U.S. patent number 10,385,303 [Application Number 15/363,300] was granted by the patent office on 2019-08-20 for methods of selective cell attachment/detachment, cell patternization and cell harvesting by means of near infrared rays.
This patent grant is currently assigned to Industry-Academic Cooperation Foundation, Yonsei University. The grantee listed for this patent is Industry-Academic Cooperation Foundation, Yonsei University. Invention is credited to June Seok Heo, Byeong Gwan Kim, Eun Kyoung Kim, Han Soo Kim, Hyun Ok Kim, Jeong Hun Kim, Tea Hoon Park, Jung Mok You.
View All Diagrams
United States Patent |
10,385,303 |
Kim , et al. |
August 20, 2019 |
Methods of selective cell attachment/detachment, cell
patternization and cell harvesting by means of near infrared
rays
Abstract
The present invention relates to a method for selective cell
attachment/detachment, cell patternization and cell harvesting by
means of near infrared rays. More particularly, conducting polymers
or metal oxides having exothermic characteristics upon irradiation
of near infrared light is used as a cell culture scaffold, thus
selectively attaching/detaching cells without an enzyme treatment.
The scaffold has an effect of promoting proliferation or
differentiation of stem cells, and therefore, can be used as a stem
cell culture scaffold. The scaffold enables cell
attachment/detachment without temporal or spatial restrictions,
thus enabling cell patternization.
Inventors: |
Kim; Eun Kyoung (Seoul,
KR), Kim; Hyun Ok (Gyeonggi-do, KR), You;
Jung Mok (Seoul, KR), Kim; Jeong Hun (Seoul,
KR), Park; Tea Hoon (Gyeongnam, KR), Kim;
Byeong Gwan (Gyeonggi-do, KR), Heo; June Seok
(Seoul, KR), Kim; Han Soo (Gyeonggi-do,
KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Industry-Academic Cooperation Foundation, Yonsei
University |
Seoul |
N/A |
KR |
|
|
Assignee: |
Industry-Academic Cooperation
Foundation, Yonsei University (Seoul, KR)
|
Family
ID: |
58257242 |
Appl.
No.: |
15/363,300 |
Filed: |
November 29, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20170073627 A1 |
Mar 16, 2017 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
14342451 |
|
|
|
|
|
PCT/KR2013/003079 |
Apr 12, 2013 |
|
|
|
|
Foreign Application Priority Data
|
|
|
|
|
Apr 12, 2012 [KR] |
|
|
10-2012-0037954 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
23/20 (20130101); C08J 5/18 (20130101); C12N
5/0068 (20130101); C12M 33/00 (20130101); C12N
2535/10 (20130101); C12N 2539/10 (20130101); C08J
2333/16 (20130101); C08J 2335/02 (20130101); C08J
2379/02 (20130101); C08J 2333/14 (20130101); C08J
2365/00 (20130101); C12N 2533/30 (20130101); C12N
2529/10 (20130101) |
Current International
Class: |
C12M
1/26 (20060101); C12M 1/00 (20060101); C08J
5/18 (20060101); C12N 5/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1994-335381 |
|
Dec 1994 |
|
JP |
|
2007-075034 |
|
Mar 2007 |
|
JP |
|
2008-228585 |
|
Oct 2008 |
|
JP |
|
Other References
You J, et al "Noninvasive Photodetachnnent of Stem Cells on Tunable
Conductive Polymer Nano Thin Films: Selective Harvesting and
Preserved Differentiation Capacity" ACSNano, Apr. 12, 2013,7(5),pp.
4119-4128; doi: 10.1021/nn400405t. (Year: 2013). cited by
examiner.
|
Primary Examiner: Kosar; Aaron J
Attorney, Agent or Firm: Hammer & Associates, P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of co-pending U.S.
application Ser. No. 14/342,451, filed Mar. 3, 2014.
Claims
What is claimed is:
1. A method for detaching cultured cells comprising irradiating
cells cultured on a cell culture container with near-infrared
radiation to detach the cells from the cell culture container,
wherein the cell culture container comprises a cell culture region
in which a polymer or copolymer film of at least one monomer
selected from the group consisting of compounds of Formulas 1a to
1j having an absorbance in a near-infrared region is formed:
##STR00006## ##STR00007##
2. The method for detaching cultured cells according to claim 1,
wherein the cell culture region is formed of any one of
polycarbonate, polypropylene, polyethylene, polystyrene,
polyurethane, polyethylene terephthalate, polyester, polyimide,
polyethylene glycol, polydimethylsiloxane, and a copolymer or
composite thereof; Nylon; paper; cotton; and glass.
3. The method for detaching cultured cells according to claim 1,
wherein the polymer or copolymer has a weight average molecular
weight of 1,000 to 1,000,000 Da.
4. The method for detaching cultured cells according to claim 1,
wherein the polymer or copolymer film has a thickness of 10 nm to 1
mm.
5. The method for detaching cultured cells according to claim 1,
wherein the cells include adult stem cells obtained from breasts,
bone marrow, cord blood, blood, liver, skin, gastrointestine,
placenta, or womb.
Description
BACKGROUND
1. Field of the Invention
The present invention relates to methods for selectively detaching,
patterning, and harvesting cells using near-infrared capable of
being used in cell culture and detaching cells without trypsin.
2. Discussion of Related Art
Stem cells are cells having capabilities of self-replication and
differentiation into at least two cells, and may be classified into
totipotent stem cells, pluripotent stem cells, and multipotent stem
cells.
Recently, therapeutic methods using such stem cells capable of
being continuously self-replicated and differentiated into various
tissues in the body are widely used, boosted by development of
biotechnology. Particularly, such methods start to be used to treat
incurable diseases such as Parkinson's disease, cancer, diabetes,
etc. as well as human organ regeneration (Miyahara Y. et al.,
Nature Medicine, 12(4), 459-465, 2006; Kang, K. S. et al., Stem
Cells, 24(6), 1620-1626, 2006; Silva, G. V. et al., Circulation,
18, 111, 2005). While various therapeutic methods using stem cells
have been developed so far, there is still less research on the
characteristics of stem cells, and there is a limit to treatment
using stem cells due to limits to proliferation and differentiation
of stem cells.
Generally, it is known that a fate of differentiated stem cells is
often influenced by a cell to cell, and a cell to extracellular
matrix (ECM) including growth factors, and also by an instructive
environment (Nakayama et al, Neurosci Res, 46, 241-249, 2003).
Recently, as research on interaction between an environment of stem
cells and the stem cells, a bioengineering field is emerging. It is
not a method of controlling a hormone, growth factor, or serum
included in a cell culture, which is conventionally used in
research or induction of the function of a cell, but a method of
controlling attachment, proliferation, differentiation, and
secretion to an extracellular matrix, which are characteristics of
a cell, through interaction between a support to which the cell is
attached and grown and the cell (Bauer S. et al., Acta
Biomaterialia, 4, 1576-1582, 2008; Guo L. et al., Biomaterials, 29,
23-32, 2008). To this end, chemical surface modification which is
used to develop a material having biocompatibility and change a
surface characteristic is a critical factor.
The pluripotent stem cells can be differentiated into various cells
and tissues derived from an ectoderm, a mesoderm, and an endoderm.
These cells are derived from an inner cell mass located in a
blastocyst generated after 4 to 5 days of fertilization, and called
embryo stem cells. They are differentiated into various different
tissues, but do not create a new organism.
The multipotent stem cells can be only differentiated into cells
specific to tissues and organs in which these cells are included.
They are involved in growth and development of tissues and organs
in an embryonic period, a neonatal period, and an adult period, and
functions of maintaining homeostasis of adult tissues and inducing
regeneration of damaged tissues, and tissue-specific multipotent
stem cells are generally called adult stem cells.
The adult stem cells are found in a stage in which individual
organs of embryos are formed after development or at an adult
stage, and differentiated only into cells generally constituting a
specific tissue. Such adult stem cells serve to replenish the loss
of cells normally or pathologically occurring in most of organs in
an adult. Exemplary adult stem cells include hematopoietic stem
cells (HSCs) and mesenchymal stem cells (MSCs). It is known that
the HSCs are usually differentiated into blood cells in blood such
as erythrocytes, leukocytes, and thrombocytes, and the MSCs are
differentiated into cells of mesodermal tissues such as
osteoblasts, chondroblasts, adipocytes, and myoblasts.
Stems cells can be differentiated into various cells according to
how to differentiate or treat the stem cells. To control the
differentiation capability of the stem cells, it is important to
research and control the interaction between cell-to-cell and
cell-to-extracellular matrix (ECM) including growth factors.
Generally, as a conventional technique to detach cells, an enzyme
called trypsin is widely used. The trypsin chemically damages a
bond in a cell attached to a cell culture container, resulting in
damage to a cell wall or a protein present in the cell wall of a
stem cell. Accordingly, when the trypsin is used, stem cells may be
damaged, and thus degradation in proliferation capacity and
differentiation potency may occur. In addition, since the trypsin
is treated entirely to a culture container, it may be difficult to
partially obtain a desired cell.
For this reason, there is a demand for developing a new technique
to easily detach cells from a culture container, and to detach
cells only from a desired part without damage to the cells.
SUMMARY OF THE INVENTION
The present invention is directed to providing a cell culture
container for culturing cells on a surface of a conductive compound
or metal oxide film that can absorb near-infrared, and easily and
selectively detaching cells without damage to the cells using a
photothermal characteristic of the conductive compound or metal
oxide by near-infrared radiation, a cell culture kit including the
same, and a method of proliferating, differentiating, or detaching
cells using the kit.
The present invention is also directed to providing a patterned
substrate for cell culture for easily detaching cells using a
photothermal characteristic of a conductive compound or metal oxide
by near-infrared radiation.
One aspect of the present invention provides a cell culture
container including a cell culture region in which a conductive
polymer or metal oxide film having absorbance in a near-infrared
region is formed.
Another aspect of the present invention provides a kit for cell
culture including the cell culture container of the present
invention and an apparatus for irradiating near-infrared.
Still another aspect of the present invention provides a method of
proliferating or differentiating stem cells including culturing
adult stem cells in a cell culture container.
Yet another aspect of the present invention provides a method of
detaching cultured cells by irradiating a cell culture container
with near-infrared.
Yet another aspect of the present invention provides a patterned
substrate for cell culture, which includes a substrate and a cell
culture region formed on the substrate and containing a conductive
polymer or metal oxide film having an absorbance in a near-infrared
region.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features, and advantages of the
present invention will become more apparent to those of ordinary
skill in the art by describing in detail exemplary embodiments
thereof with reference to the attached drawings, in which:
FIG. 1 is an absorption spectrum of a heterocyclic compound of
Formula 1a according to the present invention;
FIG. 2 shows a photothermal effect by near-infrared absorption (808
nm) of a heterocyclic compound of Formula 1a according to the
present invention;
FIG. 3 shows a proliferation rate of stem cells confirmed using an
oxidized or reduced (reduced and thus neutral) film manufactured of
a heterocyclic compound of Formula 1 d according to the present
invention;
FIG. 4 is a microscope image showing detachment of stem cells
cultured on a film manufactured of a heterocyclic compound of
Formula 1a according to the present invention from a selective
region by near-infrared irradiation;
FIG. 5 shows an a detached area of stem cells proportional to
near-infrared irradiation time;
FIG. 6 is a microscope image of stem cells detached from a film
manufactured of a heterocyclic compound of Formula 1e according to
the present invention by near-infrared irradiation, and cultured in
a new cell culture container; and
FIG. 7 shows results of differentiation of the stem cells detached
from a film manufactured of a heterocyclic compound of Formula 1e
according to the present invention by near-infrared irradiation
into (a) osteocytes, (b) adipocytes, and (c) chondrocytes in a cell
culture container for 16 days.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
Hereinafter, exemplary embodiments of the present invention will be
described in detail. However, the present invention is not limited
to the embodiments disclosed below, but can be implemented in
various forms. The following embodiments are described in order to
enable those of ordinary skill in the art to embody and practice
the present invention.
Although the terms first, second, etc. may be used to describe
various elements, these elements are not limited by these terms.
These terms are only used to distinguish one element from another.
For example, a first element could be termed a second element, and,
similarly, a second element could be termed a first element,
without departing from the scope of exemplary embodiments. The term
"and/or" includes any and all combinations of one or more of the
associated listed items.
It will be understood that when an element is referred to as being
"connected" or "coupled" to another element, it can be directly
connected or coupled to the other element or intervening elements
may be present. In contrast, when an element is referred to as
being "directly connected" or "directly coupled" to another
element, there are no intervening elements present.
The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
exemplary embodiments. The singular forms "a," "an," and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. It will be further understood that the
terms "comprises," "comprising," "includes," and/or "including,"
when used herein, specify the presence of stated features,
integers, steps, operations, elements, components, and/or groups
thereof, but do not preclude the presence or addition of one or
more other features, integers, steps, operations, elements,
components, and/or groups thereof.
With reference to the appended drawings, exemplary embodiments of
the present invention will be described in detail below. To aid in
understanding the present invention, like numbers refer to like
elements throughout the description of the figures, and the
description of the same elements will be not reiterated.
The present invention relates to a cell culture container including
a cell culture region in which a conductive polymer or metal oxide
film having an absorbance in a near-infrared region is formed.
The "cell culture container" used herein refers to a container used
in conventional cell culture, and may be formed of a material
suitable for cell culture, for example, any one of polycarbonate,
polypropylene, polyethylene, polystyrene, polyurethane,
polyethylene terephthalate, polyester, polyimide, polyethylene
glycol, polydimethylsiloxane, or a copolymer or composite thereof;
Nylon; paper; cotton; or glass. The material is preferably
transparent to count cells under a microscope, but may be colored.
The container may have a smooth surface, and may be formed in a
round or square shape, but the present invention is not limited
thereto. The container may be manufactured in a shape suitable for
characteristics of a cell or its use, and by special treatment such
as insertion of a regular pattern on a substrate. The cell culture
container may be formed in a cylindrical, rectangular, or polygonal
structure, but the present invention is not limited thereto. The
cell culture container includes a cell culture region to which a
cell is attached to be cultured, and may be a flask, or an enclosed
structure such as a petri dish, but the present invention is not
particularly limited thereto.
The cell culture container of the present invention is
characterized by forming a conductive polymer or metal oxide film
having an absorbance in a near-infrared region above a cell culture
region in which cells are cultured in the cell culture container
having the above-described shape and formed of the above-described
material.
Since the conductive polymer or metal oxide film uses a
photothermal characteristic of the conductive polymer or metal
oxide generating heat by converting light energy into thermal
energy due to absorption of near-infrared, when the film is used as
a cell support, cells may be easily detached from the
heat-generated part during the near-infrared irradiation without
damage to a cell wall or a cell wall protein according to
conventional trypsin treatment, and therefore repetitively used in
cell culture and detachment.
In addition, according to one embodiment, when the film is used as
a stem cell culture support, a proliferation rate of the stem cells
are higher than that of stem cells in a common cell culture
container, and the selectively detached stem cells can be subjected
to additional culture and differentiation.
Accordingly, the conductive polymer or metal oxide film may be used
as a support for cell proliferation or differentiation.
The conductive polymer or metal oxide film may be manufactured of a
polymer or copolymer of a conductive monomer or a metal oxide
having an absorbance in a near-infrared region.
In the present invention, near-infrared is in a wavelength range
from 700 to 2500 nm, and conductive monomers having an absorbance
in the near-infrared region of the present invention may also have
an absorbance in the above range. According to an embodiment, in
measurement of an absorbance at a wavelength of approximately 808
nm, when the near-infrared is irradiated for up to 300 seconds, a
pyrogenic effect of approximately 25.degree. C. may be
exhibited.
The conductive monomer may be at least one selected from the group
consisting of a heterocyclic compound represented by Formula 1 and
aniline.
##STR00001##
In Formula 1, X is N, O, S, Se, or Te,
R.sub.1 and R.sub.2 are the same as or different from each other,
each of which is a hydrogen atom,
--(CH.sub.2).sub.l--O--(CH.sub.2)m-(CF.sub.2).sub.n--(CR.sub.7R.sub.8).su-
b.k--(CH.sub.2).sub.d--Z,
##STR00002## --O--CH(R.sub.3)--CH(R.sub.4)--O--, or
--O--CH.sub.2--C(R.sub.5)(R.sub.6)--CH.sub.2--O--. However, R.sub.1
and R.sub.2 are not simultaneously hydrogen. R.sub.3, R.sub.4,
R.sub.5, and R.sub.6 are the same as or different from each other,
each of which is a hydrogen atom, --(CH.sub.2).sub.d--Z,
--(CH.sub.2).sub.l--O--(CH.sub.2).sub.m--(CF.sub.2).sub.n--(CR.sub.7R.sub-
.8).sub.k--(CH.sub.2).sub.d--Z, or
##STR00003## but when R.sub.3 and R.sub.4 are simultaneously
hydrogen, R.sub.5 and R.sub.6 are not simultaneously hydrogen.
R.sub.7 and R.sub.8 are the same as or different from each other,
each of which is hydrogen, an alkyl group having 1 to 5 carbon
atoms, or --(CH.sub.2).sub.d--Z. Z is a methacrylate group or an
acrylate group, l is an integer from 0 to 2, m is an integer from 0
to 3, n is an integer from 0 to 5, k is an integer from 0 to 4, a
is an integer from 0 to 2, b is an integer from 0 to 7, and d is an
integer from 0 to 2.
Preferably, the heterocyclic compound of Formula 1 may be at least
one of Formulas 1a to 1k.
##STR00004## ##STR00005##
The conductive polymer may have a weight average molecular weight
of 1,000 to 1,000,000 Da.
The conductive polymer refers to a polymerized product produced by
polymerization of the above-described heterocyclic compound and/or
aniline, which is a polymer or copolymer polymerized using an
electrical, chemical, thermal, or optical method or an
initiator.
The conductive polymer may be prepared by polymerizing a
heterocyclic compound through solution polymerization using a
conventional catalyst, electropolymerization using electricity
[Macromolecular Research, 17, 791-796, 2009], vapor polymerization
[Macromolecules, 43, 2322-2327, 2010], solution coating
polymerization [Advanced Materials, 23, 4168-4173, 2011], or
emulsion polymerization in an aqueous phase. The
electropolymerization, vapor polymerization, solution coating
polymerization, or emulsion polymerization for preparing particles
used herein induces oxidative polymerization of the heterocyclic
compound of the present invention, and the polymerization method
using a conventionally used catalyst (acid, oxidant, etc.) is a
conventional method used in polymerization of a monomer such as
aniline as well as a heterocyclic compound.
In the method of preparing a conductive polymer film of the present
invention, the conductive polymer may be directly coated on various
substrates using the polymerization method, the conductive polymer
dissolved in a solvent may be secondarily coated using various
coating methods such as spin coating, printing coating, etc. after
synthesis, a conductive polymer particle synthesized by an emulsion
method may be dispersed in a solvent and secondarily coated to form
a film. The present invention is not limited to the coating method,
but various coating methods may be suitably used according to a
compound or process, or the range of use or application range.
For example, when a doping state of a conductive polymer thin film
is controlled, the conductive polymer thin film manufactured as
described above is put into an electrolyte solution (solvent)
without a monomer, circulated three times at a rate of 50 mV/s
between 1 to -1 V using cyclic voltammetry, washed with a deionized
solvent by removing power after the circulation is stopped for
several seconds at a voltage (between 1 to -1 V) in a desired
doping state, and dried.
The metal oxide may be magnesium oxide, strontium oxide, zinc
oxide, aluminum oxide, or arsenic oxide, which is used alone or in
combination of at least two thereof.
The conductive polymer or metal oxide film may have a thickness of
10 nm to 1 mm. When the thickness is less than 10 nm, the film is
not easily formed and a photothermal phenomenon or an effect
thereof occurring in the film is low. When the thickness of the
film is more than 1 mm, it is difficult to form the film likewise,
and when the absorbance of the material is high, it is necessary to
transfer heat generated by the photothermal phenomenon from a part
adjacent to a substrate, and here, time necessary to detach cells
may be quite long. In addition, since the cell culture container of
the present invention may be applied to a conventional cell
culture, it is not limited to the kind of the cells, and for
example, may be used in adult stem cell culture.
The "adult stem cells" used herein refer to stem cells shown in a
stage in which an organ of an embryo is formed after development or
an adult stage, and are only limited to cells generally
differentiated into a specific tissue.
The adult stem cells of the present invention may be separated to
use from adult stem cells derived from the breast, bone marrow,
cord blood, blood, liver, skin, gastrointestine, placenta, or womb.
The adult stem cells include neural stem cells capable of being
differentiated into astrocytes, hematopoietic stem cells capable of
being differentiated into myelocytes, mesenchymal stem cells
capable of being differentiated into a bone, cartilage, lipid,
muscle, etc., and liver stem cells capable of being differentiated
into hepatocytes. Among these, the mesenchymal stem cells are cells
having the ability of differentiation into various musculoskeletal
cells such as osteocytes, chondrocytes, adipocytes, muscle cells,
and fibrocytes.
Since the mesenchymal stem cells are present in cord blood
(umbilical cord) and a bone marrow, they are more easily separated
than other adult tissues, and there is an endeavor to use of the
mesenchymal stem cells in treatment of various diseases including
such musculoskeletal diseases. Unlike other stem cells, the
mesenchymal stem cells are easily cultured to amplify in a bone
marrow, unlike that has been known so far, the stem cells can be
differentiated into mesoderm-, endoderm-, or ectoderm-derived
cells, do not have rejection to immunity due to use of a self cell,
and there is a bare chance that cells not differentiated in a
desired direction, unlike embryonic stem cells, induce a cancer,
which are very important in clinic.
The term "differentiation" used herein refers to a phenomenon in
which a structure or function of cells is specified while the cells
are divided, proliferated, and then developed, that is, a change in
a shape or function of cells or tissues of an organism to execute a
work given thereto. Generally, the differentiation is a phenomenon
of dividing a system into at least two subsystems having different
properties.
The term "proliferation" used herein refers an increase in the same
kind of cells by division, that is, generally, an increase in cell
counts in a multicellular organism. When a cell count reaches a
certain level due to proliferation of cells, a trait (or
characteristics) is generally differentiated and controlled. The
increase in cells in the body and neogenesis of cytoplasms in cells
are generally classified as growth. However, since the cell count
increases in a biological aspect, it is appropriate that a period
in which differentiation does not occur in an embryo stage of the
multicellular organism is considered proliferation.
When adult stem cells are cultured on a conductive polymer or metal
oxide film that is reduced and in a neutral state in the cell
culture container of the present invention, proliferation of the
cells increases, and when the cell culture container is irradiated
with near-infrared, the cells are detached without damage due to a
pyrogenic effect of the conductive polymer or metal oxide film, and
the detached stem cells are transferred to a new cell culture
container for normal proliferation and differentiation.
The present invention also relates to a cell culture kit including
the cell culture container of the present invention, and an
apparatus for irradiating near-infrared.
Since the kit of the present invention includes the cell culture
container using a polymer film as a cell support, proliferation
during cell culture is stimulated, cells are detached in an
irradiated region by near-infrared, and the polymer film at the
cell-detached part is not removed and thus repeatedly used in cell
culture and detachment. Particularly, the kit may be effectively
used in harvest of stem cells, individual separation of stem cells,
or research on a characteristic of one stem cell.
Generally, to detach stem cells in a cell culture container (tissue
culture polystyrene) during the harvest of the stem cells, the stem
cells being proliferated in the container should be entirely
detached using a trypsin enzyme. According to the present
invention, when the stem cells are cultured on a surface of a
conductive polymer film, the cells may be simply harvested by
irradiating near-infrared not harmful to the cells without trypsin,
and stem cells having a desired size and in a desired region may be
selectively detached. That is, the conventional method is difficult
to individually separate stem cells or research a characteristic of
an individual stem cell, but the present invention can control a
size of detachment region, that is, the number of harvested cells,
and individually detach stem cells one by one.
Rays for detaching the cells may be laser beams, and radiation may
be performed for 30 seconds to 10 hours at 1 .mu.W/cm.sup.2 to 300
W/cm.sup.2, and preferably 100 mW/cm.sup.2 to 250 W/cm.sup.2.
Accordingly, the present invention provides a method of detaching
cultured cells by irradiating the cell culture container with
near-infrared.
In addition, when a conductive polymer film prepared by reducing an
oxidized conductive film into a neutral state is used, stem cell
proliferation increases compared with that in a common cell culture
container, and therefore the conductive polymer film may be useful
in cell therapy using stem cells.
Since the stem cells harvested using the conductive film can be
normally cultured and proliferated, and when the stem cells are
induced to be differentiated into predetermined cells, adult stem
cells may be efficiently differentiated into osteocytes,
adipocytes, or chondrocytes.
In addition, the conductive film is manufactured and then a doping
degree thereof may be controlled as shown in Examples 19 and 20 to
be described later. For example, when the doping degree is
controlled thereby reducing the conductive film manufactured in an
oxidized state into a neutral state as shown in Example 19, as
shown as reduced-PEDOT in FIG. 3, cell culture efficiency may be
enhanced, compared with the control TCPS used in a conventional
cell culture container.
Accordingly, the present invention provides a method of
proliferating or differentiating stem cells including culturing
adult stem cells in the cell culture container of the present
invention.
The present invention also relates to a patterned substrate for
cell culture, which includes a substrate, and a cell culture region
formed on the substrate and containing a conductive polymer or
metal oxide film having an absorbance in a near-infrared
region.
The patterned substrate for cell culture is used in culture of
cells of blood vessels capable of forming tissues, and may
efficiently arrange cells in regularity.
Since the patterned substrate for cell culture uses the film as a
cell support, the cells can be detached without enzyme treatment
during near-infrared irradiation, and cells may be still cultured
in a cell culture region that is not subject to near-infrared
irradiation.
The conductive polymer or metal oxide film having an absorbance in
the near-infrared region has excellent cell adhesion, and thus
cells are possibly attached to a cell culture region without a
separate cell adhesive layer.
The substrate may be at least one of insulating substrates such as
metal, glass, silicon, or plastic.
The patterned substrate for cell culture may have a patterned
non-cell culture region in which a layer inhibiting cell attachment
to a cell culture region.
Hereinafter, the present invention will be described in detail by
means of Examples. However, it should be understood that the
following Example are given by way of illustration of the present
invention only, and are not intended to limit the scope of the
present invention.
<Preparation Example 1> Culture of Bone Marrow Mesenchymal
Stem Cells
The human bone marrow used herein was normally obtained by consent
of a patient approved by Institutional Review Board (IRB) of
Severance Hospital, and the experiment was approved by
Institutional Review Board (IRB) of Severance Hospital in Korea.
Blood obtained from a human bone marrow was subject to Ficoll
gradient separation in a ratio of Ficoll-pague:bone marrow
blood=1:1.5. A blood sample was slowly poured into a 15 mL Ficoll
solution to separate layers, and centrifuged, thereby confirming
formation of a thin buffy coat layer on an intermediate layer of a
tube, and then the buffy coat layer was separated and transferred
to a new tube. Phosphate buffered saline (PBS) was added to the
tube to prepare a total 50 ml solution, the solution was
centrifuged at 2000 rpm for 10 minutes, a supernatant was
discarded, 50 mL PBS was added to a precipitate, the tube was
stirred to uniformly mix the contents and then centrifuged again at
1500 rpm for 5 minutes, and a supernatant was discarded, resulting
in obtaining cells. The cells were suspended in a medium [DMEM (low
glucose)+1% P/S+10% FBS], and then diluted with a medium such that
1.times.10.sup.7 cells were included in a 100 mm petri dish (an
amount of the medium in the petri dish was designed to 10 mL).
After the cells were cultured for one day in a CO.sub.2 incubator,
a supernatant was transferred to a new petri dish, and a culture
medium having the same components as the medium used in the initial
culture was filled on the cells attached to a bottom of the petri
dish. After 7 to 10 days, the cells were maintained by being
detached using trypsin and seeded in a new flask at
2.times.10.sup.5 per T75-flask, resulting in culture and
maintenance of adult stem cells.
<Example> Manufacture of Film Using Conductive Compound
Films were manufactured using conductive polymers of the present
invention prepared by polymerizing conductive monomers of Formulas
1a to 1k described above by a method such as solution coating
polymerization, vapor polymerization, electropolymerization, or
chemical polymerization according to the conditions shown in Table
1. The electropolymerization, vapor polymerization, solution
coating polymerization, or emulsion polymerization for preparing
particles was used to induce oxidative polymerization of the
conductive monomers of the present invention described above, and a
polymerization method using a conventionally used catalyst (acid,
oxidant, etc.) is a conventional method used in polymerization of a
monomer such as a heterocyclic compound or aniline.
To manufacture the conductive polymer film, the conductive polymer
could be directly coated on various substrates using the
above-described polymerization method. However, a conductive
polymer dissolved in a solvent was secondarily coated by spin
coating after being synthesized, and conductive polymer particles
synthesized by an emulsion method are dispersed in a solvent and
then secondarily coated.
In Table 1, a solvent used in electropolymerization is an
electrolyte. In addition, when a doping state of the conductive
polymer thin film was controlled, the conductive polymer thin film
manufactured as described above was put into an electrolyte
solution without a monomer, and circulated three times between 1
and -1 V at a rate of 50 mV/s through cyclic voltammetry. At a
desired doping voltage (a voltage between 1 to -1 V), the
circulation was stopped for several seconds, power was removed, and
then a resulting analyte was washed with a pure solvent and dried.
In the emulsion polymerization, a value specified at a thickness of
the polymer refers to a diameter of a particle. A cell detachment
efficiency shown below is a value obtained by converting a ratio of
an area of a part from which a cell is detached to an area of a
near-infrared radiation region with 100.
TABLE-US-00001 TABLE 1 near- near- Preparation method Thickness
infrared infrared Cell & conditions of absorbance radiation
detachment (solvent, temperature polymer (wavelength: time
efficiency Example Compound (C. .degree.)) (nm) 808 nm) (minutes)
(%) 1 1a Solution coating 150 0.72 5 100 polymerization (butanol,
50) 2 1a Solution coating 50 0.48 10 110 polymerization
(isopropanol, 50) 3 1a Vapor 160 0.75 3 100 polymerization
(isopropanol, 70) 4 1a Electropolymerization 250 0.88 2 90
(n-Bu.sub.2NClO.sub.4(0.1M)) 5 1b Solution coating 130 0.65 5 80
polymerization (butanol, isopropanol, 70) 6 1b Vapor 150 0.7 6 100
polymerization (isopropanol, 80) 7 1c Solution coating 150 0.68 10
110 polymerization (butanol, 80) 8 1d Solution coating 150 0.7 20
110 polymerization (butanol, isopropanol, 60) 9 1e Solution coating
150 0.75 30 105 polymerization (ethanol, 40) 10 1e
Electropolymerization 140 0.7 10 93 (n-Bu.sub.2NClO.sub.4(0.1M)) 11
1f Solution coating 350 0.9 15 108 polymerization (butanol, 80) 12
1g Solution coating 160 0.62 10 90 polymerization (butanol,
isopropanol, 60) 13 1h Solution coating 150 0.58 10 87
polymerization (butanol, 90) 14 1i Solution coating 500 0.85 5 90
polymerization (butanol, isopropanol, 70) 15 1j Solution coating
160 0.6 25 89 polymerization (butanol, 80) 16 Aniline Solution
coating 250 0.78 40 115 polymerization (isopropanol, 50) 17 Aniline
Spin coating after 170 0.66 5 90 chemical polymerization (methylene
chloride) 18 1k Emulsion 150 0.72 5 95 polymerization 19 1a
Reduction after 110 0.28 30 70 solution coating polymerization
(-0.2 V, 30 sec) (isopropanol, 50) 20 1b Partial doping after 120
0.55 10 100 vapor polymerization (0.4 V)(isopropanol, 80)
<Experimental Example 1> Near-Infrared Absorbance Test for
Film Using Conductive Compound
An absorbance of the conductive polymer film prepared in Example 1
(or 2) was obtained at a range from 200 to 3300 nm using a
UV-Visible spectrum. Within the range, the absorbance was shown at
808 nm corresponding to a wavelength of a near-infrared laser in
Table 1.
<Experimental Example 2> Measurement of Photothermal Effect
Through Near-Infrared of Film Using Conductive Compound
A conductive polymer film prepared in Example 3 (or 4) was placed
on a stand set such that near-infrared was radiated from a bottom
thereof, and the photothermal effect was measured. A near-infrared
laser at 808 nm was fixed to output energy at 230 mW, and radiated
to a bottom of the prepared conductive polymer film. The
photothermal effect was confirmed by measuring a temperature of a
top of the conductive polymer film through a T-type thermocouple.
In the corresponding step, the photothermal effect of the
conductive polymer film could be shown as a temperature value
measured according to near-infrared laser irradiation time.
As shown in FIG. 2, it was known that the temperature was increased
by 25.degree. C. or more by the near-infrared irradiation.
<Experimental Example 3> Method of Culturing Stem Cells on
Film Using Conductive Compound and Selectively Detaching the Stem
Cells
The conductive polymer film prepared in Example 8 was sterilized
using weak UV rays for approximately 2 minutes, and used as a
support in culture of stem cells. Culture of stem cells was
performed by putting bone marrow-derived mesenchymal stem cells
into a 6-well plate containing a conductive polymer film, and 230
mW of near-infrared was radiated from a bottom of the 6-well plate
for selective detachment.
As shown from reduced-PEDOT in FIG. 3, it was noted that, as the
stem cells were cultured by using a reduced neutral conductive film
as a support, a proliferation rate of the stem cells was higher
than that of the stem cells in a common cell culture container, and
thus could be effective in cell therapy using the stem cells.
In addition, as shown in FIGS. 4 and 5, a cell detachment area and
a cell count were possibly controlled by near-infrared irradiation
time.
<Experimental Example 4> Confirmation of Stem Cell
Differentiation
The conductive polymer film prepared in Example 9 (or 10) was
sterilized using weak UV rays for approximately 2 minutes, and used
as a support in culture of stem cells. Culture of stem cells was
performed by putting bone marrow-derived mesenchymal stem cells
into a 6-well plate containing a conductive polymer film, and 230
mW of near-infrared was radiated from a bottom of the 6-well plate
for selective detachment. FIG. 6 shows various microscope images
taken after detached stem cells are transferred to a cell
container. Afterward, differentiation was induced for 16 days
through conditions for differentiating into osteocytes, adipocytes,
and chondrocytes. Here, the group of stem cells cultured on TCPS
without a conductive film and then differentiated was determined as
a control.
To confirm osteocyte differentiation, after 16 days of the culture,
a medium was removed from the control, the cell pellet was washed
with PBS, and then the PBS was removed. After the removal,
distilled water was added to the cell pellet and then removed,
which was repeated three times. A 3% silver nitrate solution
filtered through a filter paper was added to the cell pellet, and
then stored at room temperature for 30 minutes by covering it with
a foil. After 30 minutes, color change in the cell pellet was
induced by removing the foil and the added silver nitrate solution
and exposing the cell pellet to fluorescent light, and then
observed under an optical microscope.
To confirm adipocyte differentiation, after 16 days of the culture,
a medium was removed from the control, the cell pellet was washed
with PBS, and then the PBS was removed. Here, the cell pellet was
treated with 10% formalin, and stayed at room temperature for 30
minutes. Afterward, the formalin was removed, and then the cell
pellet was washed with distilled water. After the removal, the cell
pellet was treated with 60% isopropanol, and stayed at room
temperature for 5 minutes. The isopropanol was removed, and then
the cell pellet was treated with oil red-O filtrated through a
filter paper and stayed for 10 minutes. After 10 minutes, the cell
pellet was washed with tap water until the water became clean, and
a degree of dying was observed under an optical microscope.
To confirm chondrocyte differentiation, after 16 days of the
culture, a medium was removed from the control, the cell pellet was
washed with PBS, and then the PBS was removed. The cell pellet was
treated with 1% Safranin-O solution filtrated through a filter
paper and stayed for 5 minutes. Afterward, the cell pellet was
washed three to four times with 1% acetic acid and then the acid
was removed. A degree of dying was observed under an optical
microscope.
As shown in FIG. 7, it was confirmed that the stem cells detached
by near-infrared irradiation were differentiated into osteocytes,
adipocytes, or chondrocytes after 16 days like the control.
The present invention is characterized by near-infrared absorption
characteristics depending on oxidation and reduction states, and
can be used in proliferation, selective detachment, and patterning
of cells, particularly, adult stem cells, from a desired location
without limitation to time or location using a conductive polymer
or metal oxide having a photothermal characteristic during
near-infrared irradiation as a support for cell attachment.
The present invention may be used in cell culture.
While the invention has been shown and described with reference to
certain exemplary embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details
may be made therein without departing from the spirit and scope of
the invention as defined by the appended claims.
* * * * *