, Shimizu et al.,
Biomaterials., 2009 October; 30 (30): 5943-9). The laminate of the
disclosure can be used for regenerative medicine and thus is also
applicable as a product for regenerative medicine. In addition, the
laminate of the disclosure can also be used as a graft.
[0074] In an exemplary embodiment, the laminate of the disclosure
is substantially aseptic. In an exemplary embodiment, the laminate
of the disclosure is aseptic. In an exemplary embodiment, the
sheet-shaped cell culture contained in the laminate of the
disclosure is not genetically engineered. In another exemplary
embodiment, the sheet-shaped cell culture contained in the laminate
of the disclosure is genetically engineered. Genetic engineering is
not limited and may be, for example, the introduction of genes that
enhance the viability, engraftment ability, and function of the
sheet-shaped cell culture and/or genes useful for the treatment of
a disease. Genes to be introduced are not limited, and examples
thereof include cytokine genes, such as the HGF gene and the VEGF
gene.
[0075] Another aspect of the disclosure relates to a pharmaceutical
composition containing the laminate of the disclosure. The
pharmaceutical composition of the disclosure may contain, in
addition to the laminate of the disclosure, various additional
components such as pharmacologically acceptable supports,
components that enhance the viability, engraftment ability, and/or
function of the sheet-shaped cell culture, and other active
ingredients useful for the treatment of the target disease. Such
additional components may be any known components, and one of
ordinary skill in the art is familiar with these additional
components. In addition, the pharmaceutical composition of the
disclosure can be used together with components that enhance the
viability, engraftment ability, and/or function of the sheet-shaped
cell culture, and other active ingredients useful for the treatment
of the target disease. In an exemplary embodiment, the
pharmaceutical composition of the disclosure is for use in the
treatment of a disease associated with tissue abnormality.
Treatment target tissues and diseases are as described above with
respect to the laminate of the disclosure.
[0076] Another aspect of the disclosure relates to a method for
treating a disease associated with tissue abnormality in a subject,
including administering an effective amount of the laminate or
pharmaceutical composition of the disclosure to a subject in need
of treating a disease associated with tissue abnormality. Tissues
and diseases targeted by the treatment method of the disclosure are
as described above with respect to the laminate of the disclosure.
In addition, in the treatment method of the disclosure, the
laminate or pharmaceutical composition of the disclosure can be
used together with components that enhance the viability,
engraftment ability, and/or function of the sheet-shaped cell
culture, and other active ingredients useful for the treatment of
the target disease.
[0077] The treatment method of the disclosure may further include a
step of producing a laminate in accordance with the production
method of the disclosure. The treatment method of the disclosure
may further include, before the step of producing a laminate, a
step of producing a sheet-shaped cell culture and a step of
collecting cells or a cell source tissue for producing a
sheet-shaped cell culture from a subject. In an exemplary
embodiment, the subject from which cells or a cell source tissue is
collected is the same individual as the subject to be treated by
the administration of the laminate or the pharmaceutical
composition. In another exemplary embodiment, the subject from
which cells or a cell source tissue is collected is a different
individual of the same species as the subject to be treated by the
administration of the laminate or the pharmaceutical composition.
In another exemplary embodiment, the subject from which cells or a
cell source tissue is collected is an individual of a different
species from the subject to be treated by the administration of the
laminate or the pharmaceutical composition.
[0078] In the disclosure, the term "subject" means any individual
organism, preferably an individual animal, still more preferably an
individual mammal, and yet more preferably an individual human. In
the disclosure, the subject may be healthy or may have a certain
disease. However, in the case where it is intended to treat a
disease associated with tissue abnormality, the term typically
means a subject with the disease or at risk of having the
disease.
[0079] In addition, the term "treatment" encompasses all
pharmaceutically acceptable prophylactic and/or therapeutic
interventions aimed at the cure, temporary remission, or prevention
of a disease, for example. For example, the term "treatment"
encompasses various desired medically acceptable interventions
including the delay or suspension of the progression of a disease
associated with tissue abnormality, the regression or disappearance
of the lesion, and the prevention of the occurrence or recurrence
of the disease.
[0080] In the disclosure, an effective amount is an amount (for
example, the size or weight of the laminate) that can suppress the
occurrence or recurrence of a disease, alleviate the symptoms, or
delay or suspend the progression, for example, and is preferably an
amount that prevents the occurrence and recurrence of the disease
or cures the disease. In addition, an amount that does not cause an
adverse effect exceeding the benefit of administration is
preferable. Such an amount can be suitably determined, for example,
by a test using experimental animals or disease model animals such
as mice, rats, dogs, or pigs, and such a test method is well known
to those skilled in the art. In addition, the size of the lesion in
the treatment target tissue can be an important index to determine
the effective amount.
[0081] In accordance with an exemplary embodiment, the
administration method may be direct application to a tissue. The
administration frequency can be once per treatment. However, in the
case where a desired effect is not obtained, it is also possible to
perform administration several times. When applied to a tissue, the
laminate or pharmaceutical composition of the disclosure may be
fixed to the target tissue by a locking means such as a suture or a
staple. In the case where the reinforced laminate is used, the
locking means may be applied to the reinforcing portion.
[0082] The disclosure will be described in further detail with
reference to the following examples. However, they show certain
specific examples of the disclosure, and the disclosure is not
limited thereto.
Example 1
Production of Laminate of Fibrin Gel and Sheet-Shaped Cell
Culture
[0083] Skeletal myoblasts (CD56 positive) cryopreserved in a
preservation liquid for cell freezing (MCDB culture medium
containing 10% DMSO) were thawed at 37.degree. C., and washed twice
using a physiological buffer containing 0.5% serum albumin.
6.0.times.10.sup.7 washed cells were suspended in a DMEM culture
medium containing 20% human blood serum (10 mL), and seeded on a
10-cm-diameter cell culture dish (UpCell.RTM. 10-cm dish, CS3005,
manufactured by CellSeed Inc.). After seeding, the cells were
cultured in an incubator set at 37.degree. C. and 5% CO.sub.2
(BNA-121D, manufactured by Espec Corp.) for 20 hours. After
culturing, the culture dish was taken out from the incubator. After
confirming the presence of a sheet-shaped cell culture adhering to
cover the entire bottom of the culture dish, the culture medium was
discarded. Subsequently, the sheet-shaped cell culture was
separated from the culture dish by a temperature treatment
(allowing to stand at room temperature (20 to 25.degree. C.) for 5
to 30 minutes) and pipetting. The size of the obtained sheet-shaped
cell culture was 47 mm.times.47 mm.
[0084] The culture solution in the culture dish was removed, and
the sheet-shaped cell culture was shaped as necessary (FIG. 1).
Subsequently, onto the sheet-shaped cell culture, 500 .mu.L of a
fibrinogen liquid (a solution of the content of Vial 1 (a
lyophilized powder of fibrinogen) of tissue adhesive BOLHEAL.RTM.
(manufactured by Teijin Pharma Ltd.) dissolved in the content of
Vial 2 (a fibrinogen solution), fibrinogen concentration: 80 mg/mL;
the same hereinafter) was dripped using the two-component mixing
set of the preparation set attached to tissue adhesive BOLHEAL.RTM.
(equipped with an application nozzle about 6 cm in length and about
1 mm in inner diameter, manufactured by Nipro Corporation) (FIG.
2). Next, 800 .mu.L of a thrombin liquid (a solution of the content
of Vial 3 (a lyophilized powder of thrombin) of tissue adhesive
BOLHEAL.RTM. (manufactured by Teijin Pharma Ltd.) dissolved in the
content of Vial 4 (a thrombin solution), thrombin concentration:
250 units/mL; the same hereinafter) was sprayed using BOLHEAL.RTM.
spray set (manufactured by Akita Sumitomo Bakelite Co., Ltd) from a
spray nozzle placed about 7 cm away from the cell sheet at a
pressure of 0.03 MPa (FIG. 3). A sheet-shaped cell culture
contracts as separated from a culture dish and becomes smaller than
the bottom of the culture dish (FIG. 1). Thus, it is estimated that
out of 800 .mu.L sprayed, about 5004 of the thrombin liquid adhered
onto the sheet-shaped cell culture. Incidentally, the estimated
adhesion amount of the thrombin liquid was calculated as follows. A
predetermined amount of thrombin liquid (in this case, 800 .mu.L)
was actually sprayed employing a predetermined spray (in the case
of this example, BOLHEAL.RTM. spray set), spray pressure (in the
case of this example, 0.03 MPa), height (in the case of this
example, about 7 cm), and spray angle (in the case of this example,
45.degree.), and the weight of the thrombin liquid adhering to a
predetermined region (in the case of this example, within a circle
about 45 mm in diameter) was measured with an electronic balance
and divided by the density of the thrombin liquid.
[0085] A fibrin gel is formed by a reaction between the fibrinogen
liquid and the thrombin liquid. After allowing to stand for about 5
minutes, 24 mL of Hanks' balanced salt solution (HBSS (+), Cat No.
14025, manufactured by Life Technologies Corporation; the same
hereinafter) was added to the culture dish and immediately removed,
thereby washing the culture dish containing the sheet-shaped cell
culture. Unreacted fibrinogen liquid and thrombin liquid can thus
be removed. Next, 24 mL of Hanks' balanced salt solution was added
again to the culture dish and allowed to stand for about 15
minutes. Subsequently, the solution in the culture dish was
removed, the fibrin gel solidified other than on the sheet-shaped
cell culture was trimmed with a scalpel (FIG. 4), and a laminate of
the fibrin gel and the sheet-shaped culture was isolated (Laminate
1). The isolated laminate was stored until use in a culture dish
filled with 24 mL of Hanks' balanced salt solution.
[0086] Laminate 2 was produced by the same procedure except that
the dripping amount of the fibrinogen liquid was 300 .mu.L, and the
spraying amount of the thrombin liquid was about 600 .mu.L
(estimated adhesion amount: about 300 .mu.L), Laminate 3 was
produced by the same procedure except that the dripping amount of
the fibrinogen liquid was 300 .mu.L, and the spraying amount of the
thrombin liquid was about 900 .mu.L (estimated adhesion amount:
about 450 .mu.L), Laminate 4 was produced by the same procedure
except that the dripping amount of the fibrinogen liquid was 300
.mu.L, and the spraying amount of the thrombin liquid was about 300
.mu.L (estimated adhesion amount: about 100 .mu.L), and Laminate 5
was produced by the same procedure except that the dripping amount
of the fibrinogen liquid was 500 .mu.L, and the spraying amount of
the thrombin liquid was about 500 .mu.L (estimated adhesion amount:
about 180 .mu.L).
[0087] In addition, for comparison, an attempt was made to produce
a laminate of a fibrin gel and a sheet-shaped cell culture by a
technique of simultaneously spraying a fibrinogen liquid and a
thrombin liquid.
[0088] In the same manner as for Laminate 1, a sheet-shaped cell
culture was formed and separated from the culture dish. The culture
solution in the culture dish was removed, and the sheet-shaped cell
culture was shaped as necessary (FIG. 1). Subsequently, 800 .mu.L
of a fibrinogen liquid and 800 .mu.L of a thrombin liquid were
simultaneously sprayed onto the sheet-shaped cell culture using
BOLHEAL.RTM. spray set (manufactured by Akita Sumitomo Bakelite
Co., Ltd) from a spray nozzle placed about 7 cm away from the cell
sheet at a pressure of 0.03 MPa (the estimated adhesion amounts to
the sheet-shaped cell culture were each about 500 .mu.L). After
allowing to stand for about 5 minutes, 24 mL of Hanks' balanced
salt solution was added to the culture dish and immediately
removed, thereby washing the culture dish containing the
sheet-shaped cell culture. Next, 24 mL of Hanks' balanced salt
solution was added again to the culture dish and allowed to stand
for about 15 minutes, and then solution in the culture dish was
removed (FIG. 5). The fibrin gel solidified other than on the
sheet-shaped cell culture was trimmed with a scalpel as an attempt
to isolate a laminate of the fibrin gel and the sheet-shaped cell
culture. However, the fibrin gel came off the sheet-shaped cell
culture, and a laminate was not obtained (FIG. 6).
Example 2
Evaluation of Laminate of Fibrin Gel and Sheet-Shaped Cell
Culture
[0089] Laminates 1 to 5 obtained in Example 1 were evaluated in
terms of size, weight, strength, thickness, operability, and
properties. Size was measured with a ruler, weight was measured
with an electronic non-automatic scale (AT201, manufactured by
Mettler-Toledo), and thickness was measured with a dial thickness
gauge (SM-124, manufactured by Teclock Corporation). Strength was
measured as follows. First, a laminate extended in a liquid was
scooped up with an intestinal spatula made of stainless steel (45
mm in width; the same hereinafter) and placed out of the liquid
with the laminate adhering to the surface of the intestinal
spatula. A suture equipped with a needle (6-0 proline) was inserted
between the laminate and the intestinal spatula, and passed through
the laminate from the lower surface to the upper surface. Both ends
of the thread were tied together to form a ring, which was then
connected to a gauge (a general-purpose digital force gage, FGC-1B,
manufactured by Nidec-Shimpo Corporation). The thread locked to the
laminate was horizontally pulled through the gauge, and the maximum
load before the laminate broke (tensile breaking load) was
measured. Measurement was performed at three different points on a
laminate (n=3). Operability was comprehensively evaluated based on
ease of operation in the operation of placing a submerged laminate
on an intestinal spatula (FIG. 7) and transferring the same to the
side of a bottle simulating the heart (ease of placement on the
intestinal spatula, resistance to falling from the intestinal
spatula during transfer, and ease of transfer from the intestinal
spatula to the bottle), and the condition of the laminate (whether
wrinkling or tearing occurs during the operation). Operability was
rated on a five-point scale from 5 (the highest) to 1 (the lowest).
For properties, the condition of a laminate observed during the
evaluation of operability was qualitatively evaluated. In addition,
for comparison, a sheet-shaped cell culture having no fibrin gel
laminated (produced under the same conditions as for Laminate 1)
was also subjected to the same evaluation. The results are shown in
Table 1.
TABLE-US-00001 TABLE 1 Evaluation Results of Laminates 1 to 5
Sheet- Shaped Laminate Laminate Laminate Laminate Laminate Cell 1 2
3 4 5 Culture FN:TN (.mu.L)* 500:500 300:300 300:450 300:100
500:180 -- FN:TN Ratio** 1:1 1:1 1:1.5 3:1 3:1 -- Size (mm) 45
.times. 42 43 .times. 42 50 .times. 46 42 .times. 45 45 .times. 40
43 .times. 41 Weight (g) 1.92995 1.26149 1.24936 0.98233 1.5484 --
Strength (N) 0.047 0.020 0.023 0.033 0.043 0.007 (n = 3) Thickness
1.250 0.467 0.433 0.615 1.337 0.073 (mm) Operability 5 4 3 1 1 --
Properties Flexible and moderately Rigid and slippery -- adhesive
*FN represents the dripping amount of fibrinogen liquid, and TN
represents the estimated adhesion amount of thrombin liquid. **The
ratio between the dripping amount of fibrinogen liquid and the
estimated adhesion amount of thrombin liquid.
[0090] The results in Table 1 show that all the laminates have much
higher strength than does the sheet-shaped cell culture having no
fibrin gel laminated, and also that an increase in the amount of
fibrinogen liquid leads to an increase in the weight, strength, and
thickness of the resulting laminate. In addition, although all the
laminates allowed for the simulated implantation operation, there
was a tendency that operability improved as the volume ratio
between the fibrinogen liquid and thrombin liquid adhering to a
sheet-shaped cell culture approached 1:1.
[0091] By the same method as for Laminate 1, Laminates 6 to 9 that
are different in the number of cells, and the number of
sheet-shaped cell cultures laminated were produced, and evaluated
in the same manner as above. The results are shown in Table 2.
Incidentally, the formation of a sheet-shaped cell culture was
performed in a 3.5-cm-diameter cell culture dish (UpCell.RTM.
3.5-cm dish, manufactured by CellSeed Inc.). The dry weight of a
laminate was determined by weighing a freeze-dried laminate with an
electronic balance. Water content was determined by the following
equation: water content=(wet weight of laminate-dry weight of
laminate)/wet weight of laminate.times.100(%). In addition, the
lamination of sheet-shaped cell cultures was performed as follows.
First, a support (CellShifter.TM., manufactured by CellSeed Inc.)
was placed on a sheet-shaped cell culture without introducing air
bubbles. The support having adhering thereto the sheet-shaped cell
culture was peeled from the end to recover the sheet-shaped cell
culture together with the support, and then placed on another
sheet-shaped cell culture after removing the culture medium. After
allowing to stand at 37.degree. C. for about 30 minutes, and after
confirming that the sheet-shaped cell cultures adhered to each
other, the support was removed from the laminated sheet-shaped cell
cultures. As necessary, the same procedure was repeated to perform
further lamination.
TABLE-US-00002 TABLE 2 Evaluation Results of Laminates 6 to 9
Laminate Laminate Laminate Laminate 6 7 8 9 Number of Seeded 9.3
.times. 10.sup.6 9.3 .times. 10.sup.6 4.56 .times. 10.sup.6 4.56
.times. 10.sup.6 Cells (per sheet) Number of 1 1 4 3 Laminated
Sheets Size of 13 .times. 11 13 .times. 12 13 .times. 12 13 .times.
11 Sheet-Shaped Cell Culture (mm) Weight of 0.0847 0.04073 0.06468
0.04864 Sheet-Shaped Cell Culture (g) FN:TN (.mu.L)* 25:25 50:50
50:50 50:50 Size of 16 .times. 15 18 .times. 13 18 .times. 13 15
.times. 13 Laminate (mm) Wet Weight of 0.13878 0.25828 0.15574
0.14036 Laminate (g) Strength of 0.02 0.06 0.08 0.07 Laminate (N)
Thickness of 0.31 0.27 0.37 0.35 Laminate (mm) Dry Weight of
0.00184 0.00422 0.00534 0.00428 Laminate (g) Water Content (%)
98.67416 98.36611 96.57121 96.95070 *FN represents the dripping
amount of fibrinogen liquid, and TN represents the estimated
adhesion amount of thrombin liquid.
[0092] The results in Table 2 show that the contribution of a
fibrin gel to the weight and strength of a laminate is greater than
that of the lamination of sheet-shaped cell cultures.
Example 3
Production of Laminate Having Reinforcing Portion
[0093] Onto an end of Laminate 2, 50 .mu.L of a thrombin liquid was
dripped, and then 50 .mu.L of a fibrinogen liquid was dripped.
After allowing to stand for 5 minutes, 24 mL of Hanks' balanced
salt solution was added to the culture dish, and unreacted
fibrinogen and thrombin were removed. A reinforcing portion was
thus formed on Laminate 2. Incidentally, the thrombin liquid and
fibrinogen liquid used were the same as those used for the
formation of a fibrin gel of a non-reinforcing portion. In
addition, onto an end of Laminate 3, 100 .mu.L of a thrombin liquid
was dripped, and then 100 .mu.L of a fibrinogen liquid was dripped,
thereby forming a reinforcing portion in the same manner (FIG. 8).
Strength at the reinforcing portion of Laminate 2 and that of
Laminate 3, each provided with a reinforcing portion, were
evaluated in the same manner as in Example 2. The results are shown
in Table 3.
TABLE-US-00003 TABLE 3 Evaluation Results of Reinforcing Portions
of Laminates 2 and 3 Laminate 2 Laminate 3 Dripping Amount to
Reinforcing Portion 50:50 100:100 (FN:TN, .mu.L) Strength of
Non-reinforcing 0.020 0.023 Laminate (N) portion Reinforcing
portion 0.09 0.17
[0094] By the same method as above, Laminate 10 having a
reinforcing portion was produced using a 3.5-cm-diameter cell
culture dish, and evaluated in the same manner as above. The
results are shown in Table 4.
TABLE-US-00004 TABLE 4 Evaluation Results of Laminate 10 Laminate
10 Number of Seeded Cells 4.56 .times. 10.sup.6 Size of
Sheet-Shaped Cell Culture (mm) 15 .times. 11 Weight of Sheet-Shaped
Cell Culture (g) 0.03558 FN:TN (.mu.L) Non-reinforcing 50:50
portion* Reinforcing 25:25 portion** Size of Laminate (mm) 16
.times. 12 Wet Weight of Laminate (g) 0.13837 Strength of Laminate
Non-reinforcing 0.02 (N) portion Reinforcing portion 0.17 Thickness
of Laminate Non-reinforcing 0.29 (mm) portion Reinforcing portion
1.04 Dry Weight of Laminate (g) 0.00508 Water Content (%) 96.32868
*FN represents the dripping amount of fibrinogen liquid, and TN
represents the estimated adhesion amount of thrombin liquid. **FN
represents the dripping amount of fibrinogen liquid, and TN
represents the dripping amount of thrombin liquid.
[0095] The results shown in Tables 3 and 4 show that a reinforcing
portion can have strength that is about 4.5 to 8.5 times that of a
non-reinforcing portion. It is also shown that the strength of a
reinforcing portion is correlated with the amounts of thrombin
liquid and fibrinogen liquid dripped for the formation of the
reinforcing portion.
Example 4
Fixing of Laminate with Suture
[0096] For the evaluation of the operability of a laminate during
implantation, the following test was performed using a laminate
produced under the same conditions as for Laminate 3 (dripping 300
.mu.L of a fibrinogen liquid+spraying about 100 .mu.L of a thrombin
liquid (estimated adhesion amount)) (n=2). A submerged laminate was
placed on an intestinal spatula, a suture (7-0 proline) was hung on
the end of the laminate (FIG. 9), and the laminate was transferred
from the intestinal spatula to a surgically exposed pig's heart
(FIGS. 10 to 11) and fixed with the suture. After the implantation
of the laminate, the chest was closed, and the subsequent condition
of the animal was observed. Five laminates were implanted per pig.
In each test, in one of the five laminates implanted, after the
laminate was fixed to the surface of the heart with a suture, the
laminate could not withstand the tension of the suture, tearing
occurred from the pass-through part, whereby the suture was
detached (FIG. 12), and suturing was performed again (FIG. 13).
However, the laminates were all eventually fixed to the surface of
the heart.
[0097] In addition, the following test was performed using a
laminate produced under the same conditions as for Laminate 1
(dripping 500 .mu.L of a fibrinogen liquid+spraying about 500 .mu.L
of a thrombin liquid (estimated adhesion amount)). A submerged
laminate was placed on an intestinal spatula (FIG. 14), a suture
(7-0 proline) was hung on a marginal part (FIGS. 15 to 17), and the
operation of transferring the same to the side of a bottle
simulating the heart (FIG. 18) was performed. The laminate had
moderate flexibility and stickiness, was easy to place on an
intestinal spatula, allowed for smooth transfer from the intestinal
spatula to the bottle, and also fitted well on the curving surface
of the bottle. In addition, during the series of operations, the
suture remained firmly hung on the laminate, and it did not happen
that the laminate was broken to detach the suture.
Example 5
Treatment with Laminate
[0098] Laminates were used to treat human patients having serious
myocarditis (ischaemic cardiomyopathy, and dilated cardiomyopathy).
A laminate was produced under the same conditions as for Laminate 1
(dripping 500 .mu.L of a fibrinogen liquid+spraying about 500 .mu.L
of a thrombin liquid (estimated adhesion amount)). The laminate was
placed on an intestinal spatula, transferred onto the patient's
heart exposed by thoracotomy, and fixed with a suture. The chest
was then closed, and the condition of the patient was observed. In
all the patients, improvement was seen in cardiac performance,
exercise tolerance, QOL, morbidity, and prognosis.
[0099] The detailed description above describes a method for
producing a laminate of a sheet-shaped cell culture and a fibrin
gel, a laminate produced by the method, a pharmaceutical
composition containing the laminate, and a method for treating a
disease using the laminate. The invention is not limited, however,
to the precise embodiments and variations described. Various
changes, modifications and equivalents can be effected by one
skilled in the art without departing from the spirit and scope of
the invention as defined in the accompanying claims. It is
expressly intended that all such changes, modifications and
equivalents which fall within the scope of the claims are embraced
by the claims.
* * * * *