U.S. patent application number 12/227688 was filed with the patent office on 2009-11-05 for method of culturing vascular smooth muscle cells, culture device and medical material obtained by the culture.
Invention is credited to Takashi Horiuchi, Keiichi Miyamoto.
Application Number | 20090274664 12/227688 |
Document ID | / |
Family ID | 38778493 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090274664 |
Kind Code |
A1 |
Miyamoto; Keiichi ; et
al. |
November 5, 2009 |
Method of culturing vascular smooth muscle cells, culture device
and medical material obtained by the culture
Abstract
There is provided a method for culturing vascular smooth muscle
cells while maintaining their normal function, and a culture device
and regenerative medical material for the same. The method takes
advantage of vascular smooth muscle cell recognition of elastin as
an extracellular matrix. The invention provides a method for
culturing vascular smooth muscle cells on elastin, a culture device
having elastin anchored on the cell-growing surface, a culture
device wherein the cell-growing surface is composed of an elastin
molded article, and medical materials obtained by culturing
vascular smooth muscle cells using such culture devices.
Inventors: |
Miyamoto; Keiichi; (Mie,
JP) ; Horiuchi; Takashi; (Mie, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
1030 15th Street, N.W.,, Suite 400 East
Washington
DC
20005-1503
US
|
Family ID: |
38778493 |
Appl. No.: |
12/227688 |
Filed: |
May 24, 2007 |
PCT Filed: |
May 24, 2007 |
PCT NO: |
PCT/JP2007/060604 |
371 Date: |
November 25, 2008 |
Current U.S.
Class: |
424/93.7 ;
435/395 |
Current CPC
Class: |
C12N 2533/50 20130101;
C12M 25/02 20130101; C12N 5/0691 20130101; A61L 27/227 20130101;
A61L 27/383 20130101; A61L 27/3826 20130101; A61K 35/12 20130101;
A61L 27/3895 20130101; A61L 27/507 20130101 |
Class at
Publication: |
424/93.7 ;
435/395 |
International
Class: |
A61K 35/34 20060101
A61K035/34; C12N 5/02 20060101 C12N005/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2006 |
JP |
2006-144909 |
Claims
1. A method of culturing vascular smooth muscle cells,
characterized by culturing vascular smooth muscle cells on
elastin.
2. The culturing method of claim 1, wherein the vascular smooth
muscle cells are cultured under static conditions in which the
elastin neither expands nor contracts, until the vascular smooth
muscle cells have become bound to the elastin, and then the
vascular smooth muscle cells are cultured under dynamic conditions
in which the elastin expands and contracts.
3. A method of culturing vascular smooth muscle cells by selective
growth of the vascular smooth muscle cells, characterized by
culturing a mixture of vascular smooth muscle cells and other
vascular cells on elastin.
4. A culture device for vascular smooth muscle cells, having
elastin anchored on a cell-growing surface.
5. A culture device for vascular smooth muscle cells according to
claim 4, wherein the elastin is anchored on a non-elastic material
surface.
6. A culture device for vascular smooth muscle cells according to
claim 4, wherein the elastin is anchored on an elastic material
surface.
7. A culture device for vascular smooth muscle cells according to
claim 6, wherein the elastic material is a silicon rubber
sheet.
8. A culture device for vascular smooth muscle cells, wherein the
cell-growing surface is composed of a molded crosslinked elastin
article.
9. A cell culture device for vascular smooth muscle cells according
to claim 4, wherein the cell-growing surface has a visible light or
ultraviolet light transmittance of 50-100%.
10. A medical material obtained by culturing vascular smooth muscle
cells using a culture device for vascular smooth muscle cells that
has elastin anchored on a cell-growing surface.
11. A medical material obtained by culturing vascular smooth muscle
cells on a molded crosslinked elastin article obtained by
crosslinking water-soluble elastin.
12. A cell culture device for vascular smooth muscle cells
according to claim 5, wherein the cell-growing surface has a
visible light or ultraviolet light transmittance of 50-100%.
13. A cell culture device for vascular smooth muscle cells
according to claim 6, wherein the cell-growing surface has a
visible light or ultraviolet light transmittance of 50-100%.
14. A cell culture device for vascular smooth muscle cells
according to claim 7, wherein the cell-growing surface has a
visible light or ultraviolet light transmittance of 50-100%.
15. A cell culture device for vascular smooth muscle cells
according to claim 8, wherein the cell-growing surface has a
visible light or ultraviolet light transmittance of 50-100%.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of culturing
vascular smooth muscle cells, to a culture device therefor and to a
medical material obtained by the culture.
BACKGROUND ART
[0002] Vascular graft replacement has come to be performed in
recent years as surgical treatment for trauma and vascular diseases
such as atherosclerosis, but artificial blood vessels have not been
realized for small blood vessels with diameters of 5 mm and less.
The reason is that obstruction due to thickening of the tunica
intima of vascular grafts or thrombus formation cannot be
prevented, because of the poor biocompatibility of artificial
materials. The major bypass or vascular replacement technique
currently used for elastic arteries such as coronary artery and
lower leg peripheral arteries is autologous vein grafting. However,
approximately 20-30% of all patients requiring vascular replacement
do not have suitable blood vessels and are not candidates for
autologous vein grafting.
[0003] In recent years, it has been attempted to coat extracellular
matrixes such as collagen onto the lumen surfaces of artificial
vessels made of synthetic fibers worked into small diameters
(Dacron/polyurethane/drawn polytetrafluoroethylene), or onto the
lumen surfaces of tubular structures formed from bioabsorbable
synthetic polymers (for example, polyglycolic acid, polylactic
acid, poly .epsilon.-caprolactam and the like), in order to provide
them with a cell adhesion property, and to culture endothelial
cells on those extracellular matrixes. However, small-diameter
artificial vessels have not been realized because the endothelial
cells do not become stably anchored to the extracellular matrix, or
the artificial vessels undergo intimal thickening.
[0004] The blood vessel wall damage that occurs due to endothelial
thickening causes smooth muscle cells near the endothelium to
undergo a phenotypic change from the normal contractile to
synthetic type, thus promoting growth and organization and
extending the lesion. Numerous methods of culturing vascular smooth
muscle cells have been investigated, as basic research on vascular
smooth muscle. Examples thereof include a method of culturing
solution containing insulin-like growth factor-I on a
laminin-coated culture dish (for example, Patent document 1), and a
method of fixing smooth muscle cells onto fibronectin-coated glass
and applying shear flow stress for culturing, as a method that
takes into account the orientation of the smooth muscle cells (for
example, Patent document 2).
[0005] The present inventors have also developed a crosslinked
elastin article having water-soluble elastin crosslinked with a
water-soluble crosslinking agent, which can be applied as a
biomaterial for regenerative medicine (Patent document 3). In this
method, the crosslinked elastin article is utilized as a culture
substrate for regenerative medicine, and embryonic stem (ES) cells,
somatic stem cells, mesenchymal stem cells or the like are cultured
on a film surface or tube interior made therefrom, to form organs
having desired shapes. Culturing of neuroblastoma cells has been
carried out using elastin films produced from crosslinked elastin
articles, but no biomaterial has been disclosed with special
affinity between elastin and vascular smooth muscle cells.
[0006] Since most cells in the body are present at sites that are
subject to mechanical forces, dynamic culturing methods are also
known whereby the cell-growing surface is expanded and contracted
in order to accomplish cell culturing with growth under conditions
similar to in vivo conditions (Non-patent document 1).
[Patent document 1] Japanese Unexamined Patent Publication No.
2002-335955 [Patent document 2] Japanese Patent Public Inspection
No. 2002-531118 [Patent document 3] WO 02/096978 [Non-patent
document 1] K. Naruse, T. Yamada, M. Sokabe, Am. J. Physiol. 274:
H1532-1538 (1998) Involvement of SA channels in orienting response
of cultured endothelial cells to cyclic stretch
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0007] A very strong demand exists for culturing methods and
culture devices for vascular smooth muscle cells, to be used in
basic research toward elucidation of the mechanism of endothelial
thickening associated with vascular smooth muscle cells and
observation of the reaction of vascular smooth muscle cells to
drugs, and in the development of vascular grafts. It is an object
of the present invention to provide a culturing method which
facilitates growth of vascular smooth muscle cells, for example,
and particularly a method and device for culturing of vascular
smooth muscle cells while maintaining the normal types
(differentiated types) of the vascular smooth muscle cells.
Means for Solving the Problems
[0008] The present invention is an application of the knowledge
that vascular smooth muscle cells grow on elastin as a scaffold.
The invention therefore relates to a method of culturing vascular
smooth muscle cells characterized by culturing vascular smooth
muscle cells on elastin. Vascular smooth muscle cells are subjected
to mechanical stress in the body. In order to obtain cells with
properties similar to in vivo vascular smooth muscle cells,
therefore, it is preferred for the culturing to be carried out
under dynamic conditions similar to in vivo conditions. Growth of
vascular smooth muscle cells, however, requires initial growth
under static conditions until the cells have become bound to the
growing surface. According to a preferred mode of the culturing
method, therefore, the vascular smooth muscle cells are cultured
under static conditions in which the elastin neither expands nor
contracts, until the vascular smooth muscle cells have become bound
to the elastin, and then the vascular smooth muscle cells are
cultured under dynamic conditions in which the elastin expands and
contracts.
[0009] The invention also incorporates the knowledge that vascular
smooth muscle cells grow more specifically on elastin, compared to
other vascular cells. The invention therefore further relates to a
method of culturing vascular smooth muscle cells by selective
growth of the vascular smooth muscle cells, characterized by
culturing a mixture of vascular smooth muscle cells and other
vascular cells on elastin.
[0010] The invention still further relates to a culture device for
vascular smooth muscle cells, having elastin anchored on a
cell-growing surface. One mode thereof is a culture device for
vascular smooth muscle cells wherein the elastin is anchored on a
non-elastic material surface. Another mode thereof is a culture
device for vascular smooth muscle cells wherein the elastin is
anchored on an elastic material surface. Preferred is a culture
device for vascular smooth muscle cells wherein the elastin is
anchored onto a silicon rubber sheet. Since such culture devices
are usually placed under microscope observation, the transmittance
of the cell-growing surface for visible light or ultraviolet light
is preferably 50-100%.
[0011] The invention still further relates to a culture device for
vascular smooth muscle cells, wherein the cell-growing surface is
composed of a molded crosslinked elastin article. The transmittance
of the cell-growing surface of the culture device for visible light
or ultraviolet light is preferably 50-100%.
[0012] The invention yet further relates to a medical material
obtained by culturing vascular smooth muscle cells using a culture
device for vascular smooth muscle cells that has elastin anchored
on a cell-growing surface, and to a medical material obtained by
culturing vascular smooth muscle cells on a molded crosslinked
elastin article obtained by crosslinking water-soluble elastin.
EFFECT OF THE INVENTION
[0013] By culturing vascular smooth muscle cells on elastin, and
especially by using a culture device having elastin anchored on the
cell growth surface for the culturing, it is possible to accomplish
selective culturing of vascular smooth muscle cells, and to
accomplish the cell culturing while maintaining the normal type
(differentiated type) of the vascular smooth muscle cells. In
addition, it is possible to produce a medical material by culturing
the vascular smooth muscle cells on the elastin-anchored culture
device or molded crosslinked elastin article.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a bar graph showing the cytotoxicity test results
for water-soluble elastin using MTT. The water-soluble elastin was
judged to have no cytotoxicity.
[0015] FIG. 2 is a bar graph showing the cytotoxicity test results
for a crosslinking agent (DODE-DSP) using MTT. The crosslinking
agent (DODE-DSP) was judged to have no cytotoxicity.
[0016] FIG. 3 is a bar graph showing the cytotoxicity test results
for active ester group (DSP) using MTT. The active ester group
(DSP) was judged to have no cytotoxicity.
[0017] FIG. 4 is a graph showing the results of measuring the
coacervation temperature (elastin concentration: 0.1-70 wt %). No
coacervation temperature is exhibited at 60% and greater because of
the non-agglutinating liquid.
[0018] FIG. 5 is a bar graph showing the results of measuring the
water droplet contact angle on the surface of a sample subjected
only to corona discharge (Control), a sample subjected to corona
discharge and then elastin treatment (Elastin) and a sample
subjected to corona discharge and then collagen treatment
(Collagen), in a polystyrene culturing dish. (**P<0.0001 vs.
control)
[0019] FIG. 6 is a bar graph showing the results of measuring the
water droplet contact angle on the surface of a sample subjected
only to corona discharge (Control), a sample subjected to corona
discharge and then elastin treatment (Elastin) and a sample
subjected to corona discharge and then collagen treatment
(Collagen), in a culturing dish with a silicon sheet bottom.
[0020] FIG. 7 is a set of graphs showing the results of measuring
the adhesion and distensibility of vascular cells on a
surface-treated polystyrene culturing dish. It shows the a)
adhesion and b) distensibility of vascular smooth muscle cells, the
c) adhesion and d) distensibility of fibroblasts and the e)
adhesion and f) distensibility of vascular arterial endothelial
cells. .largecircle. represents samples subjected to corona
discharge and then collagen treatment, .box-solid. represents
samples subjected to corona discharge and then elastin treatment,
and X represents samples subjected only to corona discharge.
[0021] FIG. 8 is a bar graph showing the results of measuring the
change in orientation angle (0-4 hour) during static culturing.
[0022] FIG. 9 is a bar graph showing the results of measuring the
change in orientation angle (0-4 hour) during dynamic culturing.
The stretch conditions were a cycle of 0.5 Hz and a distensibility
of 10%.
[0023] FIG. 10 is a bar graph showing the results of assaying
.alpha.-actin and .beta.-actin in smooth muscle cells cultured on
an elastin-coated dish.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] Arterial vascular walls have a three-layer structure
comprising an intima, media and adventitia, and it is a muscular
tissue structure with smooth muscle cells as the major components.
The intima which directly contacts with the blood has a monolayer
squamous epithelium of endothelial cells and, directly under it, a
subendothelial layer consisting of a basement membrane, loose
connective tissue and a small number of longitudinal smooth muscle
cells. The endothelial cell layer covers the vascular lumen, and is
oriented in such a manner the cell long axes are parallel to the
direction of blood flow. The media is the blood vessel wall and the
thickest layer, and the concentric cell layers composing it are
made of smooth muscle cells oriented parallel to the pulsating
direction. The smooth muscle cells perform the important function
of maintaining blood pressure and controlling the distribution of
blood flow. Between the unstriated muscle layers there are found
elastin fibers, type III collagen and proteoglycans, confering
arteries with the additional property of contracting during cardiac
systole due to the abundant elastin in the arterial walls. Between
the intima and media lies a structure composed of a fenestrated
homogeneous elastin layer, known as the internal elastic lamina.
The adventitia is composed mainly of fibroblasts, collagen fibers
and longitudinal elastic fibers, and a fenestrated homogeneous
elastin layer known as the external elastic lamina is likewise
present between the media and adventitia.
[0025] Thus, the vascular smooth muscle is in an intimate
relationship with elastin layers, and therefore when vascular
smooth muscle cells are cultured with a culture device comprising
an elastin layer, the elastin acts as a scaffold for the vascular
smooth muscle cells. In addition, since elastin forms tissue that
surrounds vascular smooth muscle cells in the body, it is
conjectured that the binding of the vascular smooth muscle cells to
elastin in the vascular wall media structure and their presence in
the elastin matrix while being subjected to mechanical stimulation
in the body, are the factors responsible for the normal type of
smooth muscle cells. Consequently, culturing of vascular smooth
muscle cells on elastin can be accomplished under conditions
similar to in vivo tissue, thus suitably maintaining the normal
type (differentiated type).
[0026] The first aspect of the invention relates to a method of
culturing vascular smooth muscle cells, characterized by culturing
vascular smooth muscle cells on elastin.
[0027] One mode thereof comprises culturing vascular smooth muscle
cells with a culture device having elastin anchored on a
cell-growing surface. Another mode employs a molded crosslinked
elastin article as the cell-growing surface.
[0028] Any elastin may be used for the invention, including
water-soluble elastin, water-insoluble elastin and crosslinked
elastin obtained by crosslinking water-soluble elastin with a
crosslinking agent. According to the invention, water-soluble
elastin and crosslinked elastin according to the invention are
preferable for ease of use.
[0029] Cell culturing methods include dynamic culturing and static
culturing. Dynamic culturing involves culturing while contracting
and expanding the surface on which the cells grow. Dynamic
culturing according to the invention is therefore a method of
culturing wherein elastin is anchored onto the cell-growing
surface, i.e. the surface on which the culture solution is placed,
and the surface is repeatedly contracted and expanded. Most cells
in the body are located in areas subject to dynamic forces, and
function while being exposed to dynamic environmental changes. In
the case of blood vessels, shear flow stress is produced in the
endothelial cells composing the vascular wall, and periodic tensile
load is exerted on the vascular smooth muscle cells and fibroblasts
that do not contact with the blood flow. Culturing under conditions
similar to vascular smooth muscle cells in the body is dynamic
culturing. Dynamic culturing with vascular smooth muscle cells
placed under mechanical stress is effective for observing whether
dedifferentiation occurs, or for observing the phenomenon of
cellular orientation.
[0030] Static culturing is a common culturing method with the
cell-growing surface in a static state, i.e. without expansion or
contraction. This culturing method is carried out under conditions
that are different from the in vivo environment, but it is still
useful as a method for examining the basic properties of vascular
smooth muscle cells. However, ordinary static culturing is known to
cause vascular smooth muscle cells to dedifferentiate to the
synthetic form, and to promote proliferation. Since cells in this
state are not the normal in vivo type, it must be kept in mind that
false conclusions can be drawn when they are utilized for drug
screening or as a cell culture model for tissue regeneration.
[0031] During culturing of vascular smooth muscle cells, the cells
proliferation after attaching to the bottom of the culture device.
Even with dynamic culturing, static culturing must be carried out
until the vascular smooth muscle cells attach to the bottom of the
culture device. Therefore, the most advantageous culturing method
for vascular smooth muscle cells according to the invention is a
method in which the vascular smooth muscle cells are statically
cultured until they bind to elastin, and then dynamically cultured
by expanding and contracting the cell-growing surface to which the
elastin is anchored.
[0032] The culturing temperature and carbon dioxide gas
concentration for static culturing and dynamic culturing may be
according to ordinary conditions, preferably with a temperature of
approximately 37.degree. C. and a carbon dioxide gas concentration
of approximately 5%. A standard frequency for expansion and
contraction for dynamic culturing is 0.5 Hz, i.e. one cycle every 2
seconds, but this frequency may be higher or lower. The expansion
and contraction may be unaxial stretch in a single direction or
biaxial stretch in two directions. The degree of expansion and
contraction is generally about 5-50% of the length of the
cell-growing surface, but this may be varied depending on the needs
of the study. The degree of expansion and contraction is determined
in relation to its frequency.
[0033] The second aspect of the invention is to provide a method of
selectively culturing vascular smooth muscle cells, characterized
by culturing a mixture of vascular smooth muscle cells and other
vascular cells (vascular endothelial cells, vascular smooth muscle
cells, fibroblasts, hemocytes, macrophages or the like) on elastin.
Using elastin as the surface substance allows specific growth of
vascular smooth muscle cells, as opposed to most of the major
extracellular matrix substances such as collagen, fibronectin and
laminin. It is believed that only the smooth muscle cells recognize
elastin among the vascular cells. On the other hand, all cell types
bind and distend on collagen without specificity. Utilizing this
property, a mixture of vascular smooth muscle cells with other
vascular cells on elastin can accomplish growth of the vascular
smooth muscle cells alone. Presumably, elastin-recognizing
receptors on the vascular smooth muscle cell surfaces play an
important role in this growth.
[0034] The third aspect of the invention is to provide a culture
device for vascular smooth muscle cells by the culturing method
described above. As a feature of the culture device of the
invention, elastin is used on the surface on which the vascular
smooth muscle cells are to be grown (the surface on which the
culture solution is placed). The elastin anchored to the
cell-growing surface acts through cell surface receptors to
participate in extracellular matrix recognition in signal
transduction of vascular smooth muscle cell activity, thus
providing a very favorable environment for growth of the vascular
smooth muscle cells.
[0035] The material of the cell-growing surface on the culture
device may be elastin anchored on a plastic or silicon rubber
sheet, or an elastin molded article produced by crosslinking of
water-soluble elastin.
[0036] For static culturing alone, a non-elastic plastic material
such as polystyrene, polycarbonate, polypropylene or
polyacrylonitrile may be employed. For dynamic culturing, any
material that can withstand repeated expansion and contraction may
be employed, such as silicon rubber, natural rubber, acrylic
rubber, polyurethane rubber or the like. An elastin molded article
produced by crosslinking water-soluble elastin is elastic and can
therefore be used for dynamic culturing. Materials that can be used
for dynamic culturing can, of course, also be used for static
culturing.
[0037] For basic research on vascular smooth muscle cells carried
out under microscope observation, it is preferred for the material
of the cell-growing surface to be transparent. The transmittance of
the cell-growing surface for visible light or ultraviolet light is
most preferably 50-100%. In some cases, however, the material will
not need to be transparent, and the material may therefore be
selected without regard to transparency.
[0038] The water-soluble elastin used for the invention can be
obtained by hydrolysis of elastin, and specifically, it may be one
or more types of elastin such as a-elastin or .beta.-elastin
obtained by hot oxalic acid treatment of animal cervical ligaments,
K-elastin obtained by alkali ethanol treatment of elastin,
water-soluble elastin obtained by enzyme treatment with elastase,
or tropoelastin as a precursor of the elastin biosynthetic pathway.
Water-insoluble elastin is commercially available, and it may be
subjected to hot oxalic acid treatment to obtain water-soluble
elastin.
[0039] For anchoring of the elastin onto the culture device, the
cell-growing surface, i.e. the surface on which the culture
solution is to be placed, may be chemically or physically
activated, a water-soluble elastin solution placed thereover and
the activated surface allowed to react with the water-soluble
elastin for a fixed period of time, and then the excess elastin
solution discarded and the surface dried.
[0040] The anchoring reaction may be based on a commonly known
chemical reaction (hydrogen bonding, ionic bonding, covalent
bonding), or a radical bonding reaction.
[0041] The method of activating the surface of the culture device
may be any method that generates highly reactive functional groups
(amino, aldehyde, epoxy, carboxyl, hydroxyl, thiol groups, etc.) or
radicals. For example, the inactive surface may be treated by
carbonylation or carboxyl group introduction with a chemical agent
such as chromic acid, sulfuric acid or hypochlorous acid, or by
coating with a silane coupling agent containing functional groups,
for activation of the culture substrate surface. As examples of
silane coupling agents there may be mentioned
.gamma.-aminopropyltriethoxysilane,
N-.beta.-(aminoethyl)-.gamma.-aminopropyltrimethoxysilane and
N-.beta.-(aminoethyl)-.gamma.-aminopropylmethyldimethoxysilane.
There may also be employed a method of polymer coating, such as the
method of covering the inactive surface with, for example,
polyacrylic acid containing added azide groups to introduce active
groups, as described in Japanese Unexamined Patent Publication No.
2003-189843. This allows anchoring by photoreaction to be
accomplished by pre-azidation of the elastin to be anchored. It is
also effective to accomplish anchoring by the surface activation
method via introduction of functional groups (mainly amine groups)
using ammonia plasma as described by Miyamoto et al. in Int. Biol.
Macromol., 30, 75-80 (2002). The water-soluble elastin is
preferably reacted immediately after activation of the inactive
surface by corona discharge treatment. For corona discharge
treatment, the material with the inactive surface is placed on a
glass base, and the tip of the corona flame blow-out point is
brought to a position about 5-10 mm distant, and the corona flame
is discharged onto the material surface (approximately 9 kV) for
about 10-40 seconds. Within several hours, active radicals
disappear from the surface that has been activated by corona
discharge at the activation sites other than the locations where
the water-soluble elastin has been anchored, thus restoring the
inactive surface. This prevents anchoring of other substances
present in the culture solution during growth of the cells, in
order to facilitate a continuous effect of the elastin-anchored
surface.
[0042] Since the water-soluble elastin is anchored by reaction with
the surface-treated material as described above, it does not elute
into the culture solution when subsequently contacted with the
culture solution.
[0043] Another preferred mode of the culture device of the
invention is a culture device for vascular smooth muscle cells
employing a molded crosslinked elastin article having water-soluble
elastin crosslinked on the cell-growing surface (bottom). A
crosslinked elastin article having water-soluble elastin
crosslinked with a crosslinking agent can be produced by the method
described in WO 02/096978. The molded crosslinked elastin article
produced by this process is contractile and porous (spongy). The
crosslinked elastin article can then be shaped into a transparent
sheet. The culture device having such a material as the
cell-growing surface is suitable for static culturing and dynamic
culturing of vascular smooth muscle cells. By forming it to an
elastin concentration of at least 50% as the weight ratio, it is
possible to inhibit the coacervation phenomenon that is a
characteristic property of water-soluble elastin, and thus obtain a
gel sheet with high optical transparency. Since the elastin molded
article is porous, the three-dimensional culturing condition of the
vascular smooth muscle cells can be observed under a
microscope.
[0044] A preferred crosslinking agent for the water-soluble elastin
is one represented by the following general formula.
##STR00001##
[0045] In the formula,
R.sub.1 and R.sub.3 are <A> or <B> represented by the
following structural formula, and R.sub.1 and R.sub.3 may be either
the same or different,
##STR00002##
(wherein R.sub.4 and R.sub.5 are H, CH.sub.3 or C.sub.2H.sub.5, and
R.sub.4 and R.sub.5 may be either the same or different)
##STR00003##
(wherein R.sub.2 is a compound <C> or <D> represented
by the following structural formula)
##STR00004##
(wherein n is an integer of 1-20)
##STR00005##
(wherein m and 1 are integers of 0-15, X and Y are CH.sub.2 or 0, X
and Y being either the same or different, Z is C or N, and R.sub.6,
R.sub.7, R.sub.8 and R.sub.9 are H, CH.sub.3 or C.sub.2H.sub.5, and
may be either the same or different).
[0046] The apparatus used for dynamic culturing is made of an
elastic material capable of expanding and contracting the entire
culture device containing the culture solution. It employs an
elastic material having elastin attached to the cell-growing
surface, or an elastic elastin molded article composed of
crosslinked elastin. Any desired shape may be used. When expansion
and contraction are applied uniaxially, a quadrilateral shape is
most advantageous. A hexagonal shape may be employed to obtain
non-uniformity for the directions of expansion and contraction. A
quadrilateral culture device is produced by forming a rectangular
box-like enclosure using a relatively thick material that can
withstand repeated expansion and contraction, such as a thick
natural rubber or silicon rubber sheet. According to one mode, a
thin silicon rubber sheet is attached to the enclosure with an
adhesive, and then the cell-growing surface is activated by corona
discharge or the like to anchor the elastin. According to another
mode, a crosslinked elastin sheet is attached to the bottom of the
enclosure. For expansion and contraction of the entire culture
device, a tensile tool is loaded at the opposing enclosure
sections.
[0047] The apparatus for dynamic culturing may be cylindrical with
stopped ends. A hole is formed at one of the stopped ends, a tool
that applies expansive force and contractive force is fitted near
the opening, and the culture solution is added through the opening
for rotated culturing, to allow growth of the vascular smooth
muscle cells only inside the cylindrical section. When cylindrical
silicon rubber is used, only the inner surface is surface treated
and elastin is anchored thereto. Alternatively, a cylinder may be
formed with crosslinked elastin and the vascular smooth muscle
cells grown on the inner surface.
[0048] A commercially available apparatus such as one by Strex,
Inc., may be used as a dynamic culturing apparatus that expands and
contracts the cell-growing surface. There may also be used a
combination of apparatuses employing a linear actuator and linear
slider.
[0049] The fourth aspect of the invention is a medical material
obtained by culturing vascular smooth muscle cells on elastin. One
mode thereof is obtained by culturing vascular smooth muscle cells
using a culture device having elastin anchored on a cell-growing
surface. For example, only the inner surface of a silicon rubber
tube is activated by a chemical method and elastin is anchored
thereto, and the tube is rotated in the culture solution while
repeating expansion and contraction, so that only the inner surface
becomes coated with vascular smooth muscle cells and can be used as
a vascular graft.
[0050] Another mode is a medical material obtained by culturing
vascular smooth muscle cells on a molded crosslinked elastin
article obtained by crosslinking water-soluble elastin. A sheet is
formed of elastin gel comprising crosslinked water-soluble elastin,
and is attached to the bottom of a rectangular box-like enclosure
formed of, for example, natural rubber or silicon rubber, to
construct box-shaped culture device. After culturing the vascular
smooth muscle cells, the section on which the vascular smooth
muscle cells have grown may be cut out and directly applied as a
material for in vivo implantation. As another example of using
molded crosslinked elastin, a tube of approximately the size of a
small-diameter artery may be formed of crosslinked elastin, and
vascular smooth muscular tissue uniformly cultured and grown on the
inner surface thereof, to produce a tissue engineering graft as a
substitute small-diameter artery. A wide variety of uses are
possible for biomedical materials obtained by culturing and growing
vascular smooth muscle cells on elastin. A material obtained by a
method of culturing vascular smooth muscle cells using crosslinked
elastin according to the invention can be utilized for treatment of
patients that are normally not candidates for vascular replacement
techniques.
EXAMPLES
[0051] The present invention will now be explained in greater
detail by examples, but the methods described hereunder were
employed merely to confirm the effect and are not intended to be
restrictive, while they may also be carried out with various
modifications so long as the gist is maintained.
Production Example 1
Production of Water-Soluble Elastin
[0052] Defatted porcine aorta was washed with water and then
immersed in 5-10 wt % aqueous sodium chloride and allowed to stand
for 24 hours in a refrigerator, for dissolution and removal of the
collagen. After washing, water was added and the mixture was boiled
for 1 hour with a pressure cooker, after which the vascular
accretions were removed and the mixture was placed in a mixer for
crushing to a tissue fragment size of 1-5 mm. It was then heated
for 1 hour in the pressure cooker and washed with water. After
transfer to a meshed bag and washing with an electric washer,
ethanol was added to a final concentration of 50 vol % and the
mixture was allowed to stand for 30 minutes. Ethanol was then added
to a final concentration of 70 vol % and the mixture was allowed to
stand for 90 minutes. Ethanol was then further added to a final
concentration of 90 vol % and the mixture was allowed to stand for
10 hours. Finally, the ethanol was removed and the product was
dried under reduced pressure in a desiccator to obtain insoluble
elastin (yield: approximately 330 g by dry weight from 2 kg of
blood vessels). After adding 45 mL of 0.25 M oxalic acid to 10 g of
the insoluble elastin, the mixture was heated at 100.degree. C. for
1 hour. This was cooled with ice water and subjected to centrifugal
separation, and the supernatant was collected and dialyzed against
deionized water in a dialysis tube with a molecular weight of
10,000-14,000 to an external solution pH of about 5-6 for removal
of the oxalic acid. The supernatant was subjected to suction
filtration with a glass filter, and the filtrate was lyophilized to
obtain water-soluble elastin (approximately 3 g).
Production Example 2
Production of Crosslinking Agent
[0053] A chemical crosslinking agent was prepared by the method
described in WO 02/096978, for production of an elastin structure
using water-soluble elastin. The method involves active
esterification of the carboxyl groups of dodecanedicarboxylic acid
with 4-hydroxyphenyldimethyl-sulfonium methyl sulfate (hereunder,
DSP). Specifically, 1 mol of dodecanedicarboxylic acid, 2 mol of
DSP and 2 mol of dicyclohexylcarbodiimide (hereunder, DCCD) are
dissolved in acetonitrile and the mixture is stirred at 25.degree.
C. for 5 hours for reaction. The dicyclohexylurea produced during
the reaction process was removed with a glass filter. The reaction
mixture (filtrate) was then solidified by dropwise addition into
ether. The solid product was dried under reduced pressure to obtain
the target crosslinking agent. The chemical structure and purity
was confirmed by .sup.1H-NMR.
Test Example 1
Cytotoxicity Test Using Water-Soluble Elastin and Crosslinking
Agent
[0054] The MTT method, which is based on the principle that viable
cells convert
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(hereunder, MTT) to a formazan product, was used as a toxicity test
for the water-soluble elastin and crosslinking agent with respect
to vascular smooth muscle cells. Since tetrazolium salts of MTT and
the like can serve as substrates of dehydrogenase in the TCA cycle,
which is an oxidative phosphorylation reaction in mitochondria,
they allow measurement of the inhibition of sample substances on
cell activity. A Cell Proliferation Kit I (Roche) was used for the
measurement.
[0055] As the specific measuring method, CSC culture medium
(vascular smooth muscle cell culturing medium: product of Dainippon
Pharmaceutical Co., Ltd.) containing 10 vol % fetal bovine serum
was added to a 96 well cell culture plate at 100 .mu.L/well and
incubated, and then a smooth muscle cell suspension (cell density:
1.5.times.10.sup.5 cells/mL) was seeded at 10 .mu.L/well and
culturing was carried out at 37.degree. C. in a 5 vol % CO.sub.2
incubator. Upon reaching subconfluence, the medium was aspirated,
100 .mu.L of CSC medium containing 0.1 vol % fetal bovine serum was
added, and culturing was continued for 24 hours in the incubator
until cell growth ceased. After rinsing with PBS, the water-soluble
elastin aqueous solution (concentration: 0.1-5 wt %) produced in
Production Example 1, the DSP activated dodecanedicarboxylic acid
crosslinking agent (50-500 .mu.M) produced in Production Example 2
and the DSP (0.05-20 mM) used in Production Example 2 were added at
100 .mu.L/well each, and culturing was continued for 24 hours in
the incubator. The sample solution was then aspirated and rinsed
with PBS, CSC medium containing 10 vol % fetal bovine serum was
added to 100 .mu.L/well, a specified amount of MTT solution was
added prior to stationing overnight in the incubator, and a
microplate reader was used for measurement of the absorbance at a
measuring wavelength of 550 nm and a reference wavelength of 655
nm. A control was prepared without addition of sample and compared
with the measured values. The results are shown in FIG. 1, FIG. 2
and FIG. 3. The water-soluble elastin, crosslinking agent and
activeester group portion (DSP) were all essentially unchanged with
respect to the control within the measured concentration range, or
in other words, they had no effect on cell activation and exhibited
no toxicity.
Test Example 2
Measurement of Coacervation Temperature of Water-Soluble
Elastin
[0056] Water-soluble elastin exhibited a heat-sensitive phenomenon
of hydrophobic interaction and aggregation within the molecules or
between molecules with increasing solution temperature. This is
referred to as "coacervation", and is known as an important
phenomenon in water-soluble elastin. The coacervation temperature
can be determined by measuring the solution turbidity.
Specifically, a spectrophotometer with a temperature-variable
device was used for irradiation of the sample solution with a
specific wavelength (640 nm), and the proportion of transmitted
light intensity with respect to incident light intensity was
calculated as the transmittance. The transmittance was measured
while varying the temperature from 10-80.degree. C., and the
temperature at which the transmittance reduction was 50% of the
total was defined as the coacervation temperature. The coacervation
temperature was measured while varying the elastin concentration
from 0.1-70 wt %. The results are shown in FIG. 4. It was confirmed
that the coacervation temperature increased with increasing
water-soluble elastin concentration, with no aggregation at 60 wt %
and higher. These results demonstrated that with a water-soluble
elastin concentration of 60 wt % or greater, the water-soluble
elastin crosslinked at a temperature of 100.degree. C. or above to
obtain a transparent molded article.
Example 1
Preparation of Elastin-Coated Dish for Static Cell Culturing
[0057] The following procedure was followed under aseptic
conditions on a clean bench. The surface of a 35.phi. dish for
suspension culture (product of Sumitomo Bakelite Co., Ltd.,
polystyrene material) was subjected to discharge (9 kV) treatment
for 45 seconds with the corona flame blow-out hole of a corona
discharge apparatus (CoronaFit: product of Shinko Electric &
Instrumentation Co., Ltd.) kept at a distance of 10 mm from the
culturing dish surface, and then 1 mL of a 100 .mu.g/mL
water-soluble elastin solution was allowed to stand therein for 8
hours. The elastin solution was then discarded and the dish was
dried at room temperature for about 1 hour. This treatment results
in reaction and anchoring of elastin onto the dish surface that has
been activated by the corona discharge, so that it becomes coated
with elastin. As a control experiment there was also prepared a
collagen-coated dish. The coated state was evaluated by measuring
the water droplet contact angle on the surface. Specifically, a
35.phi. suspension culture dish was placed on slide glass situated
on the placement stage of a contact angle measuring instrument (by
ERMA Corp.), 20 .mu.L of deionized water was added with a
microburet, and the contact angle was measured after allowing it to
stand for several seconds. The average value of 5 contact angle
measurements was recorded as the measured value.
[0058] Elastin and collagen were anchored onto the surface by
activating the hydrophobic surface by corona discharge and reacting
it with protein in water. Restoration to the original
hydrophobicity in air was observed at approximately 8 hours after
discharge (contact angle: approximately 80.degree.). When water
alone was added immediately after discharge (control), a portion of
the water molecules slowly reacted during restoration to the
original hydrophobicity producing a hydrophilic state, and surface
change was stabilized at about 8 hours, with a contact angle of
about 42.degree.. With immersion in an elastin aqueous solution or
collagen aqueous solution immediately after discharge, the contact
angles were 25.degree. and 34.degree. respectively after 8 hours,
and thus a stabilized surface had been formed in a more hydrophilic
state than the control (FIG. 5). These results confirmed that
elastin and collagen had been coated onto the corona discharge
treated dish surface.
Example 2
Production of Dynamic Culture Device Using Elastin-Coated Silicon
Sheet
[0059] A 10 mm-thick silicon rubber sheet was prepared and used to
form a box-like enclosure for a culture dish with dimensions of 3
cm length.times.3 cm width.times.1 cm height. A 50 .mu.m-thick
silicon rubber sheet was attached to the bottom using Bathcoke
(HJ-132 by Cemedine Co., Ltd.), and this was allowed to stand for a
day for complete attachment. After thorough washing with deionized
water, autoclave treatment was carried out at 121.degree. C. for 30
minutes for sterilization. The same corona discharge apparatus of
Example 1 was used for 45 seconds of surface treatment, and after
adding 1 mL of a 100 .mu.g/mL water-soluble elastin solution and
collagen solution, the dish was allowed to stand for 8 hours. As a
control experiment there was also prepared a collagen-coated dish.
The coating was evaluated based on the measured contact angle (FIG.
6). Since a significant reduction in contact angle is also seen
with a silicon sheet as for the suspension culture dish, it was
confirmed that elastin and collagen had been coated.
Example 3
Production of Dynamic Culture Device Using Elastin Gel Sheet
[0060] After adding 240 .mu.L of a 340 mM aqueous solution of the
crosslinking agent produced in Production Example 2 to 600 mg of
water-soluble elastin, deionized water was further added to prepare
a solution with a final elastin concentration of 65 wt %. A
template was formed with a glass plate treated for water-repellency
on both sides, and the elastin solution was cast into a sheet form
and subjected to heat crosslinking at 100.degree. C. for 30 minutes
to obtain an optically transparent elastin gel sheet with a film
thickness of about 100 .mu.m. The conditions were such as to avoid
coacervation, and the transmittance of the elastin gel sheet for
visible light was about 60-90%, allowing observation under an
optical microscope. An enclosure for a culture dish with dimensions
of 3 cm length.times.3 cm width.times.1 cm height was produced
using a 10 mm-thick silicon rubber sheet, similar to the one
prepared in Example 2, and the elastin gel sheet was attached
thereto with an adhesive. After thorough washing with deionized
water, autoclave treatment was carried out at 121.degree. C. for 30
minutes for sterilization.
Example 4
Vascular Cell Culturing Adhesion/Stretch Test
[0061] Commercially available normal human vascular smooth muscle
cells, normal human fibroblasts and normal human vascular
endothelial cells were seeded at 1.0.times.10.sup.4 (cells/mL),
together with special serum-free medium, onto an elastin-coated
dish and a collagen-coated dish, as prepared in Example 1.
Culturing was carried out with a 5% CO.sub.2 culturing apparatus
(OLYMPUS MI-IBC) while inputting an image from an optical
microscope (OLYMPUS IMT-2) to personal computer XCAP software, with
a set number of photographs and photographing interval. The time at
seeding was defined as 0, and the photographed images were analyzed
with Adobe Photoshop. The cell adhesion and stretch of the attached
cells were evaluated based on analysis of the photographed images.
This the total number of adhering cells at each time point and the
number of distended cells were measured from the chronologically
photographed cell images, and values for the adhesion (%)=(number
of adhering cells/total number of cells).times.100 and the stretch
(%)=(number of distended cells/total number of cells).times.100
were calculated.
[0062] The adhesion was judged in the following manner. The cells
immediately after seeding are spherical, but generate desmosomes at
the first change. When the cells extend pseudopods to the matrix
and the surface receptor integrin binds to the matrix, integrin
aggregates to form desmosomes at the high density region points.
The shapes of the cells are essentially spherical. This was defined
as the attached state. As the second change, when all of the
integrin at the sections between the cells and matrix has become
attached, desmosomes form over the entire cell membrane such that
the cells undergo a change in shape to a non-spherical shape. This
is the distended state. The adhesion and stretch is evaluated based
on these two changes.
[0063] The results of determining the adhesion and stretch of each
cell type for the elastin- (or collagen)-coated dish are shown in
FIG. 7. The vascular smooth muscle cells were observed to exhibit
high adhesion (.gtoreq.63% after 1 hour) and stretch (.gtoreq.62%
after 4 hours) for the elastin surface and collagen surface. In
particular, it was found that adhesion and stretch were essentially
complete with about 1 hour and within 4 hours, respectively. With
fibroblasts, high adhesion (.gtoreq.80% after 1 hour) and stretch
(.gtoreq.80% after 4 hours) were confirmed with respect to the
collagen surface, but with respect to the elastin surface, low
adhesion (34.8% after 1 hour) and stretch (13.0% after 4 hours)
were exhibited similar to the dish that had only been corona
discharge treated. With vascular endothelial cells, the same
tendency was exhibited as with fibroblasts. Specifically, high
adhesion (.gtoreq.72% after 1 hour) and stretch (.gtoreq.72% after
4 hours) were exhibited with respect to the collagen surface, but
with respect to the elastin surface, low adhesion (20.0% after 1
hour) and stretch (13.3% after 4 hours) were exhibited.
[0064] These results are shown in Table 1, wherein adhesion of 50%
or greater after 1 hour and stretch of 50% or greater after 4 hours
is represented by .largecircle., and adhesion of up to 50% after 1
hour and stretch of up to 50% after 4 hours is represented by X.
The cell instrument having elastin on the surface, as the invention
product, clearly has a high degree of recognition as an
extracellular matrix for vascular smooth muscle cells.
TABLE-US-00001 TABLE 1 Vascular smooth Fibro- Vascular muscle cells
blasts endothelial cells Discharge only X X X Elastin .largecircle.
X X Collagen .largecircle. .largecircle. .largecircle.
Example 5
Dynamic Culturing Test with Dynamic Culture Device Using
Elastin-Coated Silicon Sheet
[0065] In a dynamic culture device employing the elastin-coated
silicon sheet prepared in Example 2, with CSC medium (vascular
smooth muscle cell medium: product of Dainippon Pharmaceutical Co.,
Ltd.), there was seeded 1 mL of a vascular smooth muscle cell
suspension prepared to a cell density of 1.0.times.10.sup.5
(cells/mL). At 20 hours after seeding the cells, static culturing
was carried out at 37.degree. C. in a 5% CO.sub.2 culturing
apparatus (OLYMPUS MI-IBC). Upon removing the dynamic culture
device and confirming adhesion of the cells using a phase contrast
microscope, the medium was exchanged with 3 mL of CSC medium and
static culturing was continued for 4 hours at 37.degree. C., 5%
CO.sub.2 for incubation. Next, the static-cultured dynamic culture
device was set in a dynamic cell culturing apparatus fixed to a
microscope stage, and dynamic cell culturing (uniaxial stretch
culturing) was carried out for 4 hours with stretching conditions
of 1 cycle every 2 seconds (0.5 Hz) at 10% stretch, with repeated
photographing of the same location. Analysis of the photographed
images allowed measurement of the orientation angle of the vascular
smooth muscle cells in the direction of tension application at each
time point during dynamic culturing. FIG. 8 shows the change in
orientation angle during static culturing (0-4 hour), and FIG. 9
shows the change in orientation angle during dynamic culturing (0-4
hour). Almost no change in angle occurred in the static culturing,
but based on the orientation angle distribution during the dynamic
culturing, the proportion of cells oriented at a vertical angle
with respect to the direction of tension application was found to
increase with time. Also, no change was seen under stretching
conditions of 0.5 Hz, 0.2%, similar to the static culturing.
Example 6
Vascular Smooth Muscle Differentiation Inducing Test
[0066] As an experiment to examine cytoskeleton structure changes
in vascular smooth muscle cells, immunostaining of the cytoskeletal
proteins .alpha.-actin and .beta.-actin was carried out, and the
protein was assayed by pixel analysis of the obtained images.
Ordinarily, .alpha.-actin is characterized by differentiation while
.beta.-actin is mostly dedifferentiated. The amounts of both actins
expressed in vascular smooth muscle cells cultured on an
elastin-coated dish prepared by the method of Example 1 were
divided by the amounts of both actins expressed in vascular smooth
muscle cells cultured in a common adhesive cell dish (product of
Sumitomo Bakelite Co., Ltd., hydrophilic treated polystyrene) as a
control, and the ratios were as shown in FIG. 10.
[0067] Specifically, this was carried out as follows. Smooth muscle
cells were seeded in an elastin-coated culture dish prepared by the
method of Example 1 and cultured to subconfluence, and then the
medium was removed by aspiration and rinsed with PBS, after which
it was immersed in glutaraldehyde-containing fixing solution for 30
minutes and the fixing solution was removed, prior to drying. To
the cells there was added 200 .mu.L of primary antibody (mouse
anti-human .alpha.-actin antibody) and (mouse anti-human
.beta.-actin antibody), for immersion overnight. Next, using 200
.mu.L each of FITC-labeled antimouse antibody as green secondary
antibody for .alpha.-actin, and rhodamine-labeled anti-mouse
antibody as red secondary antibody for .beta.-actin, binding was
carried out for 60 minutes in a dark location, followed by rinsing
and drying, and then observation under a fluorescent
microscope.
[0068] The smooth muscle cells cultured on elastin, even without
dynamic culturing, had increased .alpha.-actin and reduced
.beta.-actin compared with the actin in the untreated culture dish,
thus indicating that differentiation had been induced.
Example 7
Dynamic Culturing Test with Dynamic Culture Device Using Elastin
Gel Sheet
[0069] Vascular smooth muscle cells were cultured in the same
manner as Example 5, using a dynamic culture device employing the
elastin gel sheet prepared in Example 3. It was possible to perform
three-dimensional microscope observation of the cells growing on
the elastin gel sheet.
* * * * *