Technical Field
[0001]
The present invention relates to cell culturing
using a bone marrow-like structure having a three-
10 dimensional culture support. More specifically, it
relates to cell culturing using a bone marrow-like
structure with a porous polyimide film. The invention
further relates to a flexible porous sheet for healing of
bone injury sites. More specifically, it relates to a
15 porous polyimide film for healing of bone injury sites.
20
Background Art
[0002]
Cell culturing
Cells generally exist as three-dimensional
aggregates in the body, but in classical plate culturing,
cells are cultured in a monolayer fashion with the cells
attached to a vessel. Numerous reports have indicated
significant differences in cell properties with different
25 culturing environments.
[0003]
Bone marrow-related cells have a strong tendency to
be affected by the scaffold of the culturing environment
in which they grow, and many cases have been reported in
30 which cell culturing is promoted using three-dimensional
culture supports. PTL 1 discloses a bioreactor that
employs a method utilizing a nonwoven fiber matrix that
forms a three-dimensional fiber network, to increase and
maintain undifferentiated hematopoietic stem cells or
35 hematopoietic precursor cells isolated from a body, when
they are outside of the body. Use of a nonwoven fabric
is also disclosed in NPL 1 and elsewhere. The stem cell
- 2 -
properties of hematopoietic stem cells are known to be
easily lost, but NPL 2 teaches that by synthesizing a
porous hydrogel and culturing hematopoietic stem cells on
it, it is possible to maintain the stem cell properties
5 of the hematopoietic stem cells in a manner specific to
the cell source. A more direct case is reported in PTL
2, describing efficient adhesion of bone marrow stem
cells by seeding the cells on the surface of a support
composed of a calcium phosphate-based compound. In
10 addition, PTL 3 reports that Oshima et al. of the
University of Tsukuba seeded and cultured cells recovered
from mouse bone marrow on a collagen-treated threedimensional
scaffold formed of polyvinyl formal and
transplanted them into mouse dorsal regions, by which
15 they were able to function as artificial bone marrow.
However, no sufficiently practical methodology has yet
been established.
[0004]
Moreover, while spatial structure has been shown by
20 previous inventions to be in a strict proportion to
function and efficiency, demonstrating the importance of
a three-dimensional scaffold structure, this has been
approached from the point of view of the general concept
of "spatial structure", or from the viewpoint of bone
25 marrow-like composition or size control of the porous
structure, and not from the viewpoint of morphology or
anatomy, in terms of similarity of morphological
structure. It is desirable to establish a method of
culturing bone marrow cells designed from a novel
30 viewpoint that is directed toward establishing a more
efficient and highly practical methodology.
[0005]
Bone injury and healing
With a view toward patient QOL, healing of bone
35 injuries, including fractures and bone loss, has
traditionally employed techniques using a variety of
tools and means, involving methods of anchoring plates or
- 3 -
screws in the affected areas. In recent years, bone
disease treatment materials and implements with various
structural, functional and biocompatible features have
been created and utilized in medicine (NPL 3 and PTL 4).
5 In regenerative medicine as well, different methods have
been employed in attempts to promote healing of bone
injuries including fractures and bone loss, and for
example, there have been reports of methods of
supplementing affected areas with compound materials
10 obtained by culturing cells such as mesenchymal stem
cells in biomaterials such as collagen or apatite, or
bioabsorbable materials (PTLs 5 and 6) . The therapeutic
efficacy of stem cells and the extent to which they
contribute has been a matter of dispute (NPL 4).
15 [0006]
Such methods are mainly characterized in that
materials composed of biological substances, or cellcontaining
biological substances, or combinations of
cells and autolytic substances, are used in the body to
20 supplement wounds or deficient sites, but in some cases
these methods are not suitable, for sites with extensive
damage or loss or for complex shapes, and in other cases
the methods are very time consuming.
25
[0007]
In the field of dentistry, certain operations
involve leaving a space in the wound area while forming a
film on the surface (NPL 5). Such techniques are
designed to avoid rapid epithelial formation in the wound
area, but are not widely applicable for bone
30 regeneration.
[0008]
There is demand for a convenient and effective
method of treating bone injury that is suitable for a
variety of wound surfaces including sites with extensive
35 loss and sites with complex shapes.
[0009]
Porous polyimide film
- 4 -
The term "polyimide" is a general term for polymers
including imide bonds in the repeating unit. An
"aromatic polyimide" is a polymer in which aromatic
compounds are directly linked by imide bonds. An
5 aromatic polyimide has an aromatic-aromatic conjugated
structure via an imide bond, and therefore has a strong
rigid molecular structure, and since the imide bonds
provide powerful intermolecular force, it has very high
levels of thermal, mechanical and chemical properties.
10 [0010)
Porous polyimide films have been utilized in the
prior art for filters and low permittivity films, and
especially for battery-related purposes, such as fuel
cell electrolyte membranes and the like. PTLs 7 to 9
15 describe porous polyimide films with numerous macrovoids,
having excellent permeability for gases and the
like, high porosity, excellent smoothness on both
surfaces, relatively high strength and, despite high
porosity, also excellent resistance against compression
20 stress in the film thickness direction. All of these are
porous polyimide films formed via amic acid.
Citation List
[Patent Literature]
25 [0011)
[PTL 1] W02000/046349
[PTL 2] Japanese Patent No. 4393908
[PTL 3] Japanese Patent No. 3421741
[PTL 4] Japanese Patent No. 3029266
30 [PTL 5] Japanese Patent No. 4412537
[PTL 6] Japanese Patent No. 4950269
[PTL 7] W02010/038873
[PTL 8] Japanese Unexamined Patent Publication No. 2011-
219585
35 [ PTL 9] Japanese Unexamined Patent Publication No. 2011-
219586
[Non-patent literature]
[0012]
- 5 -
[NPL 1) Funakoshi News, October 1st issue in 2012
[NPL 2) A. Raic et al. I Biomaterials 35 (2014) 929-940
5 [NPL 3) F.G. Lyons et al. I Biomaterials 31 (2010) 9232-
9243
10
[NPL 4) S. Wu et al. I Materials Science and Engineering
R 80 (2014)1-36
[NPL 5) GC Corporation, GC Membrane Manual
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0013]
It is an object of the present invention to provide
15 a more efficient and highly practical method of culturing
bone marrow cells. It is another object of the invention
to provide convenient and effective means for treating
bone injury, that is applicable for many different wound
surfaces.
20
Means for Solving the Problems
[0014]
The present inventors have found that the spatial
structure of a porous polyimide film, which is an organic
25 thin-film, morphologically approximates the structure of
bone marrow. It was further found that culturing bone
marrow cells using the porous polyimide film allows
proliferation of CD45-positive cells. Surprisingly, it
was also found that when the porous polyimide film is
30 used for culturing of bone marrow cells, a cell mass is
produced having differentiating characteristics similar
to bone marrow, while following the spatial structure of
the porous polyimide film. It was yet further found that
using a differentiation-inducing accelerator can cause
35 differentiation of the cultured cells to erythroid
progenitor cells within the porous polyimide film.
[0015]
- 6 -
The present inventors still further found that a
porous polyimide film with a spatial structure
morphologically approximating bone marrow structure can
be used for healing of bone injury sites.
5 [0016]
The present invention preferably includes, but is
not limited to, the following modes.
[Mode 1]
A method of culturing bone marrow cells that
10 includes applying bone marrow cells to a porous polyimide
film and culturing them.
[Mode 2]
The method according to mode 1, wherein the bone
marrow cells are marrow stromal cells.
15 [Mode 3]
The method according to mode 1, wherein the bone
marrow cells are bone marrow-derived blood cells.
[Mode 4]
The method according to any one of modes 1 to 3,
20 wherein the bone marrow cells are cells harvested from
mammalian bone marrow.
[Mode 5]
The method according to any one of modes 1 to 3,
wherein the bone marrow cells are primary cultured cells
25 from cells harvested from mammalian bone marrow.
[Mode 6]
The method according to any one of modes 1 to 5,
further including differentiation of bone marrow cells to
hematocytes by culturing.
30 [Mode 7]
The method according to any one of modes 1 to 6,
further including applying bone marrow cells to a porous
polyimide film and then adding a differentiation-inducing
accelerating substance and culturing, to accelerate
35 differentiation from the bone marrow cells to hematocytes
in a manner specific to the spatial structure of the
porous polyimide film.
- 7 -
[Mode 8]
The method of preparing hematocytes, including
recovering hematocytes obtained using a method according
to mode 6 or 7.
5 [Mode 9]
10
The method according to any one of modes 6 to 8,
wherein the hematocytes are erythroid progenitor cells or
erythrocytes.
[Mode 10]
The method of culturing bone marrow cells including:
(1) a step of applying a first cell group to a
porous polyimide film and culturing it, and
(2) a step of applying a second cell group to the
porous polyimide film after the culturing in step (1),
15 and culturing it,
wherein the second cell group consists of bone marrow
cells.
[Mode 11]
The method according to mode 10, wherein the first
20 cell group is selected from the group consisting of
animal cells, insect cells, plant cells, yeast cells and
bacteria.
[Mode 12]
The method according to mode 11, wherein the first
25 cell group consists of bone marrow cells derived from a
mammal.
[Mode 13]
The method according to any one of modes 10 to 12,
further including differentiation of bone marrow cells to
30 hematocytes by the culturing in step (2).
[Mode 14]
The method according to any one of modes 10 to 13,
further including applying the second cell group in step
(2) to a porous polyimide film and then adding a
35 differentiation-inducing accelerating substance and
culturing, to accelerate differentiation from the bone
marrow cells, as the second cell group, to hematocytes in
- 8 -
a manner specific to the spatial structure of the porous
polyimide film.
[Mode 15]
A method of preparing hematocytes, including
5 recovering hematocytes obtained using a method according
to mode 13 or 14.
[Mode 16]
The method according to any one of modes 13 to 15,
wherein the hematocytes are erythroid progenitor cells or
10 erythrocytes.
[Mode 17]
The method according to any one of modes 1 to 16,
wherein the porous polyimide film is a porous polyimide
film including a polyimide obtained from a
15 tetracarboxylic dianhydride and a diamine.
[Mode 18]
The method according to mode 17, wherein the porous
polyimide film is a colored porous polyimide film
obtained by forming a po1yamic acid solution composition
20 including a polyamic acid solution obtained from a
tetracarboxylic dianhydride and a diamine, and a coloring
precursor, and then heat treating it at 250°C or higher.
[Mode 19]
The method according to mode 17 or 18, wherein the
25 porous polyimide film is a porous polyimide film with a
multilayer structure, having two different surface layers
and a macro-void layer.
[Mode 20]
The method according to any one of modes 1 to 19,
30 using two or more porous polyimide films layered either
above and below or left and right in the cell culture
medium.
[Mode 21]
The method according to any one of modes 1 to 20,
35 wherein the porous polyimide film is:
i) folded,
ii) wound into a roll,
- 9 -
iii) connected as sheets or fragments by a
filamentous structure, or
iv) bound into a rope,
and used by suspension or anchoring in the cell culture
5 medium in the cell culturing vessel.
[Mode 22]
10
A kit for use in the method according to any one of
modes 1 to 21, including a porous polyimide film.
[Mode 23]
Use of a porous polyimide film for the method
according to any one of modes 1 to 21.
[Mode 24]
A porous polyimide film for healing of a bone injury
site.
15 [Mode 25]
The porous polyimide film according to mode 24,
wherein the bone injury is a fracture.
[Mode 26]
The porous polyimide film according to mode 24,
20 wherein the bone injury is bone loss.
[Mode 27]
The porous polyimide film according to any one of
modes 24 to 26, which is to be used for transplantation
into the body in contact with an affected area of bone
25 injury.
[Mode 28]
The porous polyimide film according to any one of
modes 24 to 27, which is a porous polyimide film
including a polyimide obtained from a tetracarboxylic
30 dianhydride and a diamine.
[Mode 29]
The porous polyimide film according to mode 28,
which is a colored porous polyimide film obtained by
forming a polyamic acid solution composition including a
35 polyamic acid solution obtained from a tetracarboxylic
dianhydride and a diamine, and a coloring precursor, and
then heat treating it at 250°C or higher.
- 10 -
[Mode 30]
The porous polyimide film according to mode 28 or
29, which is a porous polyimide film with a multilayer
structure, having two different surface layers and a
5 macro-void layer.
[Mode 31]
The porous polyimide film according to mode 30,
which is to be used for transplantation into the body in
such a manner that of the two different surface layers,
10 the surface with the smaller mean pore size is in contact
with the affected area of bone injury.
[Mode 32]
The porous polyimide film according to any one of
modes 24 to 31, wherein the film thickness of the porous
15 polyimide film is no greater than 100 micrometers.
[Mode 33]
The porous polyimide film according to any one of
modes 24 to 32, wherein the surface of the porous
polyimide film is treated by a step of modifying its
20 physical properties.
[Mode 34]
The porous polyimide film according to mode 33,
wherein the step of modifying the physical properties of
the surface of the porous polyimide film is selected from
25 the group consisting of a step of alkali treatment of the
surface of the porous polyimide film, a step of calcium
treatment, a step of covering with a biocompatible
material, and a combination of any of these steps.
[Mode 35]
30 The method according to mode 34, wherein the
biocompatible material is selected from the group
consisting of collagen, fibronectin, laminin, polylysine,
polylactide, polyglycolide, polycaprolactone,
polyhydroxybutyrate, polylactide-co-caprolactone,
35 polycarbonate, biodegradable polyurethane, polyether
ester, polyesteramide, hydroxyapatite, collagen/~-TCP
complex, and combinations of the foregoing.
- 11 -
[Mode 36]
The porous polyimide film according to any one of
modes 24 to 35 on which cells have been supported
beforehand.
5 [Mode 37]
The porous polyimide film according to mode 36,
wherein the cells are cells selected from the group
consisting of mammal-derived pluripotent stem cells,
tissue stem cells, somatic cells, and combinations of the
10 foregoing.
[Mode 38]
The porous polyimide film according to mode 37,
wherein the cells include mammal-derived bone marrow
cells.
15 [Mode 39]
20
A kit for healing of a bone injury site, including a
porous polyimide film according to any one of modes 24 to
38.
[Mode 40]
Use of a porous polyimide film according to any one
of modes 24 to 38 for healing of a bone injury site.
[Mode 41]
A method for producing a porous polyimide film for
healing of a bone injury site, the method including
25 supporting bone marrow cells on a porous polyimide film.
[Mode 42]
The method according to mode 41, wherein the bone
marrow cells are harvested from a target of healing of a
bone injury site.
30 [Mode 43]
A method of healing a bone injury site, including
applying a porous polyimide film according to any one of
modes 24 to 38 to a bone injury site.
35 Effect of the Invention
[0017)
The present invention utilizes the spatial structure
- 12 -
of a porous polyimide film that is morphologically
similar to bone marrow structure, to allow efficient
culturing of bone marrow cells. According to the
invention, it is possible to obtain cells with
5 differentiating characteristics similar to bone marrow,
that also follow the characteristic spatial structure of
a porous polyimide film. No material with such a
function has been reported to date. According to the
invention, it is also possible to cause differentiation
10 of cultured cells to erythroid progenitor cells in a
porous polyimide film. The invention can be applied for
providing blood components with reduced infection risk
and immunological rejection risk.
[0018]
15 Moreover, by using a porous polyimide film which is
a flexible porous sheet, the invention can be applied to
a variety of wound surfaces including sites with
extensive damage or loss or sites with complex shapes, to
allow convenient and efficient healing of bone injuries.
20 The porous polyimide film to be used for the invention is
a thin-film surrounded by surfaces with a large open area
ratio having two different structures: a mesh structure
surface, referred to as the A-surface, and a large hole
structure surface, referred to as the B-surface, and
25 having a polyhedral void structure on the interior, the
film exhibiting high heat resistance and flexibility, and
also excellent shaping freedom. In addition, the porous
polyimide film to be used for the invention has a feature
whereby it can hold and grow cells inside its
30 characteristic spatial structure. By supporting cells
suited for a given purpose on the porous polyimide film
and applying them to a bone injury site, it is possible
to accelerate healing of the bone injury site.
35 Brief Description of the Drawings
[0019]
Fig. 1 is a set of scanning electron micrographs of
- 13 -
a porous polyimide film having a bone marrow-like
structure. A scanning electron micrograph shows a crosssection,
and the A-surface (mesh structure surface) and
B-surface (large hole structure surface) of the porous
5 polyimide film. For comparison, a scanning electron
micrograph of bone marrow published in NPL 1 (Funakoshi
News, October 1, 2012 Issue) is also shown (panel (b)).
Fig. 2 is a conceptual drawing of bone marrow cell
culturing. Cells harvested from bone marrow are seeded
10 and cultured on a porous polyimide film placed in a
culturing vessel, after which cells harvested from the
same bone marrow are seeded. After further culturing,
the cells become fixed on the porous polyimide film and
are analyzed by staining or the like. A differentiation-
15 inducing stimulant may also be used.
Fig. 3 shows the results of culturing cells
harvested from mouse bone marrow, using a porous
polyimide film. The upper panel shows a porous polyimide
film-fixed image after 7 days of additional culturing
20 (blue: nuclei, green: GFP), while the lower panel shows
the B-surface large hole structure and CD45-positive
cells (blue: nuclei, green: GFP, red: CD45).
Fig. 4 shows the results of twice seeding bone
marrow-derived cells, and then inducing differentiation
25 by erythropoietin. Shown is a fluorescent microscope
image taken 24 hours after having completed 7 days of
culturing following the second seeding and added 5 units
of erythropoietin.
30
Fig. 5 shows a rat cranial bone loss model.
Fig. 6 shows photographs of a technique for covering
a site of loss with a porous polyimide film sheet, in a
rat cranial bone loss model.
Fig. 7 shows healing of a bone injury, after having
applied a porous polyimide film to a bone injury site in
35 a rat cranial bone loss model.
Fig. 8 shows healing of a bone injury, after having
applied a porous polyimide film supporting bone marrow
- 14 -
cells by single seeding and culturing, to a bone injury
site in a rat cranial bone loss model.
Fig. 9 shows time-dependent change in new bone
formation volume (BV) after application of a porous
5 polyimide film supporting bone marrow cells by single or
twice seeding and culturing, to a bone injury site in a
rat cranial bone loss model.
Fig. 10 shows time-dependent change in bone mineral
content (BMC) after application of a porous polyimide
10 film supporting bone marrow cells by single or twice
seeding and culturing, to a bone injury site in a rat
cranial bone loss model.
Fig. 11 shows time-dependent change in bone mineral
density (BMD) after application of a porous polyimide
15 film supporting bone marrow cells by single or twice
seeding and culturing, to a bone injury site in a rat
cranial bone loss model.
Modes for Carrying Out the Invention
20 [0020]
Regarding a porous polyimide film
The present invention relates to I. a method of
culturing bone marrow cells, II. a method of culturing
bone marrow cells by applying bone marrow cells to a
25 porous polyimide film in two stages, and culturing them,
III. a kit to be used in the method of culturing bone
marrow cells, IV. use of a porous polyimide film for the
method of culturing bone marrow cells, V. a porous
polyimide film for healing of a bone injury site, VI. a
30 kit for healing of a bone injury site, VII. use of a
porous polyimide film for healing of a bone injury, VIII.
a method for producing a porous polyimide film for
healing of a bone injury site, and IX. a method of
healing a bone injury site. All of these aspects of the
35 invention have in common use of a porous polyimide film.
[0021]
Polyimide is a general term for polymers containing
- 15 -
imide bonds in the repeating unit, and usually it refers
to an aromatic polyimide in which aromatic compounds are
directly linked by imide bonds. An aromatic polyimide
has an aromatic-aromatic conjugated structure via an
5 imide bond, and therefore has a strong rigid molecular
structure, and since imide bonds have powerful
intermolecular force, it has very high levels of thermal,
mechanical and chemical properties.
10
[0022]
The porous polyimide film used for the invention is
preferably a porous polyimide film including (as the main
component) a polyimide obtained from a tetracarboxylic
dianhydride and a diamine, and more preferably it is a
porous polyimide film comprising a polyimide obtained
15 from a tetracarboxylic dianhydride and a diamine. The
phrase "including as the main component" means that it
essentially contains no components other than the
polyimide obtained from a tetracarboxylic dianhydride and
a diamine, as constituent components of the porous
20 polyimide film, or that it may contain them but they are
additional components that do not affect the properties
of the polyimide obtained from the tetracarboxylic
dianhydride and diamine.
25
[0023]
The porous polyimide film used for the invention
also includes colored porous polyimide films obtained by
forming a polyamic acid solution composition containing a
polyamic acid solution obtained from a tetracarboxylic
acid component and a diamine component, and a coloring
30 precursor, and then heat treating it at 250°C or higher.
[0024]
Polyamic acid
A polyamic acid is obtained by polymerization of a
tetracarboxylic acid component and a diamine component.
35 A polyamic acid is a polyimide precursor that can be
cyclized to a polyimide by thermal imidization or
chemical imidization.
- 16 -
[0025]
The polyamic acid used may be any one that does not
have an effect on the invention, even if a portion of the
amic acid is imidized. Specifically, the polyamic acid
5 may be partially thermally imidized or chemically
imidized.
[0026]
When the polyamic acid is to be thermally imidized,
there may be added to the polyamic acid solution, if
10 necessary, an imidization catalyst, an organic
phosphorus-containing compound, or fine particles such as
inorganic fine particles or organic fine particles.
Also, when the polyamic acid is to be chemically
imidized, there may be added to the polyamic acid
15 solution, if necessary, a chemical imidization agent, a
dehydrating agent, or fine particles such as inorganic
fine particles or organic fine particles. Even if such
components are added to the polyamic acid solution, they
are preferably added under conditions that do not cause
20 precipitation of the coloring precursor.
[0027]
Coloring precursor
A coloring precursor to be used for the invention is
a precursor that generates a colored substance by partial
25 or total carbonization under heat treatment at 250°C or
higher.
[0028]
Coloring precursors to be used for the invention are
preferably uniformly dissolved or dispersed in a polyamic
30 acid solution or polyimide solution and subjected to
thermal decomposition by heat treatment at 250°C or
higher, preferably 260°C or higher, even more preferably
280°C or higher and more preferably 300°C or higher, and
preferably heat treatment in the presence of oxygen such
35 as air, at 250°C, preferably 260°C or higher, even more
preferably 280°C or higher and more preferably 300°C or
- 17 -
higher, for carbonization to produce a colored substance,
more preferably producing a black colored substance, with
carbon-based coloring precursors being most preferred.
[0029]
5 The coloring precursor, when being heated, first
appears as a carbonized compound, but compositionally it
contains other elements in addition to carbon, and also
includes layered structures, aromatic crosslinked
structures and tetrahedron carbon-containing disordered
10 structures.
[0030]
Carbon-based coloring precursors are not
particularly restricted, and for example, they include
tar or pitch such as petroleum tar, petroleum pitch, coal
15 tar and coal pitch, coke, polymers obtained from
acrylonitrile-containing monomers, ferrocene compounds
(ferrocene and ferrocene derivatives), and the like. Of
these, polymers obtained from acrylonitrile-containing
monomers and/or ferrocene compounds are preferred, with
20 polyacrylnitrile being preferred as a polymer obtained
from an acrylonitrile-containing monomer.
[0031]
The tetracarboxylic dianhydride used may be any
tetracarboxylic dianhydride, selected as appropriate
25 according to the properties desired. Specific examples
of tetracarboxylic dianhydrides include
biphenyltetracarboxylic dianhydrides such as pyromellitic
dianhydride, 3,3',4,4'-biphenyltetracarboxylic
dianhydride (s-BPDA) and 2,3,3',4'-
30 biphenyltetracarboxylic dianhydride (a-BPDA),
oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'tetracarboxylic
dianhydride, bis(3,4-
dicarboxyphenyl)sulfide dianhydride, 2,2-bis(3,4-
dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane
35 dianhydride, 2,3,3',4'-benzophenonetetracarboxylic
dianhydride, 3,3',4,4'-benzophenonetetracarboxylic
dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride,
- 18 -
2,2-bis(3,4-dicarboxyphenyl)propane dianhydride, pphenylenebis(
trimellitic acid monoester acid anhydride),
p-biphenylenebis(trimellitic acid monoester acid
anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic
5 dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic
dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene
dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene
dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl
dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane
10 dianhydride, 2,3,6,7-naphthalenetetracarboxylic
dianhydride, 1,4,5,8-naphthalenetetracarboxylic
dianhydride, 4,4'-(2,2-
hexafluoroisopropylidene)diphthalic dianhydride, and the
like. Also preferably used is an aromatic
15 tetracarboxylic acid such as 2,3,3',4'diphenylsulfonetetracarboxylic
acid. These may be used
alone or in appropriate combinations of two or more.
[0032]
Particularly preferred among these are at least one
20 type of aromatic tetracarboxylic dianhydride selected
from the group consisting of biphenyltetracarboxylic
dianhydride and pyromellitic dianhydride. As a
biphenyltetracarboxylic dianhydride there may be suitably
used 3,3',4,4'-biphenyltetracarboxylic dianhydride.
25 [0033]
Any desired diamine may be used as a diamine.
Specific examples of diamines include the following.
1) Benzenediamines with one benzene nucleus, such as
1,4-diaminobenzene(paraphenylenediamine), 1,3-
30 diaminobenzene, 2,4-diaminotoluene and 2,6-
diaminotoluene;
2) diamines with two benzene nuclei, including
diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether
and 3,4'-diaminodiphenyl ether, and 4,4'-
35 diaminodiphenylmethane, 3,3'-dimethyl-4,4'diaminobiphenyl,
2,2'-dimethyl-4,4'-diaminobiphenyl,
2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-
- 19 -
dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-
4,4'-diaminodiphenylmethane, 3,3',5,5'-tetramethyl-4,4'diaminodiphenylmethane,
bis(4-aminophenyl)sulfide, 4,4'diaminobenzanilide,
3,3'-dich1orobenzidine, 3,3'-
5 dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'dimethoxybenzidine,
2,2'-dimethoxybenzidine, 3,3'diaminodiphenyl
ether, 3,4'-diaminodiphenyl ether, 4,4'diaminodiphenyl
ether, 3,3'-diaminodiphenyl sulfide,
3,4'-diaminodiphenyl sulfide, 4,4'-diaminodipheny1
10 sulfide, 3,3'-diaminodiphenylsulfone, 3,4'diaminodiphenylsu1fone,
4,4'-diaminodipheny1sulfone,
3,3'-diaminobenzophenone, 3,3'-diamino-4,4'dichlorobenzophenone,
3,3'-diamino-4,4'dimethoxybenzophenone,
3,3'-diaminodiphenylmethane, 3,4'-
15 diaminodiphenylmethane, 4,4'-diaminodiphenylmethane, 2,2-
bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane,
2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane,
2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane,
3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl
20 sulfoxide and 4,4'-diaminodiphenyl sulfoxide;
3) diamines with three benzene nuclei, including
1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-
aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-
bis(4-aminophenyl)benzene, 1,3-bis(4-
25 aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene,
1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-
4-trifluoromethylbenzene, 3,3'-diamino-4-(4-
phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-
phenylphenoxy)benzophenone, 1,3-bis(3-aminophenyl
30 sulfide)benzene, 1,3-bis(4-aminophenyl su1fide)benzene,
1,4-bis(4-aminophenyl sulfide)benzene, 1,3-bis(3-
aminopheny1sulfone)benzene, 1,3-bis(4-
aminophenylsu1fone)benzene, 1,4-bis(4-
aminophenylsu1fone)benzene, 1,3-bis[2-(4-
35 aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-
aminophenyl)isopropyl]benzene and 1,4-bis[2-(4-
aminophenyl)isopropyl]benzene;
- 20 -
4) diamines with four benzene nuclei, including
3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-
aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl,
4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-
5 aminophenoxy)phenyl]ether, bis[3-(4-
aminophenoxy)phenyl]ether, bis[4-(3-
aminophenoxy)phenyl]ether, bis[4-(4-
aminophenoxy)phenyl]ether, bis[3-(3-
aminophenoxy)phenyl]ketone, bis[3-(4-
10 aminophenoxy)phenyl]ketone, bis[4-(3-
aminophenoxy)phenyl]ketone, bis[4-(4-
aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]
sulfide, bis[3-(4-aminophenoxy)phenyl] sulfide, bis[4-(3-
aminophenoxy)phenyl] sulfide, bis[4-(4-
15 aminophenoxy)phenyl] sulfide, bis[3-(3-
aminophenoxy)phenyl]sulfone, bis[3-(4-
aminophenoxy)phenyl]sulfone, bis[4-(3-
aminophenoxy)phenyl]sulfone, bis[4-(4-
aminophenoxy)phenyl]sulfone, bis[3-(3-
20 aminophenoxy)phenyl]methane, bis[3-(4-
aminophenoxy)phenyl]methane, bis[4-(3-
aminophenoxy)phenyl]methane, bis[4-(4-
aminophenoxy)phenyl]methane, 2,2-bis[3-(3-
aminophenoxy)phenyl]propane, 2,2-bis[3-(4-
25 aminophenoxy)phenyl]propane, 2,2-bis[4-(3-
aminophenoxy)phenyl]propane, 2,2-bis[4-(4-
aminophenoxy)phenyl]propane, 2,2-bis[3-(3-
aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-
bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-
30 hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-
1,1,1,3,3,3-hexafluoropropane and 2,2-bis[4-(4-
aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane.
[0034]
These may be used alone or in mixtures of two or
35 more. The diamine used may be appropriately selected
according to the properties desired.
[0035]
- 21 -
Preferred among these are aromatic diamine
compounds, with 3,3'-diaminodiphenyl ether, 3,4'diaminodiphenyl
ether, 4,4'-diaminodiphenyl ether,
paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-
5 bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene,
1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-
aminophenoxy)benzene and 1,4-bis(3-aminophenoxy)benzene
being preferred for use. Particularly preferred is at
least one type of diamine selected from the group
10 consisting of benzenediamines, diaminodiphenyl ethers and
bis(aminophenoxy)phenyl.
[0036]
From the viewpoint of heat resistance and
dimensional stability under high temperature, the porous
15 polyimide film to be used for the invention is preferably
formed from a polyimide obtained by combination of a
tetracarboxylic dianhydride and a diamine, having a glass
transition temperature of 240°C or higher, or without a
distinct transition point at 300°C or higher.
20 [0037]
From the viewpoint of heat resistance and
dimensional stability under high temperature, the porous
polyimide film is preferably a porous polyimide film
comprising one of the following aromatic polyimides.
25 (i) An aromatic polyimide comprising at least one
tetracarboxylic acid unit selected from the group
consisting of biphenyltetracarboxylic acid units and
pyromellitic acid units, and an aromatic diamine unit,
(ii) an aromatic polyimide comprising a
30 tetracarboxylic acid unit and at least one type of
aromatic diamine unit selected from the group consisting
of benzenediamine units, diaminodiphenyl ether units and
bis(aminophenoxy)phenyl units,
and/or,
35 (iii) an aromatic polyimide comprising at least one
type of tetracarboxylic acid unit selected from the group
consisting of biphenyltetracarboxylic acid units and
- 22 -
pyromellitic acid units, and at least one type of
aromatic diamine unit selected from the group consisting
of benzenediamine units, diaminodiphenyl ether units and
bis(aminophenoxy)phenyl units.
5 [0038]
While not restrictive, the porous polyimide film for
use in the invention may be a porous polyimide film with
a multilayer structure, having at least two surface
layers (A-surface and B-surface), and a macro-void layer
10 sandwiched between the two surface layers. Preferably,
the porous polyimide film is a porous polyimide film
wherein the macro-void layer has a partition bonded to
the surface layers (A-surface and B-surface) and a
plurality of macro-voids with mean pore sizes of 10 to
15 500 ~ in the planar direction of the film, surrounded by
the partition and the surface layers (A-surface and Bsurface),
wherein the macro-void layer partition and the
surface layers (A-surface and B-surface) each have
thicknesses of 0.01 to 20 ~' with a plurality of pores
20 with mean pore sizes of 0.01 to 100 ~' the pores being
optionally communicating with each other, and also having
a partial or total multilayer structure in communication
with the macro-voids, where the total film thickness is 5
to 500 ~ and the porosity is 40% or greater and less
25 than 95%.
[0039]
The total film thickness of the porous polyimide
film used for the invention is not limited, but may be 25
to 75 ~according to one mode. Differences in the film
30 thickness may be observed as differences in cell growth
rate, cell morphology, cell saturation within the plate,
and the like.
[0040]
According to the invention, when the porous
35 polyimide film used has two different surface layers (Asurface
and B-surface), and a macro-void layer sandwiched
- 23 -
between the two surface layers, the mean pore size of the
holes in the A-surface may differ from the mean pore size
of the holes in the B-surface. Preferably, the mean pore
size of the holes in the A-surface is smaller than the
5 mean pore size of the holes in the B-surface. More
preferably, the mean pore size of the holes in the Asurface
is smaller than the mean pore size of the holes
in the B-surface, with the mean pore size of the holes in
the A-surface being 0.01 to 50 ~' 0.01 ~to 40 ~' 0.01
10 ~to 30 ~' 0.01 ~to 20 ~or 0.01 ~to 15 ~' and the
mean pore size of the holes in the B-surface being 20 ~
to 100 ~, 30 ~ to 100 ~, 40 ~ to 100 ~' 50 ~ to 100
~ or 60 ~ to 100 ~- Most preferably, the A-surface of
the porous polyimide film is a mesh structure having
15 small holes with a mean pore size of no greater than 15
~' such as 0.01 ~ to 15 ~' and the B-surface is a
large-hole structure with a mean pore size of 20 ~ or
greater, such as 20 ~ to 100 ~·
20
[0041]
The total film thickness of the porous polyimide
film used for the invention can be measured using a
contact thickness gauge.
The mean pore size of the surface of the porous
polyimide film can be determined by measuring the pore
25 area of 200 or more open holes from a scanning electron
micrograph of the porous film surface, and calculating
the mean diameter from the average value for the pore
areas according to the following formula (1), assuming
the pore shapes to be circular.
30
Mean pore size
2 xj (S a/n) (1)
(wherein Sa represents the average value for the pore
areas.)
- 24 -
[0042]
The porosity of the porous polyimide film used for
the invention can be determined by measuring the film
thickness and mass of the porous film cut out to a
5 prescribed size, and performing calculation from the
basis weight according to the following formula (2).
(1-w/ (SXdXD)) XlOO
Porosity (%) =
(2)
10 (wherein S represents the area of the porous film, d
represents the total film thickness, w represents the
measured mass, and D represents the polyimide density,
the polyimide density being defined as 1.34 g/cm3
.)
[0043]
15 For example, the porous polyimide films described in
International Patent Publication No. W02010/038873,
Japanese Unexamined Patent Publication No. 2011-219585
and Japanese Unexamined Patent Publication No. 2011-
219586 may also be used in the method of the invention.
20 [0044]
The cells that have been seeded on the surface of
the porous polyimide film can stably grow and proliferate
on the surface and/or in the interior of the film. The
cells may be in a variety of different forms, depending
25 on the location of growth and proliferation in the film.
According to one mode of the invention, growth may be
carried out while moving the surface and interior of the
porous polyimide film and changing the form, depending on
the type of cell.
30 [0045]
Naturally, the porous polyimide film to which cells
are loaded in the invention is preferably in a state
including no cells other than those that are to be
loaded, i.e. a sterilized state. The method of the
35 invention preferably includes a step of pre-sterilizing
the porous polyimide film. A porous polyimide film has
- 25 -
very excellent heat resistance and is lightweight, allows
free selection of the shape and size, and is easy to
treat for sterilization. Any desired sterilization
treatment may be conducted, such as dry heat
5 sterilization, steam sterilization, sterilization with a
disinfectant such as ethanol, or electromagnetic wave
sterilization using ultraviolet rays or gamma rays.
[0046]
I. Method of culturing bone marrow cells
10 The present invention relates to a method of
culturing bone marrow cells which includes applying bone
marrow cells to a porous polyimide film and culturing
them. The entire content of International Application
Number PCT/JP2014/070407 is incorporated herein by
15 reference.
[0047]
The method of culturing bone marrow cells of the
invention includes applying bone marrow cells to a porous
polyimide film and culturing them. The method of the
20 invention is characterized by including applying bone
marrow cells to a porous polyimide film and culturing the
bone marrow cells on the surface or in the interior of
the polyimide film.
[0048]
25 1. Bone marrow cells
Throughout the present specification, "bone marrow
cells" refers to cells present in bone marrow. Bone
marrow cells include marrow stromal cells, bone marrowderived
blood cell progenitor cells and bone marrow-
3D derived blood cells. Marrow stromal cells are cells that
support bone marrow-derived blood cells. Bone marrowderived
blood cell progenitor cells are cells that, upon
division and differentiation are capable of
differentiating to hematocytes or blood cells. Bone
35 marrow-derived blood cells are blood cells included in
bone marrow.
[0049]
- 26 -
The source of the bone marrow cells to be used for
the method of culturing bone marrow cells according to
the invention is not particularly restricted so long as
the cells are from an animal belonging to the class
5 Mammalia, or mammals, and examples include mice, rats,
humans, monkeys, pigs, dogs, sheep and goats.
[0050]
The bone marrow cells to be used in the method of
culturing bone marrow cells according to the invention
10 may be cells harvested from mammalian bone marrow. The
harvesting of the bone marrow cells from bone marrow can
be carried out using a known method, such as bone marrow
puncture or bone marrow flushing. The bone marrow cells
to be used for the invention may also be primary cultured
15 cells from cells harvested from mammalian bone marrow.
[0051]
2. Application of bone marrow cells to porous polyimide
film
In the method for culturing bone marrow cells of the
20 invention, there are no particular restrictions on the
specific steps for application of the bone marrow cells
to the porous polyimide film. It is possible to carry
out the steps described throughout the present
specification, or to employ any desired method suited for
25 applying bone marrow cells to a film-like support.
Application of bone marrow cells to the porous polyimide
film in the method of the invention includes, but is not
limited to, the following modes.
[0052]
30 (A) A mode including a step of seeding bone marrow
cells on the surface of a porous polyimide film;
(B) A mode including a step of:
placing a bone marrow cell suspension on the dried
surface of a porous polyimide film,
35 allowing it to stand, or moving the porous polyimide
film to promote efflux of the liquid, or stimulating part
of the surface to cause absorption of the bone marrow
- 27 -
cell suspension into the film, and
retaining the bone marrow cells in the bone marrow
cell suspension inside the film and allowing the water to
flow out; and
5 (C) A mode including a step of:
wetting one or both sides of a porous polyimide film
with a bone marrow cell culture solution or a sterilized
liquid,
loading a bone marrow cell suspension into the
10 wetted porous polyimide film, and
15
retaining the bone marrow cells in the bone marrow
cell suspension inside the film and allowing the water to
flow out.
[0053]
Mode (A) includes a step of directly seeding bone
marrow cells or a bone marrow cell mass on the surface of
a porous polyimide film. Alternatively, it includes a
mode of placing a porous polyimide film in a bone marrow
cell suspension and wetting the bone marrow cell culture
20 solution from the surface of the film.
[0054]
Bone marrow cells seeded on the surface of a porous
polyimide film adhere to the porous polyimide film and
infiltrate into the interiors of the pores. Preferably,
25 the bone marrow cells adhere spontaneously to the porous
polyimide film without applying any particular exterior
physical or chemical force. The bone marrow cells that
have been seeded on the surface of the porous polyimide
film can stably grow and proliferate on the surface
30 and/or in the interior of the film. The bone marrow
cells may be in a variety of different forms, depending
on the location of the film used for growth and
proliferation.
[0055]
35 For mode (B), a bone marrow cell suspension is
placed on the dried surface of a porous polyimide film.
The porous polyimide film is allowed to stand, or the
- 28 -
porous polyimide film is moved to promote efflux of the
liquid, or part of the surface is stimulated to cause
absorption of the bone marrow cell suspension into the
film, so that the bone marrow cell suspension permeates
5 into the film. While it is not our intention to be
constrained by theory, this is believed to be due to the
properties of each of the surface forms of the porous
polyimide film. According to this mode, the bone marrow
cells are absorbed and seeded in the locations of the
10 film where the cell suspension has been loaded.
[0056]
Alternatively, as according to mode (C), after all
or a portion of one or both sides of the porous polyimide
film has been wetted with the bone marrow cell culture
15 solution or sterilized liquid, the bone marrow cell
suspension may be loaded into the wetted porous polyimide
film. This will significantly increase the transit rate
of the cell suspension.
[0057]
20 For example, a method of wetting a portion of the
film edges, for the main purpose of preventing fly loss
of the film, may be used (hereunder referred to as
"single-point wetting method"). The single-point wetting
method is nearly the same as the dry method (mode (B)) in
25 which the film essentially is not wetted. However, it is
possible that bone marrow cell solution permeation
through the film is more rapid at the small wetted
portions. There may also be used a method in which all
of one or both sides of the porous polyimide film that
30 have been thoroughly wetted (hereunder this will also be
referred to as "wet film") is loaded with a bone marrow
cell suspension (this will hereunder be referred to as
"wet film method"). In this case, the entire porous
polyimide film has a greatly increased transit rate for
35 the bone marrow cell suspension.
[0058]
According to modes (B) and (C), the bone marrow
- 29 -
cells in the bone marrow cell suspension are retained in
the film, while the water flows out. This allows
treatment such as increasing the concentration of bone
marrow cells in the bone marrow cell suspension and
5 flowing out of unwanted non-cellular components together
with the water.
[0059]
Mode (A) will also be referred to as "natural
seeding", and modes (B) and (C) as "suction seeding".
10 [0060]
Preferably, but not restrictively, the viable cells
are selectively retained in the porous polyimide film.
Thus, according to a preferred mode of the invention, the
viable cells are retained in the porous polyimide film,
15 and the dead cells preferentially flow out together with
the water.
[0061]
The sterilized liquid used for mode (C) is not
particularly restricted, and may be a sterilized
20 buffering solution or sterilized water. A buffering
solution may be, for example, (+) or (-) Dulbecco's PBS,
or (+) or (-) Hank's Balanced Salt Solution. Examples of
buffering solutions are listed in Table 1 below.
[0062]
25 [Table 1)
Component Concentration Concentration
(mmol/L) (g/L)
NaCl 137 8.00
KCl 2.7 0.20
Na2HP04 10 1. 44
KHzP04 1. 76 0.24
pH (-) 7.4 7.4
[0063]
In the method for culturing bone marrow cells of the
invention, application of bone marrow cells to the porous
30 polyimide film further includes a mode of adding cells in
a floating state as a suspension together with the porous
- 30 -
polyimide film, to adhere the bone marrow cells with the
film (entangling) , For example, for application of the
bone marrow cells to the porous polyimide film in the
cell culturing method of the invention, the cell culture
5 medium, the bone marrow cells and one or more of the
porous polyimide films may be placed in the cell
culturing vessel. When the cell culture medium is a
liquid, the porous polyimide film is in a floating state
in the cell culture medium. The bone marrow cells can
10 adhere to the porous polyimide
of the porous polyimide film.
film due to the properties
Thus, even with cells that
are not suited for natural suspension culture, the porous
polyimide film allows culturing in a floating state in
the cell culture medium. The bone marrow cells
15 preferably spontaneously adhere to the porous polyimide
film. Here, "adhere spontaneously" means that the bone
marrow cells are retained on the surface or in the
interior of the porous polyimide film without applying
any particular exterior physical or chemical force.
20 [0064]
In the method for culturing bone marrow cells of the
invention, when the porous polyimide film is used in a
state suspended in the cell culture medium, two or more
fragments of the porous polyimide film may be used.
25 Since the porous polyimide film is a flexible thin-film,
using such fragments that are suspended in the culture
solution, for example, allows a porous polyimide film
with a large surface area to be added into a fixed volume
of cell culture medium. In the case of normal culturing,
30 the container base area constitutes the area limit in
which cell culture can be accomplished, but with cell
culturing using the porous polyimide film of the
invention, all of the large surface area of the
previously added porous polyimide film constitutes area
35 in which cell culturing can be accomplished. The porous
polyimide film allows the cell culture solution to pass
through, allowing supply of nutrients, oxygen and the
- 31 -
like even into the folded film, for example.
[0065)
The sizes and shapes of the porous polyimide film
fragments are not particularly restricted. The shapes
5 may be as desired, such as circular, elliptical,
quadrilateral, triangular, polygonal or string-like.
[0066)
Because the porous polyimide film used in the method
for culturing bone marrow cells of the invention is
10 flexible, it can be used with varying shapes. Instead of
a flat form, the porous polyimide film can also be used
by working into a three-dimensional shape. For example,
porous polyimide films may be: i) folded, ii) wound into
a roll, iii) connected as sheets or fragments by a
15 filamentous structure, or iv) bound into a rope, for
suspension or fixing in the cell culture medium in the
cell culturing vessel. By forming into shapes such as i)
to iv), it is possible to place a large amount of porous
polyimide films into a fixed volume of cell culture
20 medium, similar to using fragments. Furthermore, since
each fragment can be treated as an aggregate, it is
possible to aggregate and move the cell masses together,
for overall high applicability.
25
[0067)
With the same concept as fragment aggregates, two or
more porous polyimide films may be used in a layered form
either above and below or left and right in the cell
culture medium. Layering includes a mode in which
portions of the porous polyimide films overlap. Layered
30 culturing allows culturing of cells at high density in a
narrow space. It is also possible to further layer a
film on a film on which cells are already growing,
setting it to create a multilayer of different cell
types. The number of layered porous polyimide films is
35 not particularly restricted.
[0068)
Two or even more forms of the bone marrow cell
- 32 -
culturing method of the invention described above may be
used in combination. For example, using any of the
methods of modes (A) to (C), first the cells may be
applied to the porous polyimide film and then the bone
5 marrow cell-adhered porous polyimide film may be used for
suspension culture. Alternatively, the step of
application to the porous polyimide film may be a
combination of two or more of the methods of any of modes
(A) to (C).
10 [0069]
The porous polyimide film to be used in the method
of culturing bone marrow cells according to the invention
is a porous polyimide film having two different surface
layers (A-surface and B-surface), and a macro-void layer
15 sandwiched between the two surface layers, and when the
mean pore size of the holes in the A-surface is smaller
than the mean pore size of the holes in the B-surface,
the bone marrow cells may be either applied from the Asurface
or applied from the B-surface. The bone marrow
20 cells are preferably applied from the A-surface.
[0070]
In the method of culturing bone marrow cells of the
invention, preferably the bone marrow cells grow and
proliferate on the surface or in the interior of the
25 porous polyimide film.
[0071]
In the method for culturing bone marrow cells of the
invention, the bone marrow cell culturing system and
culturing conditions may be set as appropriate according
30 to the type of cells used. A person skilled in the art
may carry out culturing of cells suited for the porous
polyimide film, using any publicly known method. The
cell culture medium may also be prepared as appropriate
for the type of cells.
35 [0072]
The cell culture medium to be used in the method for
culturing bone marrow cells of the invention may be in
- 33 -
any form such as a liquid medium, semi-solid medium or
solid medium. Also, a liquid medium in droplet form may
be sprayed into the cell culturing vessel to contact the
medium with the cell-supporting porous polyimide film.
5 [0073]
10
The cell culture using a porous polyimide film may
also be combined with another suspension culture support
such as a microcarrier, cellulose sponge or the like.
[0074]
The method for culturing bone marrow cells of the
invention is not particularly restricted in terms of the
form and scale of the system used for the culturing, and
any scale from cell culturing dish to a flask, plastic
bag, test tube or large tank may be used, as appropriate.
15 These include, for example, Cell Culture Dish by BD
Falcon, and Nunc Cell Factory by Thermo Scientific. By
using a porous polyimide film according to the invention,
it has become possible to carry out culturing even of
cells that have not been capable of natural suspension
20 culture, using an apparatus intended for suspension
culture, in a state similar to suspension culturing. The
apparatus for suspension culture that is used may be, for
example, a spinner flask or rotating culturing flask by
Corning, Inc. As an environment allowing a similar
25 function to be obtained, there may be used a hollow fiber
culturing system such as the FiberCell® System by Veritas.
[0075]
The culturing in the method for culturing bone
marrow cells of the invention may be carried out in a
30 manner with continuous circulation such as continuous
addition and recovery of the medium on the porous
polyimide film, or exposure of the porous polyimide film
sheet to air using an open apparatus.
35
[0076]
In the method for culturing bone marrow cells of the
invention, cell culturing may be carried out in a system
in which a cell culture medium is continuously or
- 34 -
intermittently supplied to a cell culturing vessel from
cell culture medium supply means installed outside of the
cell culturing vessel. The system may be such that the
cell culture medium is circulated between the cell
5 culture medium supply means and the cell culturing
vessel.
[0077]
When the cell culturing is carried out in a system
in which the cell culture medium is continuously or
10 intermittently supplied to the cell culturing vessel from
cell culture medium supply means installed outside of the
cell culturing vessel, the system may be a cell culturing
apparatus including a culturing unit which is the cell
culturing vessel, and a culture medium-supply unit which
15 is the cell culture medium supply means, wherein
the culturing unit is a culturing unit that houses
one or more porous polyimide films to support cells, and
that comprises a culture medium supply port and a culture
medium discharge port, and
20 the culture medium-supply unit is a culture mediumsupply
unit comprising a culture medium housing vessel, a
culture medium supply line, and a liquid conveyance pump
that conveys the medium continuously or intermittently
through the culture medium supply line, the first end of
25 the culture medium supply line contacting the medium in
the culture medium housing vessel, and the second end of
the culture medium supply line being in communication
with the culturing unit interior via the culture medium
supply port of the culturing unit.
30 [0078]
In the cell culturing apparatus, the culturing unit
may be a culturing unit that does not comprise an air
supply port, an air discharge port and an oxygen exchange
membrane, or it may be a culturing unit that comprises an
35 air supply port and an air discharge port, or an oxygen
exchange membrane. Even if the culturing unit does not
comprise an air supply port, an air discharge port and an
- 35 -
oxygen exchange membrane, the oxygen, etc. necessary for
cell culturing is adequately supplied to the cells
through the medium. Furthermore, in the cell culturing
apparatus described above, the culturing unit may further
5 comprise a culture medium discharge line, the first end
of the culture medium discharge line being connected to
the culture medium housing vessel, the second end of the
culture medium discharge line being in communication with
the culturing unit interior via the culture medium
10 discharge port of the culturing unit, and the medium
being able to circulate through the culture medium-supply
unit and the culturing unit.
15
[0079]
3. Differentiation of bone marrow cells to hematocytes
The invention also relates to a method of culturing
bone marrow cells that further includes causing
differentiation of bone marrow cells to hematocytes by
culturing.
[0080]
20 Throughout the present specification, hematocytes
include leukocytes such as neutrophils, eosinophils,
basophils, lymphocytes, monocytes and macrophages, and
erythrocytes, platelets, mast cells and dendritic cells,
as well as precursors of the foregoing.
25 [0081]
When bone marrow cells are applied to a porous
polyimide film and culturing is continued, some of the
bone marrow cells can be induced to differentiate to
hematocytes. While it is not our intention to be
30 constrained by theory, since the spatial structure of a
porous polyimide film approximates the spatial structure
of bone marrow, cells with differentiating
characteristics similar to bone marrow become arranged
and proliferate in a manner following the spatial
35 structure of the porous polyimide film. By thus
approximately reproducing the in vivo structure of bone
marrow in the porous polyimide film, differentiation to
- 36 -
hematocytes is induced. Throughout the present
specification, inducing differentiation to hematocytes by
approximately reproducing the in vivo structure of bone
marrow in the porous polyimide film will be referred to
5 as being differentiation from bone marrow cells to
hematocytes in a manner specific to the spatial structure
of the porous polyimide film.
[0082]
In the method of culturing bone marrow cells of the
10 invention, bone marrow cells may be applied to a porous
polyimide film and then a differentiation-inducing
accelerating substance added, to accelerate
differentiation from the bone marrow cells to hematocytes
in a manner specific to the spatial structure of the
15 porous polyimide film. The differentiation-inducing
accelerating substance may be a known substance used as
appropriate for the purpose, examples of which include
colony-stimulating factor, granulocyte colony stimulating
factor, stem-cell factor, stem cell growth factor-a,
20 erythropoietin, thrombopoietin and interleukin, although
there is no limitation to these. In the method of the
invention, a single type of differentiation-inducing
accelerating substance may be used alone, or a
combination of several different differentiation-inducing
25 accelerating substances may be used.
[0083]
The invention also relates to a method of preparing
hematocytes that includes recovering hematocytes obtained
by the method of culturing bone marrow cells described
30 above.
35
[0084]
II. Method of culturing bone marrow cells in which bone
marrow cells are applied to a porous polyimide film and
cultured, in two stages.
The invention also relates to a method of culturing
bone marrow cells including:
(1) a step of applying a first cell group to a
- 37 -
porous polyimide film and culturing it, and
(2) a step of applying a second cell group to the
porous polyimide film after the culturing in step (1),
and culturing it,
5 wherein the second cell group consists of bone marrow
cells.
[0085]
The type of first cell group may be any of type of
cells, and for example, they may be selected from the
10 group consisting of animal cells, insect cells, plant
cells, yeast cells and bacteria. Animal cells, for the
purpose of the present specification, are largely divided
into cells from animals belonging to the subphylum
Vertebrata, and cells from non-vertebrates (animals other
15 than animals belonging to the subphylum Vertebrata) .
There are no particular restrictions on the source of the
animal cells, for the purpose of the present
specification. Preferably, they are cells from an animal
belonging to the subphylum Vertebrata. The subphylum
20 Vertebrata, for the purpose of the present specification,
includes the superclass Agnatha and the superclass
Gnathostomata, the superclass Gnathostomata including the
class Mammalia, the class Aves, the class Amphibia and
the class Reptilia. Preferably, they are cells from an
25 animal belonging to the class Mammalia, generally known
as mammals. Mammals, for the purpose of the present
specification, are not particularly restricted but
include, preferably, mice, rats, humans, monkeys, pigs,
dogs, sheep and goats.
30 [0086]
There are also no particular restrictions on sources
of plant cells, for the purpose of the present
specification. Suitable cells are from plants including
bryophytes, pteridophytes and spermatophytes.
35 [0087]
Plants from which spermatophyte cells are derived,
for the purpose of the present specification, include
- 38 -
both monocotyledons and dicotyledons. While not
restrictive, monocotyledons include Orchidaceae plants,
Poaceae plants (rice, corn, barley, wheat, sorghum and
the like) and Cyperaceae plants. Dicotyledons include
5 plants belonging to many subclasses including the
subclass Chrysanthemum, the subclass Magnoliidae and the
subclass Rosidae.
[0088]
For the purpose of the present specification, algae
10 may be considered cell-derived organisms. These include
different groups, from the eubacteria Cyanobacteria
(blue-green algae), to eukaryotic monocellular organisms
(diatoms, yellow-green algae, dinoflagellates and the
like) and multicellular marine algae (red algae, brown
15 algae and green algae).
[0089]
There are no particular limitations on the types of
archaebacteria or bacteria for the purpose of the present
specification. Archaebacteria are composed of groups
20 comprising methanogenic bacteria, extreme halophilic
bacteria, thermophilic acidophilic bacteria,
hyperthermophilic bacteria and the like. Bacteria are
selected from the group consisting of, for example,
lactic acid bacteria, E. coli, Bacillus subtilis and
25 cyanobacteria.
[0090]
The types of animal cells or plant cells that may be
used for the method of culturing bone marrow cells of the
invention are not particularly restricted, but are
30 preferably selected from the group consisting of
pluripotent stem cells, tissue stem cells, somatic cells
and germ cells.
[0091]
Throughout the present specification, the term
35 "pluripotent stem cells" is intended as a comprehensive
term for stem cells having the ability to differentiate
into cells of a variety of tissues (pluripotency) . While
- 39 -
not a restriction, pluripotent stem cells include
embryonic stem cells (ES cells), induced pluripotent stem
cells (iPS cells), embryonic germ cells (EG cells) and
germ stem cells (GS cells). They are preferably ES cells
5 or iPS cells. Particularly preferred are iPS cells,
which are free of ethical problems, for example. The
pluripotent stem cells used may be any publicly known
ones, and for example, the pluripotent stem cells
described in International Patent Publication No.
10 W02009/123349 (PCT/JP2009/057041) may be used.
[0092]
Throughout the present specification, the term
"tissue stem cells" refers to stem cells that are cell
lines capable of differentiation but only to limited
15 specific tissues, though having the ability to
differentiate into a variety of cell types
(multipotency). For example, hematopoietic stem cells in
the bone marrow are the source of blood cells, while
neural stem cells differentiate into neurons. Additional
20 types include hepatic stem cells from which the liver is
formed and skin stem cells that form skin tissue.
Preferably, the tissue stem cells are selected from among
mesenchymal stem cells, hepatic stem cells, pancreatic
stem cells, neural stem cells, skin stem cells and
25 hematopoietic stem cells.
[0093]
Throughout the present specification, the term
"somatic cells" refers to cells other than germ cells,
among the cells composing a multicellular organism. In
30 sexual reproduction, these are not passed on to the next
generation. Preferably, the somatic cells are selected
from among hepatocytes, pancreatic cells, muscle cells,
bone cells, osteoblasts, osteoclasts, chondrocytes,
adipocytes, skin cells, fibroblasts, pancreatic cells,
35 renal cells and lung cells, or blood cells such as
lymphocytes, erythrocytes, leukocytes, monocytes,
macrophages or megakaryocytes.
- 40 -
[0094]
Throughout the present specification, the term "germ
cells" refers to cells having the role of passing on
genetic information to the succeeding generation in
5 reproduction. These include, for example, gametes for
sexual reproduction, i.e. the ova, egg cells, sperm,
sperm cells, and spores for asexual reproduction.
[0095]
For the purpose of the present specification, the
10 cells may also be selected from the group consisting of
sarcoma cells, established cell lines and transformants.
The term "sarcoma" refers to cancer occurring in nonepithelial
cell-derived connective tissue cells, such as
the bone, cartilage, fat, muscle or blood, and includes
15 soft tissue sarcomas, malignant bone tumors and the like.
Sarcoma cells are cells derived from sarcoma. The term
"established cell line'' refers to cultured cells that are
maintained in vitro for long periods and reach a
stabilized character and can be semi-permanently
20 subcultured. Cell lines derived from various tissues of
various species including humans exist, such as PC12
cells (from rat adrenal medulla), CHO cells (from Chinese
hamster ovary), HEK293 cells (from human embryonic
kidney), HL-60 cells from (human leukocytes) and HeLa
25 cells (from human cervical cancer), Vero cells (from
African green monkey kidney epithelial cells), MOCK cells
(from canine renal tubular epithelial cells) and HepG2
cells (from human hepatic cancer). The term
"transformants" refers to cells with an altered genetic
30 nature by extracellularly introduced nucleic acid (DNA
and the like). Suitable methods are known for
transformation of animal cells, plant cells and bacteria.
[0096]
In the method of culturing bone marrow cells of the
35 invention, the first cell group preferably consists of
bone marrow cells.
[0097]
- 41 -
The method of applying the first cell group to the
porous polyimide film may be carried out in the same
manner as the method of applying the bone marrow cells to
the porous polyimide film.
5 [0098]
The method of culturing bone marrow cells in which
bone marrow cells are applied to and cultured on a porous
polyimide film in two stages, includes a step in which
the first cell group is applied to the porous polyimide
10 film and cultured, after which the second cell group is
applied to the porous polyimide film and cultured, where
the second cell group consists of bone marrow cells.
[0099]
In the method of culturing bone marrow cells in
15 which bone marrow cells are applied to and cultured on a
porous polyimide film in two stages, the bone marrow
cells may be differentiated to hematocytes by the method
described above, after application and culturing of the
second cell group. This may be followed by an additional
20 step of recovering the hematocytes.
[0100]
For example, after ordinary bone marrow cells as the
first cell group have been applied to and cultured on the
porous polyimide film, GFP transgenic mouse-derived bone
25 marrow cells, as the second cell group, may be applied to
and cultured on the porous polyimide film. After
confirming the cells visualized by GFP, differentiation
from the bone marrow cells to hematocytes may then be
monitored.
30 [0101]
III. Kit for use in method of culturing bone marrow cells
The present invention also relates to a kit for use
in the method of culturing bone marrow cells of the
invention, the kit including a porous polyimide film.
35 [0102]
The kit of the invention may include constituent
elements necessary for cell culturing in addition to the
- 42 -
porous polyimide film, as appropriate. This includes,
for example, the cells to be applied to the porous
polyimide film, the cell culture medium, the continuous
culture medium-supply apparatus, the continuous culture
5 medium-circulating apparatus, the scaffold or module for
support of the porous polyimide film, the cell culturing
apparatus, and the kit instruction manual.
[0103]
While not restrictive, one mode includes a package
10 containing either one or a plurality of sterilized porous
polyimide films stored in a transparent pouch, in a form
allowing their use for cell culturing, or a kit having a
sterile liquid encapsulated together with a porous
polyimide film in the same pouch, in the form of an
15 integrated film/liquid allowing efficient suction
seeding.
20
[0104]
IV. Use of porous polyimide film for method of culturing
bone marrow cells
The invention also relates to use of a porous
polyimide film for the aforementioned method of culturing
bone marrow cells.
[0105]
V. Porous polyimide film for healing of a bone injury
25 site.
The invention also relates to a porous polyimide
film for healing of bone injury sites.
[0106]
1. Bone injury
30 Throughout the present specification, "bone injury"
refers to a state of damage to bone tissue caused by
trauma, fatigue, disease or the like, and it includes,
for example, a state of damage only to the surface of
bone tissue, as well as fracture and bone loss.
35 Throughout the present specification, "fracture" includes
complete fracture where the bone has lost complete
continuity, and incomplete fracture where the bone has
- 43 -
not lost complete continuity. Modes of fracture include
closed fracture (simple fracture) in which the fracture
site is not released out of the body, open fracture
(complex fracture) in which the fracture site is released
5 out of the body, single fracture in which one bone is
disjoined at only one location, and compound fracture
(double fracture) in which one bone is disjoined at
multiple locations. The porous polyimide film of the
invention may be applied to any of these modes of
10 fracture.
[0107]
Throughout the present specification, "healing of a
bone injury site" means partial or complete improvement,
repair or restoration of the damaged state of a site of
15 bone injury. The term "healing of a fracture site" means
partial or complete improvement, repair or restoration of
a site where bone has been disjoined or lost. Also,
"accelerate healing of a bone injury'' means to shorten
the period for improvement, repair or restoration of the
20 state of damage at a site where bone has been damaged, or
to enlarge the area of improvement, repair or restoration
of the damaged state.
[0108]
The index for healing or acceleration of healing at
25 a bone injury site may be adhesion of disjoined bone,
decrease in the area or volume of bone loss, bone mineral
density (BMD), bone mineral content (BMC), bone mass or
new bone formation volume (BV) .
[0109]
30 2. Treatment of porous polyimide film surface
The porous polyimide film of the invention may have
its surface partially or completely treated by a step
that modifies its physical properties. The step of
modifying the physical properties of the surface of the
35 porous polyimide film may be selected as any desired step
suited for the purpose so long as it does not interfere
with healing of the bone injury site, and for example, it
- 44 -
may be a step of alkali treatment or calcium treatment of
the surface of the porous polyimide film, a step of
covering with a biocompatible material, or a combination
of any of the foregoing.
5 [0110]
A step of alkali treatment of the surface of the
porous polyimide film may be, for example, a step of
applying an alkaline substance such as sodium hydroxide
or potassium hydroxide to the surface of the porous
10 polyimide film, and modifying the physical properties of
the surface of the porous polyimide film, although this
is not limitative.
[0111]
A step of calcium treatment of the surface of the
15 porous polyimide film may be, for example, applying a
calcium-containing substance such as calcium chloride,
calcium phosphate or calcium fluoride to the surface of
the porous polyimide film, and modifying the physical
properties of the surface of the porous polyimide film,
20 although this is not limitative.
[0112]
A step of alkali treatment of the surface of the
porous polyimide film may also be followed by calcium
treatment of the surface of the porous polyimide film.
25 [0113]
Biocompatible materials that may be used in a step
of covering the surface of the porous polyimide film with
a biocompatible material include collagen, fibronectin,
laminin, polylysine, polylactide, polyglycolide,
30 polycaprolactone, polyhydroxybutyrate, polylactide-cocaprolactone,
polycarbonate, biodegradable polyurethane,
polyether ester, polyesteramide, hydroxyapatite,
collagen/~-TCP (~-tricalcium phosphate) complex, and
combinations of the foregoing, with no limitation to
35 these. The method of covering with a biocompatible
material may be a method known to those skilled in the
art, used as appropriate.
- 45 -
[0114]
After alkali treatment and/or calcium treatment of
the surface of the porous polyimide film, the surface may
be further covered with the biocompatible material.
5 [0115]
3. Porous polyimide film with cells supported beforehand
The porous polyimide film of the invention may be
one on which cells have been supported beforehand. The
porous polyimide film of the invention has a feature
10 whereby it can hold and grow cells inside its
characteristic spatial structure. By supporting cells
suited for a given purpose on the porous polyimide film
and applying them to a bone injury site, it is possible
to accelerate healing of the bone injury site.
15 [0116]
20
Although any desired method may be used as the
method of supporting the cells on the porous polyimide
film according to the invention, the following method may
be mentioned as an example.
(A) A mode including a step of seeding cells on the
surface of a porous polyimide film;
(B) A mode including a step of:
placing a cell suspension on the dried surface of
the porous polyimide film,
25 allowing it to stand, or moving the porous polyimide
film to promote efflux of the liquid, or stimulating part
of the surface to cause absorption of the cell suspension
into the film, and
retaining the cells in the cell suspension inside
30 the film and allowing the water to flow out; and
(C) A mode including a step of:
wetting one or both sides of the porous polyimide
film with a cell culture solution or a sterilized liquid,
loading a cell suspension into the wetted porous
35 polyimide film, and
retaining the cells in the cell suspension inside
the film and allowing the water to flow out.
- 46 -
[0117]
Cells seeded on the surface of the porous polyimide
film of the invention adhere to the porous polyimide film
and infiltrate into the interiors of the pores.
5 Preferably, the cells adhere spontaneously to the porous
polyimide film without applying any particular exterior
physical or chemical force. The cells that have been
seeded on the surface of the porous polyimide film can
stably grow and proliferate on the surface and/or in the
10 interior of the film. The cells may be in a variety of
different forms, depending on the location of the film
used for growth and proliferation.
[0118]
The porous polyimide film of the invention is a
15 porous polyimide film having two different surface layers
(A-surface and B-surface) , and a macro-void layer
sandwiched between the two surface layers, and when the
mean pore size of the holes in the A-surface is smaller
than the mean pore size of the holes in the B-surface,
20 the cells may be applied from the A-surface or applied
from the B-surface. The cells are preferably applied
from the A-surface.
[0119]
After the cells have been seeded on the surface of
25 the porous polyimide film of the invention, the cells may
be cultured on the porous polyimide film, according to
the purpose. The cultured cells are supported on the
porous polyimide film.
30
[ 012 OJ
There are no particular restrictions on the type of
cells to be supported on the porous polyimide film of the
invention and any desired type of cells may be used, but
it is generally preferred to use cells from an animal
belonging to the class Mammalia, known as mammals.
35 Mammals are not particularly restricted but include,
preferably, mice, rats, humans, monkeys, pigs, dogs,
sheep and goats.
- 47 -
[0121]
The type of animal cells to be supported on the
porous polyimide film of the invention is not restricted
but is preferably selected from the group consisting of
5 pluripotent stem cells, tissue stem cells, somatic cells
and combinations of the foregoing.
[0122]
The bone marrow cells to be supported on the porous
polyimide film of the invention may be cells harvested
10 from mammalian bone marrow. The harvesting of the bone
marrow cells from bone marrow can be carried out using a
known method, such as bone marrow puncture or bone marrow
flushing. The bone marrow cells to be supported on the
porous polyimide film of the invention may also be
15 primary cultured cells from cells harvested from
mammalian bone marrow.
[ 012 3]
The porous polyimide film of the invention has a
characteristic spatial structure whose form
20 morphologically approximates the structure of bone
marrow. The present inventors have found that when bone
marrow cells are seeded and cultured on a porous
polyimide film, it is possible to cause proliferation of
CD45-positive cells, and a cell mass is produced having
25 differentiating characteristics similar to bone marrow,
while following the spatial structure of the porous
polyimide film. The porous polyimide film that can be
used for the invention may be one on which bone marrow
cells have been seeded and cultured. A publicly known
30 medium may be used as appropriate for the culturing.
[0124]
One mode of the invention is a porous polyimide film
supporting bone marrow cells harvested from a target for
bone injury healing. By using such a porous polyimide
35 film for bone injury healing, it is possible to not only
provide structural supplementation at the bone injury
site, but also to restore the bone function, including
hematopoiesis.
[0125]
- 48 -
The method used to support the cells on the porous
polyimide film of the invention may also be a method
5 including the following steps, for example.
(1) A step of applying a first cell group to a
porous polyimide film and culturing it, and
(2) a step of applying a second cell group to the
porous polyimide film after the culturing in step (1),
10 and culturing and supporting the cells, wherein the
second cell group consists of bone marrow cells.
[0126]
In this cell supporting method, the type of first
cell group may be any type of cells, and for example,
15 they may be selected from the group consisting of animal
cells, insect cells, plant cells, yeast cells and
bacteria. Animal cells are largely divided into cells
from animals belonging to the subphylum Vertebrata, and
cells from non-vertebrates (animals other than animals
20 belonging to the subphylum Vertebrata) . The first cell
group preferably consists of bone marrow cells.
[0127]
4. Porous polyimide film for healing of a bone injury
site
25 The porous polyimide film of the invention may be
applied to an affected area of bone injury to heal the
bone injury site.
[0128]
The porous polyimide film of the invention has high
30 heat resistance and flexibility and also excellent
shapeability freedom, and it can therefore be cut, molded
or worked into any desired shape to match the condition
of the affected area of bone injury.
[0129]
35 Any desired method suited for the purpose may be
employed to apply the porous polyimide film of the
invention to an affected area of bone injury, but
- 49 -
preferably the porous polyimide film is transplanted into
the body in contact with the affected area of bone
injury. The porous polyimide film may be placed in
contact with the entire affected area of bone injury, or
5 the porous polyimide film may be placed in contact with a
portion of the affected area of the bone injury. When
the porous polyimide film of the invention is a porous
polyimide film with a multilayer structure having two
different surface layer sides and a macro-void layer, it
10 is preferably used by being transplanted into the body so
that of the two different surface layers, the surface
with the smaller mean pore size is in contact with the
affected area of bone injury.
15
[ 013 01
The porous polyimide film of the invention may also
be effectively used for sites with extensive bone loss or
sites with complex fracturing, for which conventional
methods have not been applicable. The porous polyimide
film of the invention may be applied so as to cover all
20 or a portion of the affected area of bone injury, and
there is no particular need for the porous polyimide film
to be anchored with another material. Depending on the
purpose, however, suture thread, staples, biocompatible
screws or the like may be used to anchor the porous
25 polyimide film in the biological tissue.
[0131]
VI. Kit for healing of bone injury site
The present invention further relates to a kit for
healing of a bone injury site, the kit including the
30 porous polyimide film described above. The kit of the
invention may include, in addition to the porous
polyimide film, also the materials necessary for bone
injury healing surgery, and an instruction manual for the
kit.
35 [0132]
While not restrictive, one mode includes a package
containing either one or a plurality of sterilized porous
- 50 -
polyimide films stored in a transparent pouch, in a form
allowing their use for bone injury healing, or a kit
having a sterile liquid encapsulated together with the
porous polyimide film in the same pouch, in the form of
5 an integrated film/liquid allowing efficient cell
seeding.
10
[0133]
VII. Use of porous polyimide film for healing of bone
injury
The invention also relates to use of the
aforementioned porous polyimide film for healing of a
bone injury.
[0134]
VIII. Method for producing porous polyimide film for
15 healing of bone injury site
The present invention further relates to a method
for producing a porous polyimide film for healing of a
bone injury site, the method including supporting bone
marrow cells on a porous polyimide film. The bone marrow
20 cells used may be ones harvested from the target of bone
injury site healing.
[0135]
IX. Method of healing bone injury site
The present invention further relates to a method of
25 healing a bone injury site that includes applying the
aforementioned porous polyimide film to a bone injury
site.
[0136]
The present invention will now be explained in
30 greater detail by examples. It is to be understood,
however, that the invention is not limited to these
examples. A person skilled in the art may easily
implement modifications and changes to the invention
based on the description in the present specification,
35 and these are also encompassed within the technical scope
of the invention. Unless otherwise specified, the term
"porous polyimide film" refers to a porous polyimide film
- 51 -
with a total film thickness of 25 ~m and a porosity of
73%. Each porous polyimide film had at least two
different surface layers (A-surface and B-surface), and a
macro-void layer sandwiched between the two surface
5 layers. The mean pore size of the holes in the A-surface
was 6 ~m, and the mean pore size of the holes in the Bsurface
was 46 ~m.
[0137]
The porous polyimide films used in the following
10 examples were prepared by forming a polyamic acid
solution composition including a polyamic acid solution
obtained from 3,3' ,4,4'-biphenyltetracarboxylic
dianhydride (s-BPDA) as a tetracarboxylic acid component
and 4,4'-diaminodiphenyl ether (ODA) as a diamine
15 component, and polyacrylamide as a coloring precursor,
and performing heat treatment at 250°C or higher.
Examples
[0138]
20 Example 1
The femora and tibia of 11- to 14-week-old male
C57BL/6 mice were extracted, the mesiodistal epiphysis
portions were excised, a 10% FBS-containing DMEM solution
was used for rinsing, and the bone marrow was harvested.
25 The cell count was 5.0 X 106
• The cells were seeded from
the A-surface of a dry heat-sterilized 1 em-square porous
polyimide film, with the A-surface facing upward (Fig.
2) . After 5 days of culturing, the medium was exchanged
and culturing was continued for 2 days. A second seeding
30 from GFP transgenic mice of the same species, age and
gender was performed on a porous polyimide film sheet
growing cells that had already been cultured for 7 days,
by the same method as described above. After an
additional 7 days of culturing, a 4% formalin solution
35 was used to fix the cells, and the specimen was analyzed
by immunostaining. GFP positive cells were observed in
- 52 -
the fixed specimen, while aggregates of CD45-positive
cells were observed around the large hole structure of
the B-surface of the porous polyimide film (Fig. 3).
[0139]
5 Example 2
Seeding of bone marrow-derived cells was carried out
twice by the same method as in Example 1. Upon 7 days of
culturing after the second seeding, erythropoietin was
added at 1 unit, 2 units and 5 units per ml. While there
10 was no change in the erythropoietin-free control group,
in the erythropoietin-added group the proerythroblast
cells (TER119 positive) were observed to form aggregates
at each location in a structure-specific manner,
depending on the time and volume (Fig. 4).
15 [0140]
Example 3
After general anesthesia of 9-week-old LEW rats with
2-3% isoflurane, they were subjected to infiltration
anesthesia in the operating field, with 1/10,000
20 epinephrine-containing lidocaine. A region wider than
the incision site was shaved, and then an incision was
made at the top of the head on a straight line up to the
subperiosteum, for sufficient delineation of the
operating field. After detaching the dermal periosteal
25 flap and exposing the parietal bone, a trephine bur was
used to form two 4 mm-diameter circular bone loss
sections under poured sterile physiological saline.
After covering the loss section with a sterilized porous
polyimide film, the dermal periosteal flap was reinstated
30 and sutured with a nylon thread. The mesh surface (Asurface)
of a porous polyimide film was placed in the
wound area to prepare one model, and the large-hole
surface (B-surface) was placed to prepare another model.
After 2 weeks, 4 weeks and 8 weeks, the state of healing
35 of the loss section was measured. The measured
parameters were bone mineral density (BMD) (mg/cm3
), bone
mineral content (BMC) (mg) , new bone formation volume
- 53 -
(BV) (cm3
), set ROI volume (TV) (cm3
), new bone formation
percentage (BV/TV) (%) and clinical BMD (BMC/TV) (mg/cm3
).
As regards early osteogenesis in the mesh surface-placed
model, it was confirmed that the model in which the mesh
5 surface had been placed in the wound area had
significantly accelerated osteogenesis. The results are
shown in Fig. 7. In Fig. 7, the control group is the
group without covering of the bone loss section with a
porous polyimide film.
10 [0141]
Example 4
After harvesting femora and tibia of 8-week-old GFP
transgenic rats and cutting off both ends of each bone,
bone marrow cell masses were harvested by flushing with
15 10% FBS-added DMEM medium. The cell masses were
pulverized by pipetting, and 1.0 X 106 bone marrow cells
were seeded on the mesh surface (A-surface) of a 1.5 emsquare
porous polyimide film and stationary cultured for
5 days in DMEM medium containing 10% FBS. On the 6th
20 day, the cell-adhered porous polyimide film was rinsed
with phosphate buffer and further cultured for 1 day,
after only changing the medium to DMEM. After general
anesthesia of 9-week-old nude rats with 2-3% isoflurane,
they were subjected to infiltration anesthesia in the
25 field of operation, with l/10,000 epinephrine-containing
lidocaine. A region wider than the incision site was
shaved, and then an incision was made at the top of the
head on a straight line up to the subperiosteum, for
sufficient delineation of the operating field. After
30 detaching the dermal periosteal flap and exposing the
parietal bone, a trephine bur was used to form two 4 mmdiameter
circular bone loss sections under poured sterile
physiological saline. The loss section was covered by
the porous polyimide film on which the cells had been
35 adhered and then cultured, in a manner so as to place the
mesh surface (A-surface) in the wound area. After 2
weeks, 4 weeks and 8 weeks, the condition of healing of
- 54 -
the loss section was measured, and healing of the wound
area was periodically confirmed. The results are shown
in Fig. 8.
(0142]
5 Example 5
After harvesting femora and tibia of 8-week-old GFP
transgenic rats and cutting off both ends of each bone,
bone marrow cell masses were harvested by flushing with
10% FBS-added DMEM medium. The cell masses were
10 pulverized by pipetting, and 1.0 x 106 bone marrow cells
were seeded on the mesh surface (A-surface) of a 1.5 emsquare
porous polyimide film and stationary cultured for
5 days in DMEM medium containing 10% FBS. After
exchanging the DMEM medium, culturing was continued for 1
15 day. Next, 1.0 x 106 bone marrow cells were seeded on the
mesh surface (A-surface) of a porous polyimide film and
stationary cultured for 5 days in DMEM medium containing
10% FBS. On the 6th day, the cell-adhered porous
polyimide film was rinsed with phosphate buffer and
20 further cultured for 1 day, after only changing the
medium to DMEM. After general anesthesia of 9-week-old
nude rats with 2-3% isoflurane, they were subjected to
infiltration anesthesia in the field of operation, with
1/10,000 epinephrine-containing lidocaine. A region
25 wider than the incision site was shaved, and then an
incision was made at the top of the head on a straight
line up to the subperiosteum, for sufficient delineation
of the operating field. After detaching the dermal
periosteal flap and exposing the parietal bone, a
30 trephine bur was used to form two 4 mm-diameter circular
bone loss sections under poured sterile physiological
saline. The loss section was covered by the porous
polyimide film on which the cells had been adhered and
then cultured, in a manner so as to place the mesh
35 surface (A-surface) in the wound area. After 2 weeks, 4
weeks and 8 weeks, the condition of healing of the loss
section was measured, and healing of the wound area was
-
- 55 -
periodically confirmed. The results are shown in Figs. 9
to 11.

CLAIMS
1. A method of culturing bone marrow cells that
includes applying bone marrow cells to a porous polyimide
film and culturing them.
5 2. The method according to claim 1, wherein the
bone marrow cells are marrow stromal cells.
3. The method according to claim 1, wherein the
bone marrow cells are bone marrow-derived blood cells.
4. The method according to any one of claims 1 to
10 3, wherein the bone marrow cells are cells harvested from
mammalian bone marrow.
5. The method according to any one of claims 1 to
3, wherein the bone marrow cells are primary cultured
cells from cells harvested from mammalian bone marrow.
15 6. The method according to any one of claims 1 to
5, further including differentiation of bone marrow cells
to hematocytes by culturing.
7. The method according to any one of claims 1 to
6, further including applying bone marrow cells to a
20 porous polyimide film and then adding a differentiationinducing
accelerating substance and culturing, to
accelerate differentiation from the bone marrow cells to
hematocytes in a manner specific to the spatial structure
of the porous polyimide film.
25 8. A method of preparing hematocytes, including
recovering hematocytes obtained using a method according
to claim 6 or 7.
9. The method according to any one of claims 6 to
8, wherein the hematocytes are erythroid progenitor cells
30 or erythrocytes.
10. A method of culturing bone marrow cells
including:
(1) a step of applying a first cell group to a
porous polyimide film and culturing it, and
35 (2) a step of applying a second cell group to
the porous polyimide film after the culturing in step
(1), and culturing it,
- 57 -
wherein the second cell group consists of bone marrow
cells.
11. The method according to claim 10, wherein the
first cell group is selected from the group consisting of
5 animal cells, insect cells, plant cells, yeast cells and
bacteria.
10
12. The method according to claim 11, wherein the
first cell group consists of bone marrow cells derived
from a mammal.
13. The method according to any one of claims 10 to
12, further including differentiation of bone marrow
cells to hematocytes by the culturing in step (2).
14. The method according to any one of claims 10 to
14, further including applying the second cell group in
15 step (2} to a porous polyimide film and then adding a
differentiation-inducing accelerating substance and
culturing, to accelerate differentiation from the bone
marrow cells, as the second cell group, to hematocytes in
a manner specific to the spatial structure of the porous
20 polyimide film.
15. A method of preparing hematocytes, including
recovering hematocytes obtained using a method according
to claim 13 or 14.
16. The method according to any one of claims 13 to
25 15, wherein the hematocytes are erythroid progenitor
cells or erythrocytes.
17. The method according to any one of claims 1 to
16, wherein the porous polyirnide film is a porous
polyimide film including a polyimide obtained from a
30 tetracarboxylic dianhydride and a diamine.
18. The method according to claim 17, wherein the
porous polyimide film is a colored porous polyimide film
obtained by forming a polyamic acid solution composition
including a polyamic acid solution obtained from a
35 tetracarboxylic dianhydride and a diamine, and a coloring
precursor, and then heat treating it at 250°C or higher.
19. The method according to claim 17 or 18, wherein
- 58 -
the porous polyimide film is a porous polyimide film with
a multilayer structure, having two different surface
layers and a macro-void layer.
20. The method according to any one of claims 1 to
5 19, using two or more porous polyimide films layered
either above and below or left and right in the cell
culture medium.
10
21. The method according to any one of claims 1 to
20, wherein the porous polyimide film is:
i) folded,
ii) wound into a roll,
iii) connected as sheets or fragments by a
filamentous structure, or
iv) bound into a rope,
15 and used by suspension or anchoring in the cell culture
medium in the cell culturing vessel.
22. A kit for use in the method according to any
one of claims 1 to 21, including a porous polyimide film.
23. Use of a porous polyimide film for the method
20 according to any one of claims 1 to 21.
25
24. A porous polyimide film for healing of a bone
injury site.
25. The porous polyimide film according to claim
24, wherein the bone injury is a fracture.
26. The porous polyimide film according to claim
24, wherein the bone injury is bone loss.
27. The porous polyimide film according to any one
of claims 24 to 26, which is to be used for
transplantation into the body in contact with an affected
30 area of bone injury.
28. The porous polyimide film according to any one
of claims 24 to 27, which is a porous polyimide film
including a polyimide obtained from a tetracarboxylic
dianhydride and a diamine.
35 29. The porous polyimide film according to claim
28, which is a colored porous polyimide film obtained by
forming a polyamic acid solution composition including a
- 59 -
polyamic acid solution obtained from a tetracarboxylic
dianhydride and a diamine, and a coloring precursor, and
then heat treating it at 250°C or higher.
30. The porous polyimide film according to claim 28
5 or 29, which is a porous polyimide film with a multilayer
structure, having two different surface layers and a
macro-void layer.
31. The porous polyimide film according to claim
30, which is to be used for transplantation into the body
10 in such a manner that of the two different surface
layers, the surface with the smaller mean pore size is in
contact with the affected area of bone injury.
32. The porous polyimide film according to any one
of claims 24 to 31, wherein the film thickness of the
15 porous polyimide film is no greater than 100 micrometers.
20
33. The porous polyimide film according to any one
of claims 24 to 32, wherein the surface of the porous
polyimide film is treated by a step of modifying its
physical properties.
34. The porous polyimide film according to claim
33, wherein the step of modifying the physical properties
of the surface of the porous polyimide film is selected
from the group consisting of a step of alkali treatment
of the surface of the porous polyimide film, a step of
25 calcium treatment, a step of covering with a
biocompatible material, and a combination of any of these
steps.
35. The method according to claim 34, wherein the
biocompatible material is selected from the group
30 consisting of collagen, fibronectin, laminin, polylysine,
polylactide, polyglycolide, polycaprolactone,
polyhydroxybutyrate, polylactide-co-caprolactone,
polycarbonate, biodegradable polyurethane, polyether
ester, polyesteramide, hydroxyapatite, collagen/~-TCP
35 complex, and combinations of the foregoing.
36. The porous polyimide film according to any one
of claims 24 to 35 on which cells have been supported
37. The porous polyimide film according to claim
36, wherein the cells are cells selected from the group
consisting of mammal-derived pluripotent stem cells,
5 tissue stem cells, somatic cells, and combinations of the
foregoing.
10
15
38. The porous polyimide film according to claim
37, wherein the cells include mammal-derived bone marrow
cells.
39. A kit for healing of a bone injury site,
including a porous polyimide film according to any one of
claims 24 to 38.
40. Use of a porous polyimide film according to any
one of claims 24 to 38 for healing of a bone injury site.
41. A method for producing a porous polyimide film
for healing of a bone injury site, the method including
supporting bone marrow cells on a porous polyimide film.
42. The method according to claim 41, wherein the
bone marrow cells are harvested from a target of healing
20 of a bone injury site.