U.S. patent application number 15/295534 was filed with the patent office on 2017-02-02 for cell culturing method, cell culturing apparatus and kit.
The applicant listed for this patent is UBE Industries, Ltd.. Invention is credited to Kousuke BABA, Masahiko HAGIHARA, Tetsuo KAWAGUCHI, Motohisa SHIMIZU.
Application Number | 20170029766 15/295534 |
Document ID | / |
Family ID | 52393444 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170029766 |
Kind Code |
A1 |
HAGIHARA; Masahiko ; et
al. |
February 2, 2017 |
CELL CULTURING METHOD, CELL CULTURING APPARATUS AND KIT
Abstract
The present invention relates to a cell culturing method as well
as a cell culturing apparatus and kit for use in said culturing
method. This cell culturing method comprises applying cells on a
porous polyimide film and culturing.
Inventors: |
HAGIHARA; Masahiko;
(Ube-shi, JP) ; KAWAGUCHI; Tetsuo; (Ube-shi,
JP) ; BABA; Kousuke; (Ube-shi, JP) ; SHIMIZU;
Motohisa; (Ube-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UBE Industries, Ltd. |
Ube-shi |
|
JP |
|
|
Family ID: |
52393444 |
Appl. No.: |
15/295534 |
Filed: |
October 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14907432 |
Jan 25, 2016 |
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PCT/JP2014/070407 |
Jul 25, 2014 |
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15295534 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0068 20130101;
C12N 11/08 20130101; C08L 79/08 20130101; C12M 25/02 20130101; C12N
1/20 20130101; C12N 2533/30 20130101; C12N 1/16 20130101 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2013 |
JP |
2013-155550 |
Claims
1. A cell culturing method that includes applying cells to a porous
polyimide film and culturing them.
2.-22. (canceled)
23. A kit for use in a cell culturing method according to claim 1,
including a porous polyimide film.
Description
TECHNICAL FIELD
[0001] The present application claims priority based on Japanese
Patent Application No. 2013-155550 submitted on Jul. 26, 2013, the
entire contents of which are incorporated herein by reference.
[0002] The present invention relates to a cell culturing method,
and to a cell culturing apparatus and kit.
BACKGROUND ART
Cell Culturing
[0003] 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 culturing environments. An alternative is suspension
culturing in which cells are cultured in a liquid culturing medium,
but some cells are suited for suspension culture while others are
not.
[0004] The NanoCulture.RTM. Plate (NCP) developed by SCIVAX
Corporation is an adhesive-type three-dimensional culturing
multiplate patterned with an extracellular matrix (microspheres or
microhoneycomb) on the bottom surface by nanoimprint technology. A
regular repeating structure is adopted, which is quadrilateral for
microspheres and hexagonal for microhoneycombs. The cells utilize
this micropattern as a scaffold to actively form spheroids
(numerous cells aggregated into a three-dimensional structure).
[0005] Also, there have been reports of using cell culture sheets
with regular arrangements of numerous uniform superfine cellular
projections (nanopillars) ("nanopillar cell culture sheets") for
culturing of three-dimensional interstitial tissue (spheroids)
having a structure similar to live hepatic tissue (Takahashi et
al., Tissue Engineering Part A. June 2012, Vol. 16, No. 6, p.
1983-1995).
[0006] Japanese Unexamined Patent Publication No. 2009-213421
describes a spheroid production method and spheroid production
apparatus that use a honeycomb-shaped porous film (honeycomb
film).
[0007] These cell culture methods are similar in that they utilize
a sheet (film) having a regular repeating pattern, in that the
cells grow while adhering to the sheet surface shape, and in that
what are formed by the cell culturing are spheroids in which
numerous cells of the same shape are aggregated into a
three-dimensional state, and in this sense these methods are
restricted. There has been a demand for development of a method for
more convenient, more efficient and more stable culturing of
cells.
Porous Polyimide Film
[0008] Polyimide is a general term for polymers containing 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 imide bond, and therefore has a strong rigid
molecular structure, and since imide bonds have powerful
intermolecular force, it has a very high level of thermal,
mechanical and chemical properties.
[0009] Porous polyimide films have been utilized in the prior art
for filters, low permittivity films, and especially for
battery-related purposes, such as fuel cell electrolyte membranes
and the like. International Patent Publication No. WO2010/038873,
Japanese Unexamined Patent Publication No. 2011-219585 and Japanese
Unexamined Patent Publication No. 2011-219586 describe porous
polyimide films with numerous macro-voids, 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 stress
in the film thickness direction.
CITATION LIST
Patent Literature
[0010] PTL 1: Japanese Unexamined Patent Publication No.
2009-213421 [0011] PTL 2: WO2010/038873 [0012] PTL 3: Japanese
Unexamined Patent Publication No. 2011-219585 [0013] PTL 4:
Japanese Unexamined Patent Publication No. 2011-219586
Non-Patent Literature
[0013] [0014] NPL 1: Takahashi et al., Tissue Engineering Part A.
June 2012, Vol. 16, No. 6, p. 1983-1995
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0015] The present invention relates to a cell culturing method,
and to a cell culturing apparatus and kit for use in the culturing
method.
Means for Solving the Problems
[0016] The present invention preferably includes, but is not
limited to, the following modes.
[Mode 1]
[0017] A cell culturing method which includes applying cells to a
porous polyimide film and culturing them.
[Mode 2]
[0018] The method according to mode 1, including a step of seeding
cells on the surface of a porous polyimide film.
[Mode 3]
[0019] The method according to mode 1, including a step of:
[0020] placing a cell suspension on the dried surface of the porous
polyimide film,
[0021] allowing the porous polyimide film 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
[0022] retaining the cells in the cell suspension inside the film
and allowing the water to flow out.
[Mode 4]
[0023] The method according to mode 1, including a step of:
[0024] wetting one or both sides of the porous polyimide film with
a cell culture medium or a sterilized liquid,
[0025] loading a cell suspension into the wetted porous polyimide
film, and
[0026] retaining the cells in the cell suspension inside the film
and allowing the water to flow out.
[Mode 5]
[0027] The method according to mode 4, wherein the viable cells are
retained in the porous polyimide film, and the dead cells are
allowed to flow out together with the water.
[Mode 6]
[0028] The method according to mode 4 or 5, wherein the sterilized
liquid is sterilized water or a sterilized buffering solution.
[Mode 7]
[0029] The method according to any one of modes 1 to 6, including
the aspect that the cell culture medium, cells and one or more
porous polyimide films are placed in a cell culturing vessel,
wherein the porous polyimide film is in a suspended state in the
cell culture medium.
[Mode 8]
[0030] The method according to mode 7, wherein two or more
fragments of the porous polyimide film are used.
[Mode 9]
[0031] The method according to mode 7 or 8, wherein the cells
spontaneously adhere to the porous polyimide film.
[Mode 10]
[0032] The method according to any one of modes 1 to 6, wherein the
porous polyimide film is:
[0033] i) folded,
[0034] ii) wound into a roll,
[0035] iii) connected as sheets or fragments by a filamentous
structure, or
[0036] iv) bound into a rope,
for suspension or fixing in the cell culture medium in the cell
culturing vessel.
[Mode 11]
[0037] The method according to mode 10, wherein the cells
spontaneously adhere to the porous polyimide film.
[Mode 12]
[0038] The method according to mode 1, including using two or more
porous polyimide films layered either above and below or left and
right in the cell culture medium.
[Mode 13]
[0039] The method according to mode 1, wherein a combination of two
or more of the methods according to modes 2 to 12 is used.
[Mode 14]
[0040] The method according to any one of modes 1 to 13, wherein
the cells grow and proliferate on the surface of and inside the
porous polyimide film.
[Mode 15]
[0041] The method according to any one of modes 1 to 14, wherein
the cells are selected from the group consisting of animal cells,
insect cells, plant cells, yeast cells and bacteria.
[Mode 16]
[0042] The method according to mode 15, wherein the animal cells
are cells derived from an animal belonging to the subphylum
Vertebrata.
[Mode 17]
[0043] The method according to mode 15, wherein the bacteria are
selected from the group consisting of lactic acid bacteria, E.
coli, Bacillus subtilis and cyanobacteria.
[Mode 18]
[0044] The method according to any one of modes 1 to 14,
[0045] wherein the cells are selected from the group consisting of
pluripotent stem cells, tissue stem cells, somatic cells and germ
cells.
[Mode 19]
[0046] The method according to any one of modes 1 to 13, wherein
the cells are selected from the group consisting of sarcoma cells,
established cell lines and transformants.
[Mode 20]
[0047] The method according to any one of modes 1 to 19, wherein
the porous polyimide film is a porous polyimide film including a
polyimide obtained from a tetracarboxylic dianhydride and a
diamine.
[Mode 21]
[0048] A cell culturing apparatus for use in a cell culturing
method according to any one of modes 1 to 20, including a porous
polyimide film.
[Mode 22]
[0049] A cell culturing apparatus according to mode 21, wherein two
or more porous polyimide films are layered either above and below
or left and right.
[Mode 23]
[0050] A kit for use in a cell culturing method according to any
one of modes 1 to 20, including a porous polyimide film.
Effect of the Invention
[0051] The present invention is based on the finding that when
cells are applied to a porous polyimide film, the cells grow. The
cells preferably spontaneously adhere to the porous polyimide film,
and can grow on the surface and interior of the film. By the method
of the invention it has become possible to culture cells in a
convenient, efficient and stable manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 shows natural seeding as one mode of seeding of a
cell suspension on a porous polyimide film.
[0053] FIG. 2 shows suction seeding as one mode of seeding of a
cell suspension on a porous polyimide film.
[0054] FIG. 3 shows time-dependent change according to type of
seeding surface, for natural seeding of human mesenchymal stem
cells.
[0055] FIG. 4 shows time-dependent change according to type of
seeding surface, for natural seeding of human skin fibroblasts.
[0056] FIG. 5 shows a fluorescent microscope photograph and a
stereomicroscope photograph containing the results of observation
with B-surface seeding of human skin fibroblasts (after 24 hours)
(wet film method).
[0057] FIG. 6 shows time-dependent changes with A-surface suction
seeding of human mesenchymal stem cells (single-point wetting
method).
[0058] FIG. 7 shows time-dependent changes with B-surface suction
seeding of human mesenchymal stem cells (single-point wetting
method).
[0059] FIG. 8 shows time-dependent change according to type of
observation surface, for entangled seeding of human mesenchymal
stem cells.
[0060] FIG. 9 shows low-magnification photographs and experimental
results photographs for entangled seeding of human mesenchymal stem
cells.
[0061] FIG. 10 shows time-dependent changes during suction seeding
of PC12 cells.
[0062] FIG. 11 shows cell counts for human skin fibroblasts
cultured using a porous polyimide film with a film thickness of 25
.mu.m. The abscissa represents number of days after culturing, and
the ordinate represents the cell count per square centimeter of
film.
[0063] FIG. 12 shows the results of culturing human skin
keratinocytes by the method of the present invention using a porous
polyimide film, by observation under a confocal microscope and a
stereo fluorescent microscope.
[0064] FIG. 13 shows the results of culturing human umbilical vein
endothelial cells by the method of the present invention using a
porous polyimide film, by observation under a confocal microscope
and a stereo fluorescent microscope.
[0065] FIG. 14-1 shows the results of culturing Vero cells by the
method of the present invention using a porous polyimide film (25
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0066] FIG. 14-2 shows the results of culturing Vero cells by the
method of the present invention using a porous polyimide film (40
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0067] FIG. 14-3 shows the results of culturing Vero cells by the
method of the present invention using a porous polyimide film (75
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0068] FIG. 15-1 shows the results of culturing HeLa cells by the
method of the present invention using a porous polyimide film (25
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0069] FIG. 15-2 shows the results of culturing HeLa cells by the
method of the present invention using a porous polyimide film (40
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0070] FIG. 15-3 shows the results of culturing HeLa cells by the
method of the present invention using a porous polyimide film (75
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0071] FIG. 16-1 shows the results of culturing CHO cells by the
method of the present invention using a porous polyimide film (25
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0072] FIG. 16-2 shows the results of culturing CHO cells by the
method of the present invention using a porous polyimide film (40
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
[0073] FIG. 16-3 shows the results of culturing CHO cells by the
method of the present invention using a porous polyimide film (75
.mu.m sheet), by observation under a confocal microscope and a
stereo fluorescent microscope.
MODE FOR CARRYING OUT THE INVENTION
I. Cell Culturing Method
[0074] The present invention relates to a cell culturing
method.
[0075] The cell culturing method of the invention includes applying
cells to a porous polyimide film and culturing them. The present
inventors have found that a porous polyimide film is suitable for
adhesion and culturing of cells, and have thereupon completed this
invention. The method of the invention includes applying cells to a
porous polyimide film and culturing the cells on the surface or in
the interior of the polyimide film.
Application of Cells to Porous Polyimide Film
[0076] There are no particular restrictions on the specific steps
for application of the 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 applying
cells to a film-like support. The method of the invention includes,
but is not limited to, the following modes.
[0077] (A) A mode including a step of seeding cells on the surface
of the porous polyimide film;
[0078] (B) A mode including a step of placing a cell suspension on
the dried surface of the porous polyimide film,
[0079] 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
[0080] retaining the cells in the cell suspension inside the film
and allowing the water to flow out; and
[0081] (C) A mode including a step of wetting one or both sides of
the porous polyimide film with a cell culture medium solution or a
sterilized liquid,
[0082] loading a cell suspension into the wetted porous polyimide
film, and
[0083] retaining the cells in the cell suspension inside the film
and allowing the water to flow out.
[0084] Mode (A) includes a step of directly seeding cells or a cell
mass on the surface of a porous polyimide film. Alternatively, it
includes a mode of placing a porous polyimide film in a cell
suspension and wetting the cell culture solution from the surface
of the film.
[0085] 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, 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 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.
[0086] For mode (B), a cell suspension is placed on the dried
surface of a porous polyimide film. The porous polyimide film is
allowed to stand, or the porous polyimide film is moved to promote
efflux of the liquid, or part of the surface is stimulated to cause
absorption of the cell suspension into the film, so that the cell
suspension permeates 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 surface forms of the porous polyimide film.
According to this mode, the cells are absorbed and seeded in the
locations of the film where the cell suspension has been
loaded.
[0087] 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 cell culture solution or sterilized liquid, the
cell suspension may be loaded into the wetted porous polyimide
film. This will significantly increase the transit rate of the cell
suspension.
[0088] For example, the single-point wetting method described in
Example 4 of the present specification is a method of wetting a
portion of the film edges for the main purpose of preventing fly
loss of the film, and it is nearly the same as the dry method (mode
(B)) in which the film is not essentially wetted. However, it is
possible that cell solution permeation through the film is more
rapid at the small wetted portions. Also, the wet film method
described in Example 3 of the present specification is a method in
which all of one or both sides of the porous polyimide film that
have been thoroughly wetted (hereunder this will also be referred
to as "wet film") is loaded with a cell suspension. In this case,
the entire porous polyimide film has a greatly increased transit
rate for the cell suspension.
[0089] According to modes (B) and (C), the cells in the cell
suspension are retained in the film, while the water flows out.
This allows treatment such as increasing the concentration of cells
in the cell suspension and flowing out of unwanted non-cellular
components together with the water.
[0090] Mode (A) will also be referred to as "natural seeding", and
modes (B) and (C) as "suction seeding".
[0091] 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, and the dead cells preferentially
flow out together with the water. In Example 3 of the invention,
the viable cell rate was 90% in the cell culture medium (cell
suspension) before application to the porous polyimide film of the
invention, but the viable cell rate was 65% in the liquid efflux
after application to the porous polyimide film. Compared in terms
of cell viability, for viable cells, the adsorption rate into the
film was 88% and the efflux rate was 12%, while for dead cells, the
adsorption rate into the film was 40% and the efflux rate was 60%.
This is interpreted as that viable cells are selectively adsorbed
into the porous polyimide film of the invention.
[0092] The sterilized liquid used for mode (C) is not particularly
restricted, and may be a sterilized 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.
TABLE-US-00001 TABLE 1 Concentration Concentration Component
(mmol/L) (g/L) NaCl 137 8.00 KCl 2.7 0.20 Na.sub.2HPO.sub.4 10 1.44
KH.sub.2PO.sub.4 1.76 0.24 pH (--) 7.4 7.4
[0093] The invention further includes a mode of adding adhesive
cells in a floating (suspended) state as a suspension together with
a porous polyimide film, to adhere the cells with the film
(entangling). For example, for application of the cells to the
porous polyimide film in the cell culturing method of the
invention, the cell culture medium, the 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 (suspended) state in the cell
culture medium. The cells can adhere to the porous polyimide film
due to the properties of the porous polyimide film. 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 cells preferably spontaneously adhere to
the porous polyimide film. Here, "adhere spontaneously" means that
the cells are retained on the surface or in the interior of the
porous polyimide film without applying any particular exterior
physical or chemical force.
[0094] Cell culturing can be classified into culturing where the
cultured cells are adhesion culture-type cells or suspension
culture-type cells, depending on the state in the cell culture.
Adhesion culture-type cells are cultured cells that adhere and grow
on a culturing vessel, with the medium being exchanged at the time
of subculture. Suspension culture-type cells are cultured cells
that grow in a suspended state in a medium, and generally the
medium is not exchanged at the time of subculture but dilution
culture is carried out. Because suspension culture allows culturing
in a suspended state, i.e. in a liquid, mass culturing becomes
possible, and because it is three-dimensional culturing, unlike
with adhering cells that grow only on the culturing vessel surface,
the advantage of increased culturable cell count per unit space is
afforded.
[0095] According to the invention, in conceptual terms, there is
provided a method in which it is possible to grow cells in a form
similar to suspension culture without being limited to the cell
type, so that cells can be conveniently cultured in large amounts.
According to the cell culture method 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. 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, 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 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 like even into the folded film, for
example.
[0096] The sizes and shapes of the porous polyimide film fragments
are not particularly restricted. The shapes may be as desired, such
as circular, elliptical, quadrilateral, triangular, polygonal or
string-like. This includes, for example, quadrilaterals (square,
rectangular or the like) and triangular shapes with side lengths of
about 0.1 mm to about 20 mm, preferably about 0.2 mm to about 10 mm
and more preferably about 1 mm to about 5 mm. Alternatively, for
example, they may be circular, with diameters of preferably about
0.1 mm to about 20 mm and more preferably about 0.5 mm to about 10
mm. Dispersing the fragments in the liquid results in a form
similar to a suspension culture.
[0097] Because the porous polyimide film of the invention is
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, the porous polyimide film may
be: i) folded, ii) wound into a roll, iii) connected as sheets or
fragments by a filamentous structure, or iv) bound into a rope, for
suspension or fixing in the cell culture medium in the cell
culturing vessel. By forming it into shapes such as i) to iv), it
is possible to place a large amount of porous polyimide film into a
fixed volume of cell culture 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, for overall high
applicability.
[0098] 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 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. This may also be used
for drug development, including verification of intercellular
interaction in a three-dimensional environment, or in a non-stress
cell culture method. The number of layered porous polyimide films
is not particularly restricted.
[0099] Two or even more forms of the cell 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 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).
[0100] In the method of the invention, preferably the cells grow
and proliferate on the surface or in the interior of the porous
polyimide film. No reports exist disclosing growth and
proliferation of cells inside a three-dimensional structure. By
utilization of a porous polyimide film according to the invention
it is possible to accomplish continuous three-dimensional culturing
of cells. While not restrictive, the method of the invention
carries out continuous growth of cells for 2 days or longer, more
preferably 4 days or longer and even more preferably 6 days or
longer. In Examples 1 and 4 described in the present specification,
growth of cells was observed for 21 days.
2. Cells
[0101] There are no particular restrictions on the type of cells
that can be utilized for the method of the invention, and it may be
used for growth of any type of cells.
[0102] For example, the cells 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 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 Vertebrata 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 animal belonging to the class Mammalia, generally known as
mammals. Mammals are not particularly restricted but include,
preferably, mice, rats, humans, monkeys, pigs, dogs, sheep and
goats.
[0103] 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.
[0104] Plants from which spermatophyte cells are derived include
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 plants belonging to many subclasses including
the subclass Chrysanthemum, the subclass Magnoliidae and the
subclass Rosidae.
[0105] Algae 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 algae and green algae).
[0106] There are no particular limitations on the types of
archaebacteria or bacteria for the purpose of the present
specification. Archaebacteria are composed of groups 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
cyanobacteria.
[0107] The types of animal cells or plant cells that may be used
for the method of the invention are not particularly restricted,
but are preferably selected from the group consisting of
pluripotent stem cells, tissue stem cells, somatic cells and germ
cells.
[0108] The term "pluripotent stem cells", for the purpose of the
invention, is intended as a comprehensive term for stem cells
having the ability to differentiate into cells of a variety of
tissues (pluripotent differentiating power). While not restrictive,
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 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. WO2009/123349 (PCT/JP2009/057041) may be used.
[0109] The term "tissue stem cells" refers to stem cells that are
cells lines capable of differentiation but only to limited specific
tissues, though having the ability to differentiate into a variety
of cell types (pluripotent differentiating power). For example,
hematopoietic stem cells in the bone marrow are the source of blood
cells, while neural stem cells differentiate into neurons.
Additional 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 hematopoietic stem cells.
[0110] The term "somatic cells" refers to cells other than germ
cells, among the cells composing a multicellular organism. With
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, renal cells and lung cells, or blood
cells such as lymphocytes, erythrocytes, leukocytes, monocytes,
macrophages or megakaryocytes.
[0111] The term "germ cells" refers to cells having the role of
passing on genetic information to the succeeding generation in
reproduction. These include, for example, gametes for sexual
reproduction, i.e. the ova, egg cells, sperm, sperm cells, and
spores for asexual reproduction.
[0112] The 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 non-epithelial cell-derived
connective tissue cells, such as the bone, cartilage, fat, muscle
or blood, and includes soft 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 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 cells (from
human cervical cancer). The term "transformants" refers to cells
with an altered genetic nature by extracellularly introduced
nucleic acid (DNA and the like). Suitable methods are known for
transformation of animal cells, plant cells and bacteria.
3. Porous Polyimide Film
[0113] Polyimide is a general term for polymers containing 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 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.
[0114] The porous polyimide film of 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 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 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.
[0115] This 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 precursor, and
then heat treating it at 250.degree. C. or higher.
Polyamic Acid
[0116] A polyamic acid is obtained by polymerization of a
tetracarboxylic acid component and a diamine component. A polyamic
acid is a polyimide precursor that can be cyclized to a polyimide
by thermal imidization or chemical imidization.
[0117] 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 may be partially
thermally imidized or chemically imidized.
[0118] When the polyamic acid is to be thermally imidized, there
may be added to the polyamic acid solution, if 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 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 precipitation of the coloring precursor.
Coloring Precursor
[0119] For the purpose of the invention, a coloring precursor is a
precursor that generates a colored substance by partial or total
carbonization under heat treatment at 250.degree. C. or higher.
[0120] Coloring precursors to be used for the invention are
preferably uniformly dissolved or dispersed in a polyamic acid
solution or polyimide solution and subjected to thermal
decomposition by heat treatment at 250.degree. C. or higher,
preferably 260.degree. C. or higher, even more preferably
280.degree. C. or higher and more preferably 300.degree. C. or
higher, and preferably heat treatment in the presence of oxygen
such as air, at 250.degree. C., preferably 260.degree. C. or
higher, even more preferably 280.degree. C. or higher and more
preferably 300.degree. C. or higher, for carbonization to produce a
colored substance, more preferably producing a black colored
substance, with carbon-based coloring precursors being most
preferred.
[0121] The coloring precursor, when heating, 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 structures.
[0122] Carbon-based coloring precursors are not particularly
restricted, and for example, these include tar or pitch such as
petroleum tar, petroleum pitch, coal 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 polyacrylnitrile
being preferred as a polymer obtained from an
acrylonitrile-containing monomer.
[0123] The tetracarboxylic dianhydride used may be any
tetracarboxylic dianhydride, selected as appropriate 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'-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
dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride,
3,3',4,4'-benzophenonetetracarboxylic dianhydride,
bis(3,4-dicarboxyphenyl)methane dianhydride,
2,2-bis(3,4-dicarboxyphenyl)propane dianhydride,
p-phenylenebis(trimellitic acid monoester acid anhydride),
p-biphenylenebis(trimellitic acid monoester acid anhydride),
m-terphenyl-3,4,3',4'-tetracarboxylic 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 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 tetracarboxylic acid such
as 2,3,3',4'-diphenylsulfonetetracarboxylic acid. These may be used
alone or in appropriate combinations of two or more.
[0124] Particularly preferred among these are at least one type of
aromatic tetracarboxylic dianhydride selected from the group
consisting of biphenyltetracarboxylic dianhydride and pyromellitic
acid dianhydride. As a biphenyltetracarboxylic dianhydride there
may be suitably used 3,3',4,4'-biphenyltetracarboxylic
dianhydride.
[0125] Any desired diamine may be used as a diamine. Specific
examples of diamines include the following.
[0126] 1) Benzenediamines with one benzene nucleus, such as
1,4-diaminobenzene(paraphenylenediamine), 1,3-diaminobenzene,
2,4-diaminotoluene and 2,6-diaminotoluene;
[0127] 2) diamines with two benzene nuclei, including
diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and
3,4'-diaminodiphenyl ether, and 4,4'-diaminodiphenylmethane,
3,3'-dimethyl-4,4'-diaminobiphenyl,
2,2'-dimethyl-4,4'-diaminobiphenyl,
2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl,
3,3'-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'-dichlorobenzidine, 3,3'-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'-diaminodiphenyl sulfide, 3,3'-diaminodiphenylsulfone,
3,4'-diaminodiphenylsulfone, 4,4'-diaminodiphenylsulfone,
3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone,
3,3'-diamino-4,4'-dimethoxybenzophenone,
3,3'-diaminodiphenylmethane, 3,4'-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 sulfoxide and
4,4'-diaminodiphenyl sulfoxide;
[0128] 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-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 sulfide)benzene, 1,3-bis(4-aminophenyl
sulfide)benzene, 1,4-bis(4-aminophenyl sulfide)benzene,
1,3-bis(3-aminophenylsulfone)benzene,
1,3-bis(4-aminophenylsulfone)benzene,
1,4-bis(4-aminophenylsulfone)benzene,
1,3-bis[2-(4-aminophenyl)isopropyl]benzene,
1,4-bis[2-(3-aminophenyl)isopropyl]benzene and
1,4-bis[2-(4-aminophenyl)isopropyl]benzene;
[0129] 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-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-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-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-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-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-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.
[0130] These may be used alone or in mixtures of two or more. The
diamine used may be appropriately selected according to the
properties desired.
[0131] 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-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 consisting of
benzenediamines, diaminodiphenyl ethers and
bis(aminophenoxy)phenyl.
[0132] From the viewpoint of heat resistance and dimensional
stability under high temperature, the porous polyimide film is
preferably formed from a polyimide obtained by combination of a
tetracarboxylic dianhydride and a diamine, having a glass
transition temperature of 240.degree. C. or higher, or without a
distinct transition point at 300.degree. C. or higher.
[0133] From the viewpoint of heat resistance and dimensional
stability under high temperature, the porous polyimide film of the
invention is preferably a porous polyimide film comprising one of
the following aromatic polyimides.
[0134] (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,
[0135] (ii) an aromatic polyimide comprising a 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,
[0136] (iii) an aromatic polyimide comprising at least one type of
tetracarboxylic acid unit selected from the group consisting of
biphenyltetracarboxylic acid units and 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.
[0137] While not restrictive, the porous polyimide film for use in
the method of 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 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-void with mean pore sizes of 10 to 500 .mu.m in the planar
direction of the film, surrounded by the partition and the surface
layers (A-surface and B-surface), wherein the macro-void layer
partition and the surface layers (A-surface and B-surface) each
have thicknesses of 0.01 to 20 .mu.m, with a plurality of pores
with mean pore sizes of 0.01 to 100 .mu.m, the pores being
optionally communicating with each other, and also having a partial
or total multilayer structure in communication with the macro-void,
where the total film thickness is 5 to 500 .mu.m and the porosity
is 40% or greater and less than 95%.
[0138] The total film thickness is not restricted but may be 25 to
75 .mu.m according to one mode. Differences in the film thickness
may be observed as differences in cell growth rate, cell
morphology, cell saturation within the plate, and the like.
[0139] According to one mode, the A-surface of the porous polyimide
film is a mesh structure having small holes with mean sizes of no
greater than 15 .mu.m, and the B-surface is a large-hole structure
with mean sizes of 20 .mu.m or greater.
[0140] For example, the porous polyimide films described in
International Patent Publication No. WO2010/038873, 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.
[0141] 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 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.
4. Cell Culturing Method and Cell Culture Medium
[0142] For the method of the invention, after the cells have been
applied to the porous polyimide film, the cells may be cultured
using any known method. Culturing methods suited for various cells
including animal cells, plant cells and bacteria are publicly
known, and a person skilled in the art may carry out culturing of
cells on the porous polyimide film using any publicly known method.
The cell culture medium may also be prepared as appropriate for the
type of cells.
[0143] Cell culture methods and cell culture media for animal cells
may be found in the Cell Culture Media Catalog of Lonza Group,
Ltd., for example. Cell culture methods and cell culture media for
plant cells may be found in the Plant Tissue Culturing Media Series
by Wako Corp., for example. Cell culture methods and cell culture
media for bacteria may be found in the General Bacterial Media
Catalog of BD Corp., for example.
II. Cell Culturing Apparatus
[0144] The present invention also relates to a cell culturing
apparatus for use in the culturing method of the invention, the
apparatus including a porous polyimide film. In the cell culturing
apparatus of the invention, the porous polyimide film may be used
in a fixed state, or it may be used in a floating state in the cell
culture medium. In the cell culturing apparatus, two or more porous
polyimide films may be layered either above and below or left and
right.
[0145] The cell culturing apparatus used for the invention may be a
publicly known cell culturing apparatus so long as it satisfies the
condition of including a porous polyimide film. The shape and scale
of the culturing apparatus is not particularly restricted, and any
scale from a dish or test tube to a large tank may be used, as
appropriate. 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 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 by Corning, Inc.
III. Kit for Use in Cell Culturing Method
[0146] The present invention also relates to a kit for use in the
cell culturing method of the invention, the apparatus including a
porous polyimide film.
[0147] The kit of the invention may include constituent elements
necessary for cell culturing in addition to the porous polyimide
film, as appropriate. This includes, for example, the cells applied
to the porous polyimide film, the cell culture medium and the
instruction manual for the cell culturing apparatus and the
kit.
[0148] While not restrictive, one mode includes a package
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 sterile liquid encapsulated
together with a porous polyimide film in the same pouch, in the
form of an integrated film/liquid allowing efficient suction
seeding.
EXAMPLES
[0149] The present invention will now be explained in detail by
examples, with the understanding that these examples are in no way
limitative on the invention. A person skilled in the art may easily
implement modifications and changes to the invention based on the
description in the present specification, and these are also
encompassed within the technical scope of the invention. Unless
otherwise specified, "porous polyimide film" refers to a porous
polyimide film with a film thickness of 25 .mu.m.
Example 1
Natural Seeding of Human Mesenchymal Stem Cells in Porous Polyimide
Film
[0150] For this example, human mesenchymal stem cells were used for
seeding in a porous polyimide film.
[0151] After adding 0.5 ml of cell culture medium to a 2 cm.times.2
cm sterilized square vessel, a sterilized 1.4 cm-square porous
polyimide film was dipped therein with the A-surface of the mesh
structure or the B-surface of the large-gap structure facing
upward. Separately, there was prepared a human mesenchymal stem
cell suspension, with human mesenchymal stem cells suspended at
3.6.times.10.sup.5 per 1 ml of medium (of which viable cells were
3.4.times.10.sup.5 and dead cells were 2.0.times.10.sup.4, for a
viable cell rate of 94%). Each cell suspension was added at 60
.mu.l to the cell culture medium in the square vessel.
[0152] After culturing in a cell culturing apparatus for 1 hour, 5
hours, 24 hours, 48 hours, 4 days, 7 days, 14 days and 21 days, the
cells were fixed and stained (DAPI, or actin+DAPI), and the cell
growth and proliferation were confirmed. The actin staining was
done with phalloidin. The cell proliferation and seeding
surface-specific form over time were observed. The results are
shown in FIG. 3. These results indicated that human mesenchymal
stem cells, a typical type of stem cell, can also be cultured by
the method of the present invention.
Example 2
Natural Seeding of Human Skin Fibroblasts in Porous Polyimide
Film
[0153] For this example, human skin fibroblasts were used, applying
the cells to the porous polyimide film by natural seeding.
[0154] After adding 0.5 ml of cell culture medium to a 2 cm.times.2
cm sterilized square vessel, a sterilized 1.4 cm-square porous
polyimide film was dipped therein with the A-surface of the mesh
structure or the B-surface of the large-gap structure facing
upward. Separately, there was prepared a human skin fibroblast
suspension with human skin fibroblasts suspended at
8.3.times.10.sup.5 per 1 ml of medium (of which viable cells were
8.1.times.10.sup.5 and dead cells were 2.0.times.10.sup.4, for a
viable cell rate of 98%). Each cell suspension was added at 50
.mu.l to the cell culture medium in the square vessel.
[0155] After culturing in a cell culturing apparatus for 1 hour, 5
hours, 24 hours, 48 hours, 4 days, 7 days, 14 days and 21 days, the
cells were fixed and stained (DAPI, or actin+DAPI), and the cell
growth and proliferation were confirmed. Proliferation of the cells
over time and the seeding surface-specific and observation
surface-specific forms were observed. The results are shown in FIG.
4. These results indicated that human skin fibroblasts, a typical
type of fibroblast, can also be cultured by the method of the
present invention.
Example 3
Suction Seeding of Human Skin Fibroblasts in Porous Polyimide Film
(Wet Film Method)
[0156] For this example, human skin fibroblasts were used, applying
the cells to the porous polyimide film by suction seeding onto a
wet film (a thoroughly wetted film; details are described
below).
[0157] There was prepared a human skin fibroblast suspension with
the cells suspended at 1.0.times.10.sup.6 per 1 ml of medium (of
which viable cells were 9.1.times.10.sup.5 and dead cells were
1.0.times.10.sup.5, for a viable cell rate of 90%). On a 10
cm.times.14 cm rectilinear plate there were aligned, without
overlapping, 25 1.4 cm-square porous polyimide films wetted with 1
ml of cell culture medium, and 20 .mu.l of cell suspension was
added to the B-surface of each porous polyimide film. The exuded
liquid was drained, each porous polyimide film was transferred to a
75 cm.sup.2 dish, and 12 ml of medium was added. Culturing was then
carried out under ordinary conditions, and the specimen was fixed
on the following day. FIG. 5 shows a fluorescent microscope
photograph and a stereo fluorescent microscope photograph of a
fixed and fluorescent-stained (DAPI or actin+DAPI) specimen after
24 hours.
[0158] During the seeding step, the exuded liquid was recovered,
the rectilinear plate was further rinsed with 2 ml of medium and
the cells remaining on the plate were counted, resulting in a total
cell count of 6.0.times.10.sup.4, of which 5.5.times.10.sup.4 were
viable cells and 3.0.times.10.sup.4 were dead cells, for a viable
cell rate of 65%. Compared in terms of cell viability, for the
viable cells, the adsorption rate into the film was 88% and the
efflux rate was 12%, while for the dead cells, the adsorption rate
into the film was 40% and the efflux rate was 60%.
Example 4
Suction Seeding of Human Mesenchymal Stem Cells in Porous Polyimide
Film (Single-Point Wetting Method)
[0159] For this example, human mesenchymal stem cells were used,
applying the cells to the porous polyimide film by suction seeding
onto a single-point wetted film (a simple fixed film wetted at only
the center point with a liquid droplets; details are described
below).
[0160] For this example, human mesenchymal stem cells were used,
applying the cells to the porous polyimide film by suction seeding
onto a film wetted only at the center point of the film
material.
[0161] At the center section where the film was to be placed, a
droplet of about 10 .mu.l was formed and the porous polyimide film
was placed over it, attempting to fix the film area to create an
easily seedable location, while utilizing the moisture to increase
the rapidity of cell solution film permeation.
[0162] There was prepared a human mesenchymal stem cell suspension
with the cells suspended at 1.0.times.10.sup.6 per 1 ml of medium
(of which viable cells were 9.6.times.10.sup.5 and dead cells were
4.0.times.10.sup.4, for a viable cell rate of 96%). On a 10
cm.times.14 cm rectilinear plate there were aligned, without
overlapping, 10 sterilized and dried 1.4 cm-square porous polyimide
films on their A-surfaces and B-surfaces, and 40 .mu.l of cell
suspension was added to each porous polyimide film. The exuded
liquid was drained, the porous polyimide film was transferred to a
10 cm.sup.2 dish for each seeding surface, and 2 ml of medium was
added. Culturing was then carried out under ordinary culturing
conditions, the cells were fixed and stained (DAPI, actin+DAPI or
actin) after 0.5 hour, 3 days, 7 days and 12 days and the cell
growth and proliferation were confirmed. The results are shown in
FIG. 6 and FIG. 7.
[0163] During the seeding step, the exuded liquid was recovered,
the rectilinear plate was further rinsed with 4 ml of medium and
the cells remaining on the plate were counted, resulting in a total
cell count of 6.5.times.10.sup.4, with A-surface seeding, of which
4.0.times.10.sup.4 were viable cells and 2.5.times.10.sup.4 were
dead cells, for a viable cell rate of 62%. Compared in terms of
cell viability, for the viable cells, the adsorption rate into the
film was 89% and the efflux rate was 11%, while for the dead cells,
presumably efflux occurred essentially without adsorption. With
B-surface seeding, the total cell count was 6.5.times.10.sup.4, of
which 8.0.times.10.sup.4 were viable cells and 2.0.times.10.sup.4
were dead cells, for a viable cell rate of 80%. Compared in terms
of cell viability, for the viable cells, the adsorption rate into
the film was 79% and the efflux rate was 21%, while for the dead
cells, presumably efflux occurred essentially without
adsorption.
Example 5
Entangled Seeding of Human Mesenchymal Stem Cells in Porous
Polyimide Film
[0164] For this example, human mesenchymal stem cells were used,
applying the cells to the porous polyimide film by drawing up of
the cell suspension.
[0165] A square porous polyimide film with 10 cm sides was cut into
approximately 2 to 3 mm square fragments using scissors, and after
sterilization at 160.degree. C. for 10 minutes, the fragments were
allowed to cool and wetted with 2 ml of sterilized medium. Next,
the porous polyimide film was cut with forceps and transferred to a
Falcon tube. To the film in the Falcon tube there were added 1.6 ml
of a human mesenchymal stem cell suspension, obtained by suspending
cells at 3.6.times.10.sup.5 per 1 ml of medium (of which viable
cells were 3.4.times.10.sup.5 and dead cells were
2.0.times.10.sup.4, for a viable cell rate of 94%) (total cell
count: 5.7.times.10.sup.5, of which 5.4.times.10.sup.5 were viable
cells and 3.2.times.10.sup.4 were dead cells). These were allowed
to stand for 2 hours in the culturing vessel with periodic shaking,
and upon measuring the cell count in the liquid portion, there were
observed 2.0.times.10.sup.5 cells (of which 1.2.times.10.sup.5 were
viable cells and 8.0.times.10.sup.4 were dead cells, for a viable
cell rate of 60%). The porous polyimide film fragments were cut out
and transferred to a 20 cm.sup.2 dish spread with 4 ml of medium,
and subcultured in a culturing vessel. The fragments were fixed and
stained after 48 hours, 7 days, 14 days, 21 days and 28 days (DAPI
or actin+DAPI), and the cell growth and proliferation were
observed. The results are shown in FIG. 8 and FIG. 9.
Example 6
Suction Seeding of Established Cell Line in Porous Polyimide Film
Fragments
[0166] For this example, cells of the established cell line PC12
were used, applying the cells to porous polyimide film fragments by
suction seeding.
[0167] On a 10 cm.times.14 cm rectilinear plate, 15 1.4 cm-square
porous polyimide films previously wetted with 5 ml of medium and
having the liquid thoroughly removed, were aligned in a random
fashion with partially overlapping portions above and below and
left and right. Separately, there was prepared a cell suspension of
rat adrenal pheochromocytoma PC12 cells, with the cells suspended
at 9.0.times.10.sup.5 per 1 ml of medium (of which viable cells
were 7.4.times.10.sup.5 and dead cells were 1.7.times.10.sup.5, for
a viable cell rate of 82%). A 2 ml portion of the cell suspension
was slowly added to the film while slightly tilting the plate to
cause the exudated liquid to move downward. After 5 minutes, the
exudated liquid was drained from the porous polyimide film and
transferred to 4 ml of medium spread onto a previously prepared 20
cm.sup.2 dish, and culturing was carried out in a culturing
apparatus.
[0168] After 48 hours, 4 days, 6 days and 9 days, the porous
polyimide film was fixed and stained, and proliferation of the
cells was confirmed. The staining was performed with a membrane
fluorescence stain cell mask (CellMask.TM. Orange plasma membrane
stain (Life Technologies), hereunder also referred to simply as
"CellMask")+DAPI. The results are shown in FIG. 10. With these
cells as well, there was observed a state of proliferation while
forming aggregates inside the porous polyimide film, thereby
confirming their applicability. The results indicated that PC12
cells, a typical type of established cell line, can also be
cultured by the method of the present invention.
Example 7
Measurement of Cultured Cell Count
[0169] For this example, human skin fibroblasts were cultured by
the method of the invention using a porous polyimide film, and the
cultured cell count was measured.
1. Cell Count Measurement of Ordinary Cultured Cells Using CCK8
[0170] First, the following reagent and method were used to
calculate the cell count with ordinary culturing, and the
correlation coefficient between the absorbance and actual cell
count was determined.
[Reagent] Cell Counting Kit 8; Dojindo, Solution reagent (hereunder
referred to as "CCK8"). [Method] Human skin fibroblasts cultured on
a 5 cm.sup.2 chamber dish for a prescribed period were prepared,
the culture supernatant was removed, exchange was performed with a
fixed amount of medium containing CCK8 added at 2%, and the cells
were kept in an incubator for 2 hours. Next, the colored
supernatant was extracted and the absorbance at a wavelength of 480
nm was measured (the blank used being conditions of measurement
with the medium alone). After then removing the supernatant, the
cells were rinsed twice with phosphate buffer and treated with
0.05% trypsin-EDTA solution, and the cell count was determined.
[0171] The correlation coefficient between absorbance and actual
cell count was determined for the culturing conditions and CCK8
concentration conditions by this method.
2. Measurement of Cell Count Proliferated on Porous Polyimide
Film
[0172] As in 1. above, a cell-cultured 2 cm.sup.2 (1.4 cm.times.1.4
cm) porous polyimide film was transferred to a 5 cm.sup.2 chamber
dish, a fixed amount of 5% CCK8-added medium was added, and the
film was kept in an incubator for 1 to 3 hours, after which the
supernatant was extracted and the absorbance at a wavelength of 480
nm was measured. In this case, the CCK8 was used in a 2.5-fold
amount, whereas in terms of area, the porous polyimide film area
was only an area of 2.5 times smaller, and therefore a direct
comparison can be made with the values read in 1. (When the
concentration, area or other conditions have been changed, a
conversion is necessary.) The conversion coefficient determined in
1. is used to calculate the surviving cell count on the material,
and then rinsing is performed twice with medium, and after
returning to the incubator, culturing is continued. This procedure
was repeated, and the state of proliferation of the cells on the
porous polyimide film was periodically analyzed in a quantitative
manner. Reproducibility was verified by conducting several
experiments.
[0173] The results for natural seeding and suction seeding, each
conducted 3 times, are shown in FIG. 11. As shown in FIG. 11, the
cell proliferation rate differs depending on the method of seeding,
but the difference disappears with about 1 month of culturing,
reaching similar cell counts per unit area. The dotted lines in the
graph indicate the upper limit for the cultured cell count for
ordinary adhesive culturing in a dish, carried out as a comparative
experiment. When the cell counts were compared based on area, it
was found to be possible to culture a larger amount of cells in a
unit area compared to ordinary cell culturing.
Example 8
Culturing of Human Skin Keratinocytes
[0174] For this example, human skin keratinocytes were cultured by
the method of the present invention using a porous polyimide film,
and observed under a confocal microscope and a stereo fluorescent
microscope.
[0175] After adding 0.5 ml of cell culture medium (KGM-Gold
keratinocyte growth medium, BulletKit (product of Lonza Group,
Ltd.)) to a 2 cm.times.2 cm sterilized square vessel, a sterilized
1.4 cm-square porous polyimide film was dipped therein with the
A-surface of the mesh structure facing upward. Human skin
keratinocytes were added in an amount of 4.times.10.sup.4 to the
cell culture medium in the square vessel. Specifically,
4.times.10.sup.4 human skin keratinocytes were naturally seeded for
each 1.4 cm-square porous polyimide film.
[0176] Culturing was carried out in a cell culturing apparatus, and
the cells were fixed and stained after 1 day, 3 days and 6 days.
The staining was accomplished with CellMask+DAPI or with CellMask
alone. Next, a confocal microscope (LSM700 (product of Carl Zeiss))
and a stereo fluorescent microscope (Leica M165 FC (product of
Leica Microsystems GmbH)) were used for periodic observation of the
proliferation and form of the cells.
[0177] The results are shown in FIG. 12. The results indicated that
human skin keratinocytes, as human primary cultured cells, can also
be cultured by the method of the invention.
Example 9
Culturing of Human Umbilical Vein Endothelial Cells
[0178] For this example, human umbilical vein endothelial cells
were cultured by the method of the present invention using a porous
polyimide film, and observed under a confocal microscope and a
stereo fluorescent microscope.
[0179] After adding 0.5 ml of cell culture medium (EGM-2 BulletKit
(product of Lonza Group, Ltd.)) to a 2 cm.times.2 cm sterilized
square vessel, a sterilized 1.4 cm-square porous polyimide film was
dipped therein with the A-surface of the mesh structure facing
upward. Human umbilical vein endothelial cells were added in an
amount of 4.times.10.sup.4 to the cell culture medium in the square
vessel. Specifically, 4.times.10.sup.4 human umbilical vein
endothelial cells were naturally seeded for each 1.4 cm-square
porous polyimide film.
[0180] Culturing was carried out in a cell culturing apparatus, and
the cells were fixed and stained (CellMask+DAPI and CellMask) after
3 days, 6 days and 10 days. Staining and observation under a
confocal microscope and a stereo fluorescent microscope were
carried out in the same manner as Example 8.
[0181] The results are shown in FIG. 13. The results indicated that
human umbilical vein endothelial cells, as human primary cultured
cells, can also be cultured by the method of the invention.
Example 10
Culturing of Vero Cells
[0182] For this example, Vero cells were cultured by the method of
the present invention using a porous polyimide film, and observed
under a confocal microscope and a stereo fluorescent microscope.
The porous polyimide films used were of the three types: 25 .mu.m,
40 .mu.m and 75 .mu.m. The culturing period was 1 to 15 days.
[0183] After adding 0.5 ml of cell culture medium (mixture of 10%
FBS and an antibiotic added to DMEM) to a 2 cm.times.2 cm
sterilized square vessel, a sterilized 1.4 cm-square porous
polyimide film was dipped therein with the A-surface of the mesh
structure facing upward. Vero cells were added in an amount of
4.times.10.sup.4 to the cell culture medium in the square vessel.
That is, 4.times.10.sup.4 Vero cells were naturally seeded for each
1.4 cm-square porous polyimide film.
[0184] Culturing was carried out in a cell culturing apparatus, and
the cells were fixed and stained after 1 day, 3 days, 7 days, 10
days and 15 days. The staining was accomplished with actin+DAPI,
and actin detection was with phalloidin. Observation under a
confocal microscope and a stereo fluorescent microscope were
carried out in the same manner as Example 8.
[0185] The results are shown in FIGS. 14-1 to 14-3. The results
indicated that Vero cells, a typical type of established cell line,
can also be cultured by the method of the present invention.
Example 11
Culturing of HeLa Cells
[0186] For this example, HeLa cells were cultured by the method of
the present invention using a porous polyimide film, and observed
under a confocal microscope and a stereo fluorescent microscope.
The porous polyimide films used were of the three types: 25 .mu.m,
40 .mu.m and 75 .mu.m. The culturing period, microscopes used and
specific steps were as described in Example 10.
[0187] The results are shown in FIGS. 15-1 to 15-3. The results
indicated that HeLa cells, a typical type of established cell line,
can also be cultured by the method of the present invention.
Example 12
Culturing of CHO Cells
[0188] For this example, CHO cells were cultured by the method of
the present invention using a porous polyimide film, and observed
under a confocal microscope and a stereo fluorescent microscope.
The porous polyimide films used were of the three types: 25 .mu.m,
40 .mu.m and 75 .mu.m. The culturing period was 1 to 7 days, and
the microscopes used and specific steps were as described in
Example 10.
[0189] The results are shown in FIGS. 16-1 to 16-3. The results
indicated that CHO cells, a typical type of established cell line,
can also be cultured by the method of the present invention.
* * * * *