U.S. patent application number 11/885222 was filed with the patent office on 2008-06-05 for cultured cell sheet, production method and tissue repair method using thereof.
Invention is credited to Masato Kanzaki, Akihiko Kikuchi, Tinatsu Kohno, Teruo Okano, Takamasa Onuki, Hidekazu Sekine, Masayuki Yamato.
Application Number | 20080131476 11/885222 |
Document ID | / |
Family ID | 36941182 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131476 |
Kind Code |
A1 |
Kanzaki; Masato ; et
al. |
June 5, 2008 |
Cultured Cell Sheet, Production Method and Tissue Repair Method
Using Thereof
Abstract
It is intended to provide a cultured cell sheet with excellent
tissue adherence and flexibility. The above object can be achieved
by culturing cells on a support for cell culture in which a surface
of a substrate is coated with a temperature-responsive polymer
whose lower or upper critical solution temperature against water is
in the range of 0 and 80.degree. C. along with a surfactant protein
or a crosslinking inhibitor and producing a cultured cell sheet by
detaching it by setting the temperature of the culture to the upper
critical solution temperature or higher or to the lower critical
solution temperature or lower.
Inventors: |
Kanzaki; Masato; (Saitama,
JP) ; Yamato; Masayuki; (Tokyo, JP) ; Kohno;
Tinatsu; (Tokyo, JP) ; Sekine; Hidekazu;
(Saitama, JP) ; Kikuchi; Akihiko; (Tokyo, JP)
; Okano; Teruo; (Chiba, JP) ; Onuki; Takamasa;
(Kanagawa, JP) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
36941182 |
Appl. No.: |
11/885222 |
Filed: |
February 28, 2006 |
PCT Filed: |
February 28, 2006 |
PCT NO: |
PCT/JP06/03762 |
371 Date: |
December 14, 2007 |
Current U.S.
Class: |
424/423 ;
435/395; 435/396 |
Current CPC
Class: |
A61P 9/00 20180101; C12N
2533/30 20130101; A61P 11/00 20180101; A61L 27/3839 20130101; A61L
27/3895 20130101; C12N 5/0068 20130101; A61L 27/34 20130101; A61L
27/3804 20130101; A61P 1/16 20180101; C12N 5/0688 20130101 |
Class at
Publication: |
424/423 ;
435/395; 435/396 |
International
Class: |
C12N 5/06 20060101
C12N005/06; A61F 2/00 20060101 A61F002/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 28, 2005 |
JP |
2005-096011 |
Claims
1. A cultured cell sheet, having superior tissue adhesiveness and
excellent flexibility.
2. The cultured cell sheet according to claim 1, wherein the cells
are selected from the group consisting of: fibroblasts, cells of
alveolar tissue, cells of myocardial tissue, cells of hepatic
tissue, cells of vascular tissue, mesenchymal stem cells, and
adipose derived cells.
3. The cultured cell sheet according claim 1 or 2, wherein the
cultured cell sheet is a layered cultured cell sheet.
4. The cultured cell sheet according to claim 3, wherein the
layered cell sheet is prepared by layering of the cell sheet of
claim 2.
5. The cultured cell sheet according to any one of claims 1 to 4,
wherein the superior adhesiveness allows the cultured cell sheet to
cover a site of air leakage, blood leakage or bodily fluid leakage
on tissue, and thereby to suppress such leakage.
6. The cultured cell sheet according to any one of claims 1 to 5,
wherein the cultured cell sheet has a flexibility such that the
cultured cell sheet obstructs performance of the tissue not more
than 20% after tissue has been covered by the cultured cell
sheet.
7. The cultured cell sheet according to any one of claims 1 to 6,
wherein the flexibility results from a surfactant protein.
8. The cultured cell sheet according to claim 7, wherein the
surfactant protein is produced by the cultured cell sheet.
9. The cultured cell sheet according to any one of claims 1 to 8,
wherein the flexibility results from a cross-linking inhibitor.
10. The cultured cell sheet according to claim 9, wherein a
cross-linking inhibitor is a collagen cross-linking inhibitor,
.beta.-aminopropylnitrile.
11. The cultured cell sheet according to any one of claims 1 to 10,
wherein the cultured cell sheet is used for a treatment for
suppressing air leakage, blood leakage or bodily fluid leakage from
a surface layer of an organ.
12. The cultured cell sheet according to claim 11, wherein the
treatment comprises covering the surface of an affected portion of
an organ with the cultured cell sheet.
13. The cultured cell sheet according to either claim 11 or 12,
wherein the subject to be treated is air leakage from lung tissue,
bleeding from liver tissue or bleeding from vascular tissue.
14. The cultured cell sheet according to any one of claims 11 to
13, wherein the cultured cell sheet is cut according to the size
and shape of the affected area, before covering of the surface of
the affected area.
15. A method for preparing a cultured cell sheet, wherein cells are
cultured on a cell culture support having a surface coated with a
temperature responsive polymer having an upper or lower critical
solution temperature of 0.degree. C. to 80.degree. C. in water,
and, thereafter, comprising steps of: (1) adjusting the temperature
of the culture medium to a temperature above the upper critical
solution temperature or below the lower critical solution
temperature; and (2) detaching the cultured cell sheet.
16. The method according to claim 15, wherein the temperature
responsive polymer is poly(N-isopropyl acrylamide).
17. The method according to either claim 15 or 16, wherein the cell
sheet is not treated with a proteolytic enzyme upon being
detached.
18. The method according to any one of claims 15 to 17, wherein
.beta.-aminopropylnitrile is added to the culture medium.
19. A treatment method comprising transplanting the cultured cell
sheet of any one of claims 1 to 14 to the affected area of the
surface layer of the organ from which air, blood or bodily fluid is
leaking.
20. The method according to claim 19, wherein the transplantation
is conducted by covering of the surface layer of the affected area
of the organ.
21. The method according to claim 19 or 20, wherein the cultured
cell sheet is cut according to the size and shape of the affected
portion, before covering of the surface layer of the affected area
of the organ.
22. The method according to any one of claims 19 or 21, wherein the
subject to be treated is air leakage from alveolar tissue, bleeding
from liver tissue, or bleeding from vascular tissue.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cultured cell sheet, a
production method, and an applied tissue repair method thereof, in
medical and biological fields, etc.
BACKGROUND ART
[0002] Japan is becoming an aging society with the highest average
life expectancy in the world. People are beginning to place more
emphasis on living better, i.e., "the quality of life (QOL)",
rather than merely prolonging life. Under such circumstances,
medical techniques are rapidly progressing, and techniques for
reconstruction of organs damaged by trauma or disease are also
advancing remarkably. Therefore, in recent years, a lot of
attention has been focused on regenerative medical techniques in
which organ tissue is reconstructed in the cell culture system
employing cultured cells, and then transplanted to the target
site.
[0003] To carry out such treatments, it is necessary to use tissue
adhesive agents for tissue adhesion. The tissue adhesive agents
currently used in the clinical field have been broadly classified
into cyanoacrylate adhesive agents, gelatin-aldehyde adhesive
agents, and fibrin glue adhesive agents. Cyanoacrylate adhesive
agents employ adhesiveness by a polymerization reaction of the
agents' cyanoacrylate monomers, and are superior in adhesive
intensity and bonding speed. However, cyanoacrylate adhesive agents
are synthetic agents which are not originally endogenous, and
prevent healing by producing formaldehyde by hydrolyzation of a
cured monomer, which causes toxicity to living organisms.
Therefore, the problems exist that the site to be applied was
limited, and the adhesive agents should not be applied to a site
near a central nerve or blood vessel. Gelatin-aldehyde adhesive
agents employ adhesiveness by a cross-linking reaction between a
gelatin (biopolymer of the degenerated collagen) and formaldehyde
or glutaraldehyde. However, the gelatin-aldehyde adhesive agents
are also synthetic agents that are not endogenous. Although the
gelatin-aldehyde adhesive agents also have sufficiently high
adhesive intensity, since toxic aldehyde compounds are employed as
a cross-linking agent, they are also biologically toxic. On the
other hand, fibrin glue adhesive agents are made from a
tissue-derived material, which employs adhesiveness by a reaction
of blood coagulation. Although this type of adhesive agent is less
toxic than the above described synthetic adhesive agents, the
adhesiveness is low, and large amounts of fibrin glue should be
used, since the fibrin glue itself is metabolized in vivo. Further,
recently, some problems are being indicated relating to the topical
inflammation at the site where the adhesive agent has been applied,
since the fibrin glue is prepared and purified heterogeneously or
the mechanisms of the adhesiveness of the fibrin glue are the same
as the blood coagulation reaction.
[0004] Technology that provides a cell sheet having sufficient
basal membrane-like proteins has been proposed. Conventionally,
cell culture is conducted on a glass surface, or on the surface of
a synthetic polymer compound along with a variety of surface
processing. In order to achieve this, for example, various types of
vessels made of polystyrene subjected to surface processing such as
silicone coating, gamma irradiation, etc., are commonly used as
vessels for cell culture. Cells that have been cultured and grown
with these types of cell culture vessels, are detached and
harvested from the surface of the vessel by a chemical agent
treatment or a proteinase treatment such as trypsin. However, in
cases where the cells are harvested by the above-mentioned chemical
agent treatment, some disadvantages have been pointed out: the
treatment method is cumbersome and complicated; the potential for
contamination by impurities is increasing; and examples of defects,
in which cells are caused to degenerate or are damaged by the
chemical treatment, and lose their original function.
[0005] Thus far, in order to overcome the above-mentioned
disadvantages, a number of techniques have been proposed by the
present inventors. Especially, in Japanese Patent Application No.
2001-226141, a method for producing a cultured cell sheet which
comprises steps of coating the surface of the cell culture support
with a temperature responsive polymer having a lower or upper
critical solution temperature ranging from 0.degree. C. to
80.degree. C. in water, having the cultured cell layers
multi-layered by way of a conventional method, as necessary, and
detaching the cultured cell sheet only by changing the temperature
of the culture support. As a result of application of this method,
a cultured cell sheet having sufficient strength can be produced.
Furthermore, a thus obtained cultured cell sheet also retains basal
membrane-like proteins, and also has improved adhesiveness to
tissue, when compared with a cell sheet harvested using the above
described dispase treatment. Moreover, PCT International
Publication No. WO 02/08387 discloses a method for producing a
cultured myocardial cell sheet, which comprises steps of culturing
the cells of myocardial tissue on a cell culture support having a
support surface coated or covered with a temperature responsive
polymer, preparing a myocardium-like cell sheet, and subsequently,
adjusting a temperature of the culture medium to a temperature
greater than the upper critical solution temperature or less than
the lower critical solution temperature, bringing the layered
cultured cell sheet into close contact with a polymer membrane,
detaching the cultured intact cell sheet together with the polymer
membrane, and three-dimensionally structuring by a predetermined
method. As a result of application of this method, a
myocardium-like cell sheet and a three-dimensional structure were
discovered to be constructed in vitro with reduced structural
defects and with some of the functions of myocardial tissue.
Neither of Application No. JP 2001-226141 nor WO 02/08387 have
investigated conferring the flexibility to the cell sheet, and have
discussed the use as tissue repair material for suppressing the air
leakage, blood leakage or bodily fluid leakage from the surface of
an organ. However, the use of such a cultured cell sheet as a
tissue adhesive can be employed for the patient receiving tissue
repair with an extremely high degree of safety, because the cell
sheet can be prepared from the cell of the patient himself and,
therefore, is highly safe.
DISCLOSURE OF THE INVENTION
Problem(s) to be Solved
[0006] The present invention is intended to solve the
above-mentioned problems in conventional technology. Specifically,
a purpose of the present invention is to provide a cultured cell
sheet with excellent tissue adhesiveness and excellent flexibility.
Moreover, a purpose of the present invention is to provide a method
for manufacturing the cultured cell sheet and application as tissue
repair material for suppressing the air leakage, blood leakage or
bodily fluid leakage from the surface of an organ.
Means for Solving the Problem(s)
[0007] The present inventors have conducted research and
development, by investigating various aspects, in order to solve
the above-mentioned problems. As a result, it was discovered that a
highly adhesive cultured cell sheet with excellent tissue
adhesiveness and application as tissue repair material for
suppressing the air leakage, blood leakage or bodily fluid leakage
from the surface of an organ was obtained by the steps of:
culturing cells, such as fibroblasts, cells of alveolar tissue, or
cells of myocardial tissue, on a cell culture support, the surface
of which is coated with a temperature responsive polymer; and
afterwards, adjusting a temperature of the culture medium to a
temperature greater than the upper critical solution temperature or
less than the lower critical solution temperature; and detaching
the cultured cell sheet. The present invention was completed based
on the above-mentioned knowledge.
[0008] Specifically, the present invention provides a highly
adhesive cultured cell sheet with a superior adhesiveness to the
surface of a leaking site on an organ and with an excellent
flexibility.
[0009] The present invention provides a highly adhesive cultured
cell sheet, which comprises a step of culturing at least one type
of cell selected from the group consisting of: fibroblasts, cells
of alveolar tissue, cells of myocardial tissue, cells of hepatic
tissue, cells of vascular tissue, mesenchymal stem cells, and
adipose derived cells, on a cell culture support having a surface
of the support coated with a temperature responsive polymer having
an upper or lower critical solution temperature ranging from
0.degree. C. to 80.degree. C. in water, and thereafter, comprising
the steps of: adjusting the temperature of the culture medium to a
temperature greater than the upper critical solution temperature or
less than the lower critical solution temperature; bringing the
cultured cell sheet in close contact with a carrier; and detaching
the cultured cell sheet together with the carrier. The highly
adhesive cultured cell sheet obtained in the above-mentioned manner
demonstrates superior adhesiveness to the surface of a leaking site
on an organ, and therefore, cultured cell sheets having superior
adhesiveness, such as those of the present invention, are sometimes
called, "highly adhesive cultured cell sheets".
[0010] Moreover, the present invention provides a cultured cell
sheet for application as tissue repair material for suppressing air
leakage, blood leakage or bodily fluid leakage from the surface of
an organ.
[0011] In addition, the present invention provides a treatment
method which is conducted by transplanting the highly adhesive
cultured cell sheet to the site of air leakage, blood leakage or
bodily fluid leakage from the surface of an organ.
EFFECT OF THE INVENTION
[0012] The highly adhesive cultured cell sheet obtained by the
present invention has extremely high adhesiveness to a leaking site
of the surface of an organ and an excellent flexibility. Therefore,
the use of the cell sheet of the present invention enables to
suppress air leakage, blood leakage or bodily fluid leakage from
the surface of an organ. Consequently, the present invention is
extremely useful in biological and medical fields, etc., such as
cell engineering and medical engineering.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a cultured cell sheet of alveolar cells 10 days
after the start of the culture, as indicated in Example 2;
[0014] FIG. 2 is a photograph showing the appearance of an air
leakage model, as indicated in Example 2;
[0015] FIG. 3 is a photograph showing the appearance of the
cultured cell sheet adhered to the site of air leakage, as
indicated in Example 2;
[0016] FIG. 4 is a photograph showing the appearance of the site of
air leakage which was closed by the cultured cell sheet, as
indicated in Example 2;
[0017] FIG. 5 is a photograph showing the results of a
hematoxylin-eosin stained tissue section from the site of air
leakage which was closed by the cultured cell sheet of the
invention, as described in Example 2;
[0018] FIG. 6 is a photograph showing the results of an Azan
stained tissue section from the site of air leakage which was
closed by the cultured cell sheet of the invention, as described in
Example 2;
[0019] FIG. 7 is a photograph showing the appearance of a bleeding
model, as indicated in Example 4;
[0020] FIG. 8 is a photograph showing the appearance of a site of
bleeding which was closed by the cultured cell sheet, as indicated
in Example 4;
[0021] FIG. 9 is a photograph showing the results of a
hematoxylin-eosin stained tissue section from a site of bleeding of
the liver which was closed by the cultured cell sheet, 4 weeks
after transplantation, as indicated in Example 4;
[0022] FIG. 10 is a photograph showing the results of an Azan
stained tissue section from a site of bleeding in the liver which
was closed by the cultured cell sheet of the invention, 4 weeks
after transplantation, as indicated in Example 4;
[0023] FIG. 11 is a photograph showing the appearance of a bleeding
model, as indicated in Example 5; and
[0024] FIG. 12 is a photograph showing the appearance of a site of
bleeding which was closed by the cultured cell sheet, as indicated
in Example 5.
PREFERRED MODE FOR CARRYING OUT THE INVENTION
[0025] The present invention provides a cultured cell sheet which
has superior adhesiveness to the surface of an organ and has an
excellent flexibility. Suitable cells for producing the cultured
cell sheet of the present invention may include any one type or
combination of two or more types of cells selected from the group
consisting of: fibroblasts, cells of alveolar tissue, cells of
myocardial tissue, cells of hepatic tissue, cells of vascular
tissue, mesenchymal stem cells, and adipose derived cells. However,
the present invention is not strictly limited to the
above-mentioned types of cells. In the present invention, the
highly adhesive cultured cell refers to a sheet which is prepared
by the steps of culturing each of the above-mentioned types of
cells as a single layer on the surface of the culture support, and
then, detaching the cell sheet from the support. Thus obtained cell
sheet has a lower side surface which is contacted to the culture
support at the time of culture, and an upper side surface on the
opposite side thereof. If the cells are cultured on the cell
culture support, the surface of which is coated or covered with a
temperature responsive polymer having an upper or lower critical
solution temperature ranging from 0.degree. C. to 80.degree. C. to
in water indicated in the present invention, there will be an
abundance of adhesive proteins produced on the lower side surface
of the cell sheet at the time the cells are cultured.
[0026] The cultured cell sheet of the present invention may or may
not include a scaffold other than that produced by the cultured
cells, such as collagen, fibronectin, laminin, etc. However, it is
not particularly limited.
[0027] The cultured cell sheet of the present invention is
constructed of at least one type or a combination of two or more
types of cells selected from the group consisting of: fibroblasts,
cells of alveolar tissue, cells of myocardial tissue, cells of
hepatic tissue, cells of vascular tissue, mesenchymal stem cells,
and adipose derived cells. These cells are cells that are capable
of expressing phenotype of the various types of chondroid tissue
mentioned above.
[0028] The highly adhesive cultured cell sheet of the present
invention adheres extremely well to the leaking site of the surface
of an organ. The high adhesiveness of the cultured cell sheet is
achieved by maintaining its flexibility and reducing the shrinkage
of the cultured cell sheet that is detached from the surface of the
culture support.
[0029] The flexibility as demonstrated by the present invention is
such that, after tissue has been covered by the cultured cell
sheet, the above-mentioned cultured cell sheet obstructs the
performance of the tissue no more than 20%, preferably no more than
10%, and even more preferably no more than 8%. The highly adhesive
cultured cell sheet of the present invention adheres extremely well
to the site of the leakage on the surface of the body tissue. In
cases where the cultured cell sheet that is covering the tissue is
rigid, the performance of the tissue is obstructed more than 20%,
and the organ cannot exhibit the intact function thereof.
[0030] Regarding the suppression of the shrinkage of the cultured
cell sheet, a percentage of shrinkage of the cultured cell sheet
when detached from the surface of the culture support is preferably
no more than 20%, more preferably no more than 10%, and even more
preferably no more than 5%, in either of the lengthwise directions
of the sheet. If the percentage of shrinkage is no less than 20%,
the detached cell sheet becomes flaccid, and unable to adhere to
living tissue well, even when attached to the living tissue. As a
result, the detached cell sheet with no less than 20% shrinkage
demonstrates the characteristics of the highly adhesive cell sheet
of the present invention.
[0031] A method for preventing the cultured cell sheet from
shrinking is not particularly limited in any way, and includes a
method comprising the steps of bringing a ring-shaped carrier with
a center portion cutout in close contact with the above-mentioned
cell sheets at the time of detaching the cultured cell sheet from
the culture support, and detaching the cell sheet with the
above-mentioned carrier.
[0032] The carrier that is used when detaching the highly adhesive
cultured cell sheet has a structure for keeping the cell sheet of
the present invention from shrinking, and a carrier such as that
made of a polymer membrane, or a structure formed from a polymer
membrane, or a metallic fixture, etc., may be used. For example, in
cases where a polymer is used as the carrier material,
polyvinylidene difluoride (PVDF), polypropylene, polyethylene,
cellulose and derivatives thereof, papers, chitin, chitosan,
urethane, etc., may be used as the specific material of the
carrier.
[0033] In the case of the present invention the term "close
contact" hereinafter refers to having the cell sheet in a state in
which it does not slip or slide on the carrier, in order to prevent
the cell sheet from shrinking at the boundary between the cell
sheet and carrier, and therefore, it may be in close contact by
being physically bonded, or it may be in close contact via the
fluid (for example, the culture medium, and other isotonic
solutions) existing between each of the cell sheet and the
carrier.
[0034] The shape of the carrier is not specifically limited in any
way. For example, when transplanting the highly adhesive cultured
cell sheet obtained, if a cut out portion of the carrier which is
the same size or larger than the transplantation site is utilized,
greater convenience results since the cell sheet is only fixed to
the portion surrounding the cutout portion, and only a cell sheet
with a cut-out portion that fits the transplantation site can be
transplanted.
[0035] The highly adhesive cultured cell sheet of the present
invention may be provided as a monolayered sheet, or as a layered
sheet. Here, the layered sheet can be prepared by layering sheets
consisting solely of the highly adhesive cultured cell sheet or may
be prepared by combining the highly adhesive cultured cell sheet
with a sheet of other cells. For example, a cell sheet includes,
but is not limited to, one which is prepared by layering the
above-mentioned fibroblast cell sheet and another fibroblast cell
sheet, or a cell sheet that is prepared by layering of a fibroblast
cell sheet and a cell sheet derived from cells other than those of
the fibroblast cell sheet (for example, a cell sheet made of cells
of alveolar tissue), etc. In such cases, if at least two different
types of cells are employed, the different cells intercellularly
interact with each other, and therefore, a cell sheet having the
characteristic of even higher activity can be obtained.
Furthermore, a position where the cell sheet is layered, the order
of the layered cell sheets, and the number of the layered cell
sheets are not particularly limited in any way. However, depending
on the covered tissue, the structure of the layered sheet may be
varied, by employing a highly adhesive cell sheet on the uppermost
layer, and so on. Furthermore, the number of the layered cell
sheets is preferably no more than 10, more preferably no more than
8, and even more preferably no more than 4. The fibroblasts may
also be viable even in an environment in which basic nutrients are
not fully supplied. However, a cell sheet consisting of more than
10 layers of cell sheets is undesirable, since it is difficult to
deliver oxygen and nutrients to the center portion of the layered
cell sheets.
[0036] For example, the layered sheet of the present invention can
be produced by employing a method, for example, but not limited to,
the one described below:
(1) a method of superimposing the highly adhesive cultured cell
sheet in close contact with the above-mentioned carrier by the
steps of attaching the first cell sheet in close contact with the
carrier to the cell culture support, and afterwards, removing the
carrier off the first cell sheet by the addition of a culture
medium, and layering the second cell sheet by attaching the second
cell sheet in close contact with a carrier, and repeating the
steps; (2) a method of superimposing the highly adhesive cultured
cell sheet in close contact with the above-mentioned carrier by the
steps of inverting the first cell sheet in close contact with the
carrier, fixing the carrier side of the first cell sheet to the
surface of the cell culture support, attaching a second cell sheet
to the cell sheet side of the first cell sheet, removing the
carrier off the first cell sheet by adding culture medium
afterwards, and repeating the steps by attaching another separate
cell sheet; (3) a method in which two cell sheets each in close
contact with a carrier are brought into close contact with each
other at the cell sheet side; and (4) a method in which the cell
sheet in close contact with the carrier is fitted to an affected
area of the individual, and after the cell sheet is attached to
living tissue, removing the carrier, and overlaying another
separate cell sheet on the affected area.
[0037] The highly adhesive cultured cell sheet of the present
invention is characterized in that the basal membrane-like proteins
between the cell and support formed during the culture are not
damaged by enzymes, such as proteinases like dispase, trypsin, etc.
Therefore, in order to produce a cultured cell sheet with such a
characteristic, the cell culture is preferably conducted on a
surface of the cell culture support coated with a temperature
responsive polymer.
[0038] The temperature responsive polymer that is used to coat the
cell culture support has upper or lower critical solution
temperature ranging from 0.degree. C. to 80.degree. C. in water,
and more preferably a temperature ranging from 20.degree. C. to
50.degree. C. in water. An upper or lower critical solution
temperature that exceeds 80.degree. C. is not preferable, since
cells may die. Furthermore, an upper or lower critical solution
temperature that is lower than 0.degree. C. is also not preferable,
since it typically causes an extreme decrease in cellular growth
rate or causes cell death.
[0039] The temperature responsive polymer to be used in the present
invention may be either a homopolymer or a copolymer. Examples of
such a polymer may include, for example, the polymer disclosed in
Japanese Patent Publication No. H2-211865 (JP 2-211865 A).
Specifically, for example, they may be obtained by polymerization
or copolymerization of the monomer mentioned below. Monomers that
can be used include, for example, (meth)acrylamide compound, N- (or
N,N-di)alkyl substituted (meth)acrylamide derivative, or a vinyl
ether derivative; in the case of a copolymer, at least two of these
monomers may be selected and used. Moreover, those monomers may be
copolymerized with other monomers, or polymers may be grafted
together or copolymerized, or alternatively, mixtures of polymers
and copolymers may be employed. If desired, the polymers may be
crosslinked to an extent that does not impair their properties.
[0040] The support that is to be covered with the temperature
responsive polymer may be chosen from among the glass, modified
glass, compounds such as polystyrene and poly(methyl methacrylate),
and all other substances that can generally be shaped, as
exemplified by polymer compounds other than those compounds, and
ceramics.
[0041] The method of covering the support with the temperature
responsive polymer is not limited in any particular way but one may
follow the methods described in JP 2-211865 A. Specifically, the
coating operation can be achieved by either subjecting the support
and the above-mentioned monomers or polymers to electron beam (EB)
exposure, .gamma.-ray irradiation, ultraviolet irradiation, plasma
treatment, corona treatment or organic polymerization reaction or
by means of physical adsorption as effected by application of
coating solutions or the kneading step.
[0042] The coating of the temperature responsive polymer is
suitably in the range of 0.5 to 5.0 .mu.g/cm.sup.2, preferably 1.0
to 4.0 .mu.g/cm.sup.2, and more preferably 1.2 to 3.5
.mu.g/cm.sup.2. If the coverage of the temperature responsive
polymer is less than 0.5 .mu.g/cm.sup.2, the cells on the polymer
will not easily detach even if they are given a stimulus and the
operating efficiency is considerably lowered, which is not
preferable. If, on the other hand, the coverage of the temperature
responsive polymer is greater than 5.0 .mu.g/cm.sup.2, cells will
not easily adhere to the covered area and adequate adhesion of the
cells becomes difficult to achieve. The shape of the culture
support of the present invention may include, for example, but is
not particularly limited to, a dish shape, multi-plate shape, flask
shape, cell-insert shape, etc., may be employed.
[0043] The composition of the culture medium for culturing the
above-mentioned cells of the present invention is not particularly
limited in any way, and any conventionally used may be employed at
the time the above-mentioned cells are cultured. In the cases that
fibroblasts, cells of alveolar tissue, cells of myocardial tissue,
cells of hepatic tissue, cells of vascular tissue, mesenchymal stem
cells, and adipose derived cells are cultured, the culture medium
may be prepared by, for example, supplementing an .alpha.-MEM
culture medium, an F-12 culture medium, DMEM culture medium, or any
mixture thereof, with 10% to 20% bovine serum, or optionally with
50 .mu.g/ml of ascorbic acid 2-phosphate in addition to bovine
serum.
[0044] Moreover, the cultured cell sheet of the present invention
is highly flexible, and this flexibility is achieved by culturing
cells under specific culture conditions. However, the method
thereof includes, for example, but is not limited to, a method
where a surfactant protein is added to the culture medium, a method
where alveolar cells which produce the surfactant protein are
co-cultured, a method where .beta.-aminopropylnitrile is added to
the culture medium, or a method where other types of collagen
cross-linking inhibitor is added to the culture medium. In cases
where any one of the methods where a surfactant protein,
.beta.-aminopropylnitrile, or other type of collagen cross-linking
inhibitor is added is used, the concentration thereof added to the
culture medium is preferably at least 10 .mu.M, more preferably at
least 100 .mu.M, and even more preferably at least 200 .mu.M.
Moreover, since the cell sheet is not flexible when additive
concentration is less than 10 .mu.M, and the shape of the cell
sheet cannot be retained when the additive concentration is greater
than 500 .mu.M, neither of these additive concentrations is
desirable.
[0045] The temperature of the culture medium is not particularly
limited, as long as the temperature is below the upper critical
solution temperature, or above the lower critical solution
temperature of the polymer that is coated onto the surface of the
support. However, it should be appreciated that a low temperature,
where cultured cells can not proliferate, or high temperature
region, where cultured cells undergo cell death, is of course
unsuitable. Culture conditions other than temperature are not
particularly limited, and conventional procedures may be followed.
For example, a culture medium used may be a culture medium to which
a serum such as common fetal bovine serum (FCS) has been
supplemented, or a serum-free culture medium, to which no serum has
been added.
[0046] When detaching and harvesting the cultured cells from the
cultured support material by the method of the present invention,
the highly adhesive cultured cell sheet in close contact with the
carrier can be removed by the steps of bringing the cultured cell
sheet in close with the carrier, adjusting the temperature of the
culture support attached to the cells to a temperature above the
upper critical solution temperature, or below the lower critical
solution temperature of the polymer coating the culture support,
increasing the hydrophilicity of polymer coating the surface of the
support, and detaching the cultured cell sheet in close contact
with the carrier from the culture support, due to weakening of the
attachment between the culture support and the cultured cell sheet.
Moreover, the sheet may be detached either in the culture medium
which is used to culture the cells, or in another isotonic
solution, depending on the purpose.
[0047] In order to detach and harvest the highly adhesive cultured
cell sheet at a high yield, a method in which the cell culture
support is lightly tapped and shaken, or a method in which the
culture medium is agitated using a pipet, etc., may be used alone,
or in combination. In addition, when required, cultured cells may
be washed with isotonic solution, prior to detaching and
harvesting.
[0048] The highly adhesive cultured cell sheet of the present
invention, when harvested in the above-mentioned manner, are not
damaged by proteinases like dispase, trypsin, etc., from the time
of culture to the time of detachment of the cell sheet. Therefore,
the highly adhesive cell sheet which has been detached from the
support retains the intercellular desmosome structure, has only a
few structural defects, and exhibits high strength. Furthermore,
the basal membrane-like proteins of the sheet of the present
invention between the cell and support formed at the time of
culture are not damaged by enzymes, thus providing superior
adhesion to affected area at the time of transplantation, and
allowing implementation of a very effective treatment to be
possible. More specifically, in cases where conventional
proteinases such as trypsin are used, the intercellular desmosomal
structure and the basal membrane-like proteins between the cell and
support, etc., are not retained at all; and therefore, the cells
are detached from the culture support in a individually separated
condition. Although it is commonly known that, among proteinases,
dispase can allow for detachment with about 10% to 60% of the
retained intercellular desmosomal structure, the basal
membrane-like proteins between the cell and support, etc., are
almost completely damaged, and the cell sheet obtained has only low
strength. However, the cell sheet of present invention keeps no
less 80% of the desmosomal structure and basal membrane-like
proteins intact, and therefore, provides the various effects
described above.
[0049] The leakage site of the surface on the organ is not
particularly limited as long as it is a site at which air, blood,
or bodily fluid is leaking from the organ surface. Examples of the
leakage site of the surface on the organ include the site of air
leakage from lung tissue and the site of bleeding from vascular
tissue or liver tissue. The utilization of the highly adhesive
cultured cell sheet of the present invention for the surface of the
leakage site may include, for example, but is not particularly
limited to, a method in which the affected area is covered with the
highly adhesive cultured cell sheet of the present invention. In
such cases, the cultured cell sheet may be cut to appropriately fit
the size and shape of the affected area. In this way, the highly
adhesive cultured cell sheet of the present invention is able to
adhere extremely well to the surface of leakage site on an organ
surface, which cannot be achieved by the prior art.
[0050] A method for fixing the highly adhesive cultured cell sheet
to the surface of an organ as demonstrated by the present invention
is not particularly limited in any way, and therefore, the cell
sheet may be sutured to living tissue, may be connected to living
tissue with an adhesive agent capable of being used in vivo, or may
only be attached to the affected area without using either of these
means, in order to quickly graft the highly adhesive cultured cell
sheet to living tissue as shown by the present invention.
[0051] An application of the highly adhesive cultured cell sheet as
shown in the present invention is effective, for example, but not
particularly limited to, in treatments of the air leakage from lung
tissue and the bleeding from vascular tissue or liver tissue.
[0052] In cases where the cultured cell sheet of the present
invention is used in transplantation to the surface of an organ,
the cultured cell sheet is transplanted by removing the cell sheet
off the carrier, after fitting the cell sheet to the affected area.
The method for removing the cell sheet off the carrier include, for
example, but not particularly limited to, a method of removing the
cell sheet off the carrier by wetting the carrier in order to
weaken the adhesiveness, or a method of cutting the cultured cell
sheet using a cutting tool such as a scalpel, forceps, a laserbeam,
a plasma wave, etc. For example, in cases where a cell sheet is in
close contact with a carrier with a center portion cutout is
employed, it is preferable to cut the cultured cell sheet to be
transplanted with a laserbeam, or the like, along the border of the
affected area, since it is possible to avoid attachment of the
cultured cell sheet to an undesired area outside of the affected
area.
[0053] The highly adhesive cultured cell sheet obtained by the
method mentioned above is superior when compared with one obtained
by conventional methods, due to the noninvasiveness of the cultured
cell sheet at the time of detachment, and therefore, the clinical
applications for the leakage site of the tissue for
transplantation, etc., are very promising. Especially, the highly
adhesive cultured cell sheet of the present invention shows higher
adhesiveness to living tissue than a conventional transplantation
cell sheet, and grafts to living tissue very rapidly.
[0054] Moreover, antigenic and infective problems can be solved due
to the use of autologous cells. With respect to the fixation of the
cultured cell sheet to the leakage site on the surface of an organ,
since the cultured cell sheet of the present invention is
non-invasively harvested together with the extracellular matrix
including the adhesion molecules secreted by the cultured cell
sheet, which is transplanted, the cultured cell sheet has an
advantage in early establishment of the leakage site on the surface
of an organ to be transplanted when it is transplanted to the
surface. Therefore, the present invention provides an extremely
effective technology with improvements in the treatment efficiency
of the affected area, and further alleviation of a burden on a
patient.
EXAMPLES
[0055] Hereinafter, the present invention will be explained in
further detail based on the following Examples, which are not
intended to limit the scope of the present invention in any
way.
Examples 1 and 2
[0056] To a commercial culture dish with a diameter of 3.5 cm
(Falcon 3001, manufactured by Becton Dickinson Labware), 0.07 ml of
the solution of N-isopropyl acrylamide monomer dissolved in
isopropyl alcohol at a concentration of 53% (Example 1) or 54%
(Example 2) was applied. The culture dish was exposed to electron
beams at an intensity of 0.25 MGy, and the N-isopropyl acrylamide
polymer (PIPAAm) was immobilized on a surface of the culture dish.
After irradiation, the culture dish was washed with ion-exchanged
water to remove a residual monomer and the PIPAAm that did not bind
to the culture dish, was then dried inside a clean bench, and
sterilized by ethylene oxide gas, to obtain a cell culture support
material coated with a temperature responsive polymer.
[0057] The amount of the temperature responsive polymer on the
support surface was measured. As a result, it was found that the
supports' surface was coated with the temperature responsive
polymer in an amount of 1.7 .mu.g/cm.sup.2 (Example 1), and 1.9
.mu.g/cm.sup.2 (Example 2), respectively. A lung tissue was
extracted from a GFP-transgenic neonatal rat, and the cells were
isolated with collagenase. Three days after the start of culture,
the cells were subcultured on the above described cell culture
support material, and the cell culture support was cooled for 30
minutes at 20.degree. C. to harvest the cell sheet of the fourth
passage. The cell sheet at the time of harvest is shown in FIG.
1.
[0058] The harvested cell sheet was layered, and was applied to
closing of air leakage. An air leakage model was prepared as
follows: an 8 week old F-344 nude rat was anesthetized
intraperitoneally and placed on an artificial ventilator after
endotracheal intubation; the left posterior lateral side of the
anesthetized rat was excised; the thorax was opened at the fourth
intercostal space; the lung-pleura region was excised approximately
3 cm; and the air leakage was confirmed with a Minute Volume of 400
cc. The air leakage site was then covered with a double-layered
cell sheet, the artificial ventilator was restarted after 5 minutes
of respiratory arrest, the Minute Volume was increased 100 cc every
5 minutes to a maximum of 1000 cc, and the degree of adhesion of
the cell sheet, and the presence or absence of air leakage were
evaluated.
[0059] The results showed that the above-mentioned cultured cell
sheet was attached to the air leakage site, was elongated and
contracted in agreement with the artificial ventilator, and closed
the air leakage site. The appearance is shown in FIGS. 2 to 4 (FIG.
2 shows the air leakage model, FIG. 3 shows the cultured cell sheet
attached to the air leakage site, and FIG. 4 shows air leakage
being closed by the cultured cell sheet, respectively). Moreover, a
histological evaluation was conducted by a hematoxylin-eosin
staining or an Azan staining of tissue sections of the tissue with
the closed air leakage site. The results obtained are shown in FIG.
5 (hematoxylin-eosin stain), and FIG. 6 (Azan stain).
[0060] From both figures, it was clear that the cultured cell sheet
used for covering the leakage site was in close contact with the
surface of the tissue around the air leakage site. Therefore, the
usefulness of the cultured cell sheet as a tissue repair material
could be confirmed.
Example 3
[0061] The study of this example is conducted in a similar manner
to that of Example 2, except that 250 .mu.M
.beta.-aminopropylnitrile was added to the culture medium, when
cell passage was started on the cell culture support material, 3
days after the start of culture in Example 2. The cultured cell
sheet obtained in the presence of .beta.-aminopropylnitrile was
mechanically flexible. The air leakage site was closed, and
contracture of the covered portion of the cultured cell sheet was
not found. Accordingly, the usefulness of a cultured cell sheet
having flexibility as a tissue repair material could be
confirmed.
Example 4
[0062] A lung tissue was extracted from a GFP-transgenic neonatal
rat, and the cells were isolated with collagenase, in a manner
similar to that of Example 2. Three days after the start of
culture, the cells were subcultured on the above described cell
culture support material, and the cell culture support was cooled
for 30 minutes at 20.degree. C. to harvest the cell sheet of the
fourth passage.
[0063] The harvested cell sheet was layered, and was applied to
closing the bleeding site of the liver. A model for a bleeding site
of the liver was prepared as follows: an 8 week old F-344 nude rat
was anesthetized intraperitoneally and placed on an artificial
ventilator after endotracheal intubation; the abdominal area of the
anesthetized rat was excised; the surface layer of the liver was
excised approximately 2 mm; and bleeding was confirmed. The site of
bleeding was then covered with a double-layered cultured cell
sheet, and the degree of adhesion of the cell sheet, and the
presence or absence of bleeding were evaluated.
[0064] The results showed that the above-mentioned cultured cell
sheet was attached to the bleeding site, and that the bleeding site
was closed. The appearance is shown in FIGS. 7 and 8 (FIG. 7 shows
the bleeding model, and FIG. 8 shows the cultured cell sheet
attached to the bleeding site, respectively). Moreover, a
histological evaluation was conducted by a hematoxylin-eosin
staining or an Azan staining of tissue sections of the tissue with
the closed bleeding site of the liver four weeks after
transplantation for closing the bleeding site. The results obtained
are shown in FIG. 9 (hematoxylin-eosin stain), and FIG. 10 (Azan
stain).
[0065] From both figures, it was clear that the cultured cell sheet
used for covering the bleeding site was in close contact with the
surface of the tissue around the bleeding site. Therefore, the
usefulness of the cultured cell sheet as a tissue repair material
could be confirmed.
Example 5
[0066] A lung tissue was extracted from a GFP-transgenic neonatal
rat, and the cells were isolated with collagenase, in a manner
similar to that of Example 1. Three days after the start of
culture, the cells were subcultured on the above-described cell
culture support material, and the cell culture support was cooled
for 30 minutes at 20.degree. C. to harvest the cell sheet of the
fourth passage.
[0067] The harvested cell sheet was layered, and was applied to
closing the bleeding site of the blood vessel. A model for a
bleeding site of a blood vessel was prepared as follows; an 8 week
old F-344 nude rat was anesthetized intraperitoneally and placed on
an artificial ventilator after endotracheal intubation; the
abdominal area of the anesthetized rat was excised; a blood vessel
was excised with a suture needle; and bleeding was confirmed. The
site of bleeding was then covered with a double-layered cultured
cell sheet, and the degree of adhesion of the cell sheet, and the
presence or absence of bleeding were evaluated.
[0068] The results showed that the above-mentioned cultured cell
sheet was attached to the bleeding site of the blood vessel, and
that the bleeding from the site was stopped. The appearance is
shown in FIGS. 11 and 12 (FIG. 11 shows the bleeding model, and
FIG. 12 shows the cultured cell sheet attached to the bleeding
site, respectively).
[0069] From both figures, it was clear that the cultured cell sheet
used for covering the bleeding site was in close contact with the
surface of the tissue around the bleeding site. Therefore, the
usefulness of the cultured cell sheet as a tissue repair material
could be confirmed.
INDUSTRIAL APPLICABILITY
[0070] The highly adhesive cultured cell sheet obtained by the
present invention has extremely high adhesiveness to the leakage
site of the surface the surface of an organ, and an excellent
flexibility. The use of the cell sheet of the present invention
enables suppression of air leakage, blood leakage or bodily fluid
leakage from the surface of an organ. Therefore, the present
invention is very promising in its clinical applications to air
leakage from alveolar tissue or bleeding from blood vessel tissue
or liver tissue, etc. Consequently, the present invention is
extremely useful in biological and medical fields, etc., such as
cell engineering and medical engineering.
* * * * *