U.S. patent application number 15/551595 was filed with the patent office on 2018-04-26 for in vitro fibrosis model, preparing method therefor, and use thereof.
The applicant listed for this patent is KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY. Invention is credited to Sang Heon KIM, Kang Won LEE, Kwi Deok PARK, Thanavel RAJANGAM.
Application Number | 20180112183 15/551595 |
Document ID | / |
Family ID | 57586437 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180112183 |
Kind Code |
A1 |
KIM; Sang Heon ; et
al. |
April 26, 2018 |
IN VITRO FIBROSIS MODEL, PREPARING METHOD THEREFOR, AND USE
THEREOF
Abstract
Provided are an in vitro fibrosis model, a method of preparing
the in vitro model, and use of the in vitro model, the in vitro
model including a cell cluster differentiated from mesenchymal
cells, wherein the cell cluster exhibits pathological
characteristics of fibrosis.
Inventors: |
KIM; Sang Heon; (Seoul,
KR) ; PARK; Kwi Deok; (Seoul, KR) ; LEE; Kang
Won; (Seoul, KR) ; RAJANGAM; Thanavel; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY |
Seoul |
|
KR |
|
|
Family ID: |
57586437 |
Appl. No.: |
15/551595 |
Filed: |
May 25, 2016 |
PCT Filed: |
May 25, 2016 |
PCT NO: |
PCT/KR2016/005501 |
371 Date: |
August 17, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2533/30 20130101;
G01N 33/5008 20130101; G01N 33/50 20130101; C12N 2320/10 20130101;
C12N 5/0662 20130101; C12N 2513/00 20130101; G01N 33/5073 20130101;
C12N 5/0667 20130101; C12N 2506/13 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 2015 |
KR |
10-2015-0089090 |
Claims
1. An in vitro fibrosis model comprising a cell cluster
differentiated from mesenchymal cells, wherein the cell cluster
exhibits pathological characteristics of fibrosis.
2. The model of claim 1, wherein the mesenchymal cells are selected
from the group consisting of adipose stem cells, mesenchymal stem
cells, mesenchymal stromal cells, bone marrow stem cells, and
fibroblasts.
3. The model of claim 1, wherein the cell cluster is spherical and
has a diameter in a range of about 300 .mu.m to about 2,000
.mu.m.
4. The model of claim 1, wherein the cell cluster is differentiated
by adhering mesenchymal cells to a culture container comprising a
hydrophobic surface and culturing the mesenchymal cells in the
culture container.
5. The model of claim 4, wherein the mesenchymal cells are further
cultured for at least 12 hours after the mesenchymal cells are
differentiated into the cell cluster.
6. The model of claim 1, wherein the pathological characteristics
of fibrosis comprise at least one selected from the group
consisting of: formation of excessive connective tissue; deposition
of collagen; increased expression, secretion, or synthesis of a
fibrosis-related molecule including at least one selected from the
group consisting of transforming growth factor (TGF)-beta, Smad,
laminins, and smooth muscle actin (SMA); and increased cell death,
or a combination thereof, in the cell cluster or cells constituting
the cell cluster.
7. The model of claim 1, wherein the fibrosis comprises at least
one selected from the group consisting of idiopathic pulmonary
fibrosis (IPF), pulmonary fibrosis, interstitial lung disease,
nonspecific interstitial pneumonia (NSIP), usual interstitial
pneumonia (UIP), endomyocardial fibrosis, mediastinal fibrosis,
bone marrow fibrosis, retroperitoneal fibrosis, progressive massive
fibrosis, nephrogenic systemic fibrosis, Crohn's disease, chronic
myocardial infarction, scleroderma/systemic sclerosis,
neurofibromatosis, Hermansky-Pudlak syndrome, diabetic kidney
disease, renal fibrosis, hypertrophic cardiomyopathy (HCM),
hypertension-related nephropathy, renal tubulointerstitial
fibrosis, focal segmental glomerulosclerosis (FSGS),
radiation-induced fibrosis, fibroids, alcoholic liver disease,
liver steatosis, liver fibrosis, liver cirrhosis, Hepatitis C Virus
(HCV) infection, chronic rejection of transplanted organ, fibrotic
skin disease, keloidal scar, Dupuytren's contracture, Ehlers-Danlos
syndrome, epidermolysis bullosa dystrophica, oral submucous
fibrosis, and fiber proliferative disorder.
8. A method of preparing an in vitro fibrosis model, the method
comprising: forming a cell cluster by adhering mesenchymal cells to
a culture container comprising a hydrophobic surface and culturing
the mesenchymal cells in the culture container; and establishing
pathological characteristics of fibrosis in the cell cluster by
performing additional culturing thereof for at least 12 hours.
9. The method of claim 8, wherein the forming of the cell cluster
is performed by separating the mesenchymal cells from the culture
container as a density of the mesenchymal cells increases.
10. The method of claim 8, wherein the hydrophobic surface of the
culture container is selected from the group consisting of a
silanized surface, a hydrocarbon-coated surface, a polymer surface,
and a metal surface.
11. The method of claim 8, wherein the mesenchymal cells are
adhered to the culture container by cell-substrate interactions
with the hydrophobic surface of the culture container or
interactions with a growth factor having adhesiveness to the
mesenchymal cells immobilized on the surface of the culture
container.
12. The method of claim 11, wherein the growth factor is
immobilized in the form of a polypeptide linker-growth factor
recombinant protein on the surface of the culture container by
using a polypeptide linker in such a way as to fuse an amino
terminus of the growth factor to a carboxyl terminus of the
polypeptide linker.
13. The method of claim 12, wherein the polypeptide linker is
selected from the group consisting of a maltose-binding protein
(MBP), a hydrophobin, and a hydrophobic cell penetrating peptide
(CPP).
14. A method of screening a therapeutic agent for fibrosis, the
method comprising: treating the in vitro fibrosis model of claim 1
with a test substance; and selecting, as a candidate substance for
treatment of fibrosis, the test substance which exhibits
improvement or treatment of pathological characteristics of
fibrosis in the cell cluster or cells thereof in the in vitro
fibrosis model, as compared with an untreated control group.
15. The method of claim 14, wherein the pathological
characteristics of fibrosis comprises at least one selected from
the group consisting of: formation of excessive connective tissue;
deposition of collagen; increased expression, secretion, or
synthesis of a fibrosis-related molecule including at least one of
transforming growth factor (TGF)-beta, Smad, laminins, and smooth
muscle actin (SMA); and increased cell death, or a combination
thereof, in the cell cluster or cells constituting the cell
cluster.
Description
TECHNICAL FIELD
[0001] The present invention relates to an in vitro fibrosis model,
a method of preparing the in vitro model, and use of the in vitro
model.
BACKGROUND ART
[0002] More than 5 million people worldwide currently suffer from
fibrosis, while each year 100,000 new patients are diagnosed with
fibrosis and 40,000 patients die from fibrosis.
[0003] Fibrosis is characterized by the development of excessive
connective tissue causing dysfunction and death of organs. Fibrosis
generally affects various organs, such as the kidneys, liver,
lungs, heart, skin, or bone marrow. Among these organs, renal
fibrosis including renal tubulointerstitial fibrosis or focal
segmental glomerulosclerosis is known to be difficult to treat and
irreversible.
[0004] Meanwhile, administration of bleomycin into a mouse to
prepare an experimental animal model of fibrosis or transformation
of a mouse to prepare an animal model of pulmonary fibrosis has
been known in the art. However, there is not yet a known model in
in vitro systems for studying fibrous tissue and developing
therapeutic agents. Therefore, in order to develop a therapeutic
agent for fibrosis, the development of an in vitro fibrosis model
that can mimic in vivo environments and exhibit pathological
characteristics of fibrosis is required.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0005] An aspect provides an in vitro fibrosis model including a
cell cluster differentiated from mesenchymal cells, wherein the
cell cluster exhibits pathological characteristics of fibrosis.
[0006] Another aspect provides a method of preparing an in vitro
model, the method including: forming a cell cluster by adhering
mesenchymal cells to a culture container comprising a hydrophobic
surface and culturing the mesenchymal cells in the culture
container; and establishing pathological characteristics of
fibrosis in the cell cluster by performing additional culturing
thereof for at least 12 hours.
[0007] Another aspect provides a method of screening a therapeutic
agent for fibrosis, the method including: treating the in vitro
fibrosis model with a test substance; and selecting, as a candidate
substance for treatment of fibrosis, the test substance which
exhibits improvement or treatment of pathological characteristics
of fibrosis in the cell cluster or cells thereof in the in vitro
fibrosis model, as compared with an untreated control group.
Technical Solution
[0008] An aspect provides an in vitro fibrosis model including a
cell cluster differentiated from mesenchymal cells, wherein the
cell cluster exhibits pathological characteristics of fibrosis.
[0009] The term "mesenchymal cells" used herein refers to
pluripotent stem cells that can self-proliferate and differentiate
into various lineages, and for example, refers to undifferentiated
mesodermal cells that can differentiate into loose tissue between
the mesoderm and the endoderm, connective tissue, dermis,
subcutaneous tissue, bone, cartilage, bone marrow, skeletal muscle,
smooth muscle, myocardium, blood cells, lymph nodes, lymphatic
vessels, blood vessels, spleen, stomach, or the like. The
mesenchymal cells may be separated from a subject, for example, a
mammal including a human or the like, and may include adipose stem
cells, mesenchymal stem cells, mesenchymal stromal cells, bone
marrow stem cells, or fibroblasts. The term "separated" used herein
in connection with the mesenchymal cells may refer to cells in an
environment different from an environment within which cells are
naturally produced. For example, considering that cells are
naturally produced in multicellular organs and cells removed from
the multicellular organs, cells are to be referred to as
"separated" cells.
[0010] The term "cell cluster" or "three-dimensional (3D) cell
cluster" (used interchangeably with the term `cellular tissue`)
used herein refers to a state in which two or more cells are
aggregated, and may be in the form of a tissue or in the form of
single cells. Each cell cluster may be present in the tissue itself
or in a part thereof, or may be present as a cluster of single
cells. The cell cluster may include cell-like organization of cells
differentiated from mesenchymal cells. In addition, the term
"three-dimensional (3D)" refers to a structure having a model with
three geometric parameters (for example, depth, width, and height,
or X-, Y-, and Z-axes) rather than two dimensional parameters. In
this regard, the cell cluster differentiated from the mesenchymal
cells according to an embodiment may be cultured in a 3D manner.
That is, the cell cluster may refer to a cell cluster consisting of
cells that are adhered to a culture container, cultured in a
floating state, and three-dimensionally formed into spheres,
sheets, or similar three-dimensional forms (for example, a similar
organizational body) upon the proliferation of the cells. Here, the
cell cluster may have a diameter of 300 .mu.m or more, and for
example, may have a diameter in a range of about 300 .mu.m to about
2,000 .mu.m, about 400 .mu.m to about 1,500 .mu.m, or about 400
.mu.m to about 1,000 .mu.m. In addition, the cell cluster may
include vascular cells differentiated from the mesenchymal cells,
and for example, may include vascular cells at a density in a range
of about 2.times.10.sup.4 cells/cm.sup.2 to about 1.times.10.sup.5
cells/cm.sup.2.
[0011] The differentiation of the mesenchymal cells into the cell
cluster may be performed by adhering mesenchymal cells to a culture
container including a hydrophobic surface and culturing the
mesenchymal cells in the culture container. In detail, when the
mesenchymal cells are cultured through adhesion to a culture
container including a hydrophobic surface, the adhered mesenchymal
cells may be separated from the culture container as a density of
the mesenchymal cells increases, to thereby form a cell cluster. In
addition, after the mesenchymal cells are differentiated into the
cell cluster, the culturing of the mesenchymal cells may be further
performed for at least 12 hours, at least 1 day, for example, for
12 hours to 15 days, for 1 to 15 days, for 3 to 10 days, for 3 to 7
days, or for 5 to 7 days. A detailed description of a method of
forming the cell cluster through culturing will be described
below.
[0012] The pathological characteristics of fibrosis may include
symptoms that are specific or nonspecific to fibrosis,
histomorphologic characteristics that are specific or nonspecific
to fibrosis, molecular biological characteristics, or pathological
characteristics. For example, the pathological characteristics of
fibrosis may include at least one selected from the group
consisting of: formation of excessive connective tissue as compared
with connective tissue in fibrosis-free cells or tissue; deposition
of collagen; increased expression, secretion, or synthesis of a
fibrosis-related molecule; and increased cell death, or a
combination thereof. The fibrosis-related molecule may include a
marker gene or protein that is specific or nonspecific to fibrosis,
and for example, may include at least one selected from the group
consisting of transforming growth factor-beta (TGF-beta), Smad,
laminins, and smooth muscle actin (SMA). The TGF-beta may include
TGF-.beta.1, 2, or 3, and the Smad may include any of Smads 1 to 8,
R-Smad, Co-Smad, and I-Smad. The SMA, which is a marker of a
myofibroblast, may cause the deposition of collagen in fibrosis.
Therefore, the cell cluster according to an embodiment or cells
constituting the cell cluster may exhibit such pathological
characteristics of fibrosis as described above.
[0013] The term "fibrosis" used herein refers to formation of
excessive fibrous connective tissue in an organ or tissue. The
fibrosis may include at least one selected from the group
consisting of idiopathic pulmonary fibrosis (IPF), pulmonary
fibrosis, interstitial lung disease, nonspecific interstitial
pneumonia (NSIP), usual interstitial pneumonia (UIP),
endomyocardial fibrosis, mediastinal fibrosis, bone marrow
fibrosis, retroperitoneal fibrosis, progressive massive fibrosis,
nephrogenic systemic fibrosis, Crohn's disease, chronic myocardial
infarction, scleroderma/systemic sclerosis, neurofibromatosis,
Hermansky-Pudlak syndrome, diabetic kidney disease, renal fibrosis,
hypertrophic cardiomyopathy (HCM), hypertension-related
nephropathy, renal tubulointerstitial fibrosis, focal segmental
glomerulosclerosis (FSGS), radiation-induced fibrosis, fibroids,
alcoholic liver disease, liver steatosis, liver fibrosis, liver
cirrhosis, Hepatitis C Virus (HCV) infection, chronic rejection of
a transplanted organ, fibrotic skin disease, keloidal scar,
Dupuytren's contracture, Ehlers-Danlos syndrome, epidermolysis
bullosa dystrophica, oral submucous fibrosis, and fiber
proliferative disorder.
[0014] Since the cell cluster differentiated from the mesenchymal
cells according to an embodiment is cultured three-dimensionally,
the cell cluster can mimic in vivo environments and have a
phenotype of fibrosis, i.e., pathological characteristics of
fibrosis, to be utilized in the in vitro fibrosis model. The term
"fibrosis model" used herein refers to a model configured to
schematize a structure or form of an organ, tissue, or cell having
fibrosis, or also refers to a fibrosis model configured to
elucidate the interaction between organs, tissues, or cells having
fibrosis, or the relationship of structure or morphology of organs,
tissues, or cells having fibrosis. Therefore, the fibrosis model
may have a phenotype specific or nonspecific to fibrosis, or show
expression of a marker gene or protein that is specific or
nonspecific to fibrosis.
[0015] Another aspect provides a method of preparing the in vitro
fibrosis model, the method including: forming a cell cluster by
adhering mesenchymal cells to a culture container comprising a
hydrophobic surface and culturing the mesenchymal cells in the
culture container; and; and establishing pathological
characteristics of fibrosis in the cell cluster by performing
additional culturing thereof for at least 12 hours.
[0016] The mesenchymal cells, the cell cluster, and the fibrosis
are the same as described above.
[0017] The mesenchymal cells may be adhered to the culture
container by cell-substrate interactions with the hydrophobic
surface of the culture container. The mesenchymal cells (for
example, adipose stem cells) may be, for example, separated from
human adipose tissue. The human adipose tissue may include mature
adipocytes and connective tissue surrounding the mature adipocytes,
and may be easily obtained from a patient or others having a
phenotype matching that of a patient. Here, regardless of the
position in the body, all the adipose tissues obtained by all the
methods used for collecting fat may be used, and examples of the
adipose tissues include subcutaneous fat tissue, bone marrow fat
tissue, mesenteric adipose tissue, gastrointestinal adipose tissue,
and retroperitoneal adipose tissue. The adipose stem cells may be
separated from the above-described human adipose tissue according
to a method known in the art. For example, as disclosed in
WO2000/53795 and WO2005/04273, the adipose stem cells may be
obtained from the adipose tissue by liposuction, sedimentation,
enzyme treatment using collagenase or the like, removal of floating
cells such as red blood cells by centrifugation, and the like. In
addition, the mesenchymal cells, for example, mesenchymal stem
cells, mesenchymal stromal cells, bone marrow stem cells, or
fibroblasts, may be separated from various tissues by a method
known in the art.
[0018] The separated mesenchymal cells exhibit an excellent
proliferation rate up to a passage number of 16 even after several
passages. Thus, for subsequent formation of a three-dimensional
cell cluster, the pluripotent mesenchymal cells separated from the
human tissue use cells cultured through 1 passage or cells cultured
through 10 or more passages at confluency of 60%.
[0019] When the mesenchymal cells prepared as described above are
cultured by inoculation into the culture container including a
hydrophobic surface, due to the hydrophobic surface of the culture
container, cell-substrate interactions occur between the
mesenchymal cells and the culture container. In this regard, due to
physical adsorption, the mesenchymal cells proliferate while being
adhered to the surface of the culture container. Afterwards, the
forming of the cell cluster may refer to formation of the cell
cluster such that the adhered mesenchymal cells may be separated
from the culture container as a density of the mesenchymal cells
increases.
[0020] A cell culture container that is surface-treated with a
polymer that imparts hydrophobicity to a conventional cell culture
container, or a cell culture formed of such a polymer, may be
suitable for use as the culture container including the hydrophobic
surface in the present inventive concept. Such a hydrophobic
polymer may be one selected from polystyrene, polymethyl
methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl
chloride (PVC), polyethylene (PE), polypropylene (PP),
poly(tetrafluoroethylene) (PTFE), and an aliphatic polyester-based
polymer selected from poly(L-lactic acid) (PLLA), poly(D,L-lactic
acid) (PDLLA), a poly(glycolic acid) (PGA), poly(caprolactone)
(PCL), poly(hydroxyalkanoate), polydioxanone (PDS), and
poly(trimethylene carbonate), or may be a copolymer of units above,
such as poly(lactic acid-co-glycollic acid) (PLGA), poly(L-lactic
acid-co-caprolactone) (PLCL), poly(glycolic acid-co-caprolactone)
(PGCL), or a derivative thereof. In addition, the hydrophobic
surface of the culture container may be a silanized surface, a
carbon nanotube (CNT) surface, a hydrocarbon-coated surface, or a
metallic (for example, stainless steel, titanium, gold, platinum,
or the like) surface.
[0021] In addition, in one or more embodiments of the present
invention, in order to more effectively adhere the mesenchymal
cells to the culture container than by physical adsorption through
the interaction between the mesenchymal cells and the hydrophobic
surface of the culture container, the mesenchymal cells may be
adhered to the culture container through an interaction with a
growth factor having adhesiveness to the mesenchymal cells. For
example, after such a growth factor is immobilized on the surface
of the culture container, a biochemical interaction between the
immobilized growth factor and the mesenchymal cells may be
utilized.
[0022] The growth factor may have be any substance having
adhesiveness to the mesenchymal cells, and examples thereof are
vascular endothelial growth factor (VEGF), fibroblast growth factor
(FGF), epidermal growth factor (EGF), platelet-derived endothelial
growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like
growth factor (IGF), or heparin-binding domain (HBD). The growth
factor may be immobilized on the surface of the culture container
at a concentration in a range of about 5 .mu.g/ml to about 100
.mu.g/ml.
[0023] The immobilization of the growth factor on the surface of
the culture container may be achieved by a method known in the art,
the method being used to immobilize a polypeptide on a solid
substrate surface, and for example, the immobilization may be
performed by physical adsorption or covalent bonding by a
non-selective chemical reaction. As the immobilization method, a
method of immobilizing a protein using biotin-streptavidin/avidin
bonds by binding biotin to a protein and then applying the protein
to a solid surface treated with streptavidin or avidin; a method of
immobilizing a protein by integrating an active group (a chemical
functional group for immobilizing a protein by a chemical bond)
with a substrate using plasma; a method of immobilizing a protein
by physical adsorption to a porous sol-gel thin film after forming
the porous sol-gel thin film having a sufficiently increased
specific area on a solid substrate surface according to a sol-gel
method; a method of immobilizing anticoagulant protein on a
poly(tetrafluoroethylene) (PTFE) surface by plasma reaction; a
method of immobilizing a protein by binding with an enzyme, in
which two or more cationic amino residues are fused to two enzymes
sequentially; a method of immobilizing a protein on a hydrophobic
polymer layer bonded to a solid support using a substrate; a method
of immobilizing a protein on a plastic surface using a buffering
component; or a method of immobilizing a protein by contacting a
hydrophobic solid surface with an alcohol solution, is known in the
art.
[0024] In one embodiment, a polypeptide linker that can be
subjected to recombinant mass expression and easy purification may
be used to perform immobilization in the form of a peptide
linker-growth factor recombinant protein in which an amino terminus
of the growth factor is fused to a carboxyl terminus of the
polypeptide linker.
[0025] A substance capable of being adhered to the culture
container including the hydrophobic surface through a hydrophobic
domain present at an amino terminus of a growth factor to which a
carboxyl terminus of the polypeptide linker is bonded, or a
substance that can be subjected to recombinant mass expression and
easy purification without being adversely affected, may be suitable
for use as the polypeptide linker in the present invention.
Examples of the polypeptide linker are a maltose-binding protein
(MBP), a hydrophobin, or a hydrophobic cell penetrating peptide
(CPP).
[0026] As such, when the mesenchymal cells are cultured by physical
adhesion through cell-substrate interactions with the hydrophobic
surface of the culture container, or when the mesenchymal cells are
cultured in the state of being bound to a growth factor through a
biochemical interaction with the growth factor immobilized on the
surface of the culture container, the mesenchymal cells can
proliferate while being adhered to the surface of the culture
container at the beginning. The mesenchymal cells may be seeded at
a concentration in a range of about 1.times.10.sup.3 cells/cm.sup.2
to about 1.times.10.sup.7 cells/cm.sup.2. In addition, a
temperature for the culturing of the mesenchymal cells may be in a
range of about 35.degree. C. to about 38.5.degree. C., and a
culture period required for the formation of the cell cluster may
be 4 hours to 2 days, or for example, 1 day. As a suitable medium
for the culturing of the mesenchymal cells, a serum-containing or
serum-free medium conventionally used for culturing and/or
differentiation of the mesenchymal cells may be used without
limitation, and examples thereof are a Dulbecco's modified eagle
medium (DMEM), Ham's F12, or a medium in which serum is added to a
mixture of the above substances.
[0027] Then, when the mesenchymal cells proliferate in a state in
which they are adhered to the surface of the culture container, and
the cell-cell interactions become stronger than cell-substrate
interactions at high cell densities, the mesenchymal cells become
separated from the surface of the culture container and then
proliferate while floating in the culture. The floating mesenchymal
cells then aggregate together such that a floating
three-dimensional cell cluster having a size detectable with the
naked eye may be formed.
[0028] In one embodiment, considering the hydrophobic surface of
the culture container, a non-tissue culture plate (NTCP) made of
polystyrene is used as a culture container in which cell adhesion
to such a hydrophobic surface is relatively weak, and the
mesenchymal cells are inoculated thereto to induce formation of a
three-dimensional cell cluster. The mesenchymal cells inoculated to
the polystyrene NTCP initially undergo induction of weak cell
adhesion to a surface of the polystyrene NTCP by cell-substrate
interactions, and then proliferate in a two-dimensional single
layer in a state in which they are adhered to the surface of the
polystyrene NTCP. However, as the cell density increases as the
culture time elapses, the cell-cell interactions become stronger
than the cell-substrate interactions, thereby separating the
mesenchymal cells cultured in the two-dimensional single layer from
the surface of the polystyrene NTCP. Here, the mesenchymal cells
can initially be cultured in a state in which they are adhered to
the surface of the culture container, whereas, in the case where
the mesenchymal cells are initially cultured in a floating state
without undergoing cell adhesion, a three-dimensional cell cluster
formed therefrom may have a small size and include cells that
mostly undergo cell apoptosis. When the mesenchymal cells separated
from the culture container are further cultured in a floating state
in a culture fluid, the mesenchymal cells may aggregate together
through cell-cell interactions to thereby form a three-dimensional
cell cluster. In the three-dimensional cell cluster formed
therefrom, the mesenchymal cells are initially weakly bound, but as
the culture time elapses, the cell-cell interactions enhance
adhesion between the mesenchymal cells constituting the cell
cluster to thereby form a compact three-dimensional cell
cluster.
[0029] In addition, when the formed cell cluster is further
cultured for at least 12 hours, the cell cluster or the cells of
the cell cluster may exhibit the pathological characteristics of
fibrosis. Here, the additional culture time may be at least 12
hours or at least 1 day, and for example, may be from 12 hours to
15 days, 1 to 15 days, 3 to 10 days, 3 to 7 days, or 5 to 7 days.
The pathological characteristics of fibrosis are the same as
described above.
[0030] The three-dimensional cell cluster may be differentiated
into vascular endothelial cells by proliferation in the form of the
formed three-dimensional cell cluster. When the mesenchymal cells
are cultured in the form of the three-dimensional cell cluster,
oxygen permeation into the cell cluster deceases upon the formation
of the cell cluster, and accordingly, a hypoxic state may thereby
be formed. Such a hypoxic state formed in the cell cluster induces
production of various angiogenic factors that affect the
differentiation into vascular endothelial cells, resulting in
differentiation into vascular endothelial cells.
[0031] The three-dimensional cell cluster formed by culturing the
mesenchymal cells through adhesion to the surface of the culture
container has a size detectable with the naked eye, and for
example, has a diameter in a range of about 300 .mu.m to about
2,000 .mu.m. In this regard, the formed three-dimensional cell
cluster may be easily recovered by a method such as filtration or
centrifugation. The recovered three-dimensional cell cluster may be
subjected to enzymatic treatment with collagenase, trypsin, or
dispase, mechanical treatment with pressure, or combinational
treatment, to thereby break up the cluster form into the form of
single cells, or the three-dimensional cell cluster form itself may
be used.
[0032] Another aspect provides a method of screening a therapeutic
agent for fibrosis, the method including: treating the in vitro
fibrosis model with a test substance; and selecting, as a candidate
substance for treatment of fibrosis, the test substance exhibiting
improvement or treatment of pathological characteristics of
fibrosis in the cell cluster or cells thereof in the in vitro
fibrosis model, as compared with an untreated control group.
[0033] The mesenchymal cells, the cell cluster, and the fibrosis
are the same as described above.
[0034] In the method of screening the therapeutic agent for
fibrosis, the test substance may include one selected from the
group consisting of a low-molecular weight compound, an antibody,
an antisense nucleotide, a short interfering RNA, a short hairpin
RNA, a nucleic acid, a protein, a peptide, and other extracts and
natural substances.
[0035] In addition, in the method of screening the therapeutic
agent for fibrosis, the pathological characteristics of fibrosis
include symptoms that are specific or nonspecific to fibrosis,
histomorphologic characteristics that are specific or nonspecific
to fibrosis, molecular biological characteristics, or pathological
characteristics. For example, the pathological characteristics of
fibrosis may include at least one selected from the group
consisting of: formation of excessive connective tissue as compared
with connective tissue in a fibrosis-free cell or tissue;
deposition of collagen; increased expression, secretion, or
synthesis of a fibrosis-related molecule; and increased cell death,
or a combination thereof. The fibrosis-related molecule may include
a marker gene or protein that is specific or nonspecific to
fibrosis, and for example, may include at least one selected from
the group consisting of TGF, Smad, laminins, and SMA. The TGF-beta
may include TGF-.beta.1, 2, or 3, and the Smad may include any of
Smads 1 to 8, R-Smad, Co-Smad, and I-Smad. The SMA which is a
marker of a myofibroblast may cause the deposition of collagen in
fibrosis. Therefore, for example, regarding the selecting of the
test substance for treatment of fibrosis, in a case where, as
compared to an untreated control group, connective tissue is formed
in the cell cluster or in cells constituting the cell cluster,
collagen is deposited, a thickness of collagen fibers decreases, or
viability of cells increases in a test substance-treated group, the
test substance may be selected as the candidate substance for
treatment. The phenotype of fibrosis, i.e., formation of excessive
connective tissue, deposition of collagen, or measurement of a
thickness of collagen fibers, may be identified according to the
methods known to one of ordinary skill in the art, such as a
Haematoxylin and Eosin (H&E) staining method, a
Masson-trichrome (MT) staining method, an immunofluorescence
method, or an immunohistochemical staining method. In addition, the
viability or apoptosis of cells may be identified according to an
LDH assay or a live/dead assay. In addition, in cases where the
test substance increases or decreases expression of a
fibrosis-related molecule, i.e., a marker gene or protein that is
specific or nonspecific to fibrosis, the test substance may be
selected as the candidate substance for treatment of fibrosis. For
example, in cases where the test substance decreases expression of
TGF-beta, Smad, laminin, or SMA, the test substance may be selected
as the candidate substance for treatment of fibrosis. Such
expression may be measured by one selected from the group
consisting of reverse transcriptase polymerase chain reaction
(RT-PCR), enzyme linked immunosorbent assay (ELISA),
immunohistochemistry, western blotting, and fluorescence-activated
cell sorting (FACS).
Advantageous Effects of the Invention
[0036] According to embodiments regarding the in vitro fibrosis
model and the method of preparing the same, the in vitro fibrosis
model which is formed as a three-dimensional cell cluster is able
to mimic in vivo environments and exhibit the fibrosis phenotype,
i.e., pathological characteristics of fibrosis, and thus the
disclosure of the present invention may be effectively used for
research on fibrosis or a screening method for a therapeutic
agent.
DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1A shows scanning electron microscope (SEM) images of a
three-dimensional cell cluster according to an embodiment;
[0038] FIG. 1B shows Haematoxylin and Eosin (H&E) staining
results obtained from a three-dimensional cell cluster according to
an embodiment;
[0039] FIG. 2 is a diagram showing immunofluorescence staining
results confirming a hypoxic state of a three-dimensional cell
cluster according to an embodiment;
[0040] FIG. 3 is a graph showing expression of TGF-beta in a
three-dimensional cell cluster according to an embodiment;
[0041] FIG. 4 is a graph showing expression of fibrosis-related
factors in a three-dimensional cell cluster according to an
embodiment;
[0042] FIG. 5A is a diagram showing results for deposition of
collagen in a three-dimensional cell cluster according to an
embodiment, as identified by immunofluorescence staining and
analysis of hydroxyproline contents;
[0043] FIG. 5B is a graph showing results for deposition of
collagen in a three-dimensional cell cluster according to an
embodiment, as identified by immunofluorescence staining and
analysis of hydroxyproline contents;
[0044] FIG. 6 is a diagram showing results for deposition of
collagen type I in a three-dimensional cell cluster according to an
embodiment, as identified by immunofluorescence staining;
[0045] FIG. 7 is a diagram showing results for deposition of
collagen type I in a three-dimensional cell cluster according to an
embodiment, as identified by immunofluorescence staining;
[0046] FIG. 8 shows transmission electron microscope (TEM) images
of a three-dimensional cell cluster according to an embodiment;
[0047] FIG. 9A is a graph showing results for viability and
apoptosis of cells in a three-dimensional cell cluster according to
an embodiment; and
[0048] FIG. 9B is a diagram showing results for viability and
apoptosis of cells in a three-dimensional cell cluster according to
an embodiment.
MODE OF THE INVENTION
[0049] Hereinafter, the present invention is described in detail
with reference to Examples. However, Examples shown and described
herein are illustrative examples of the present invention and are
not intended to otherwise limit the scope of the inventive concept
in any way.
EXAMPLES: PREPARATION OF IN VITRO FIBROSIS MODEL AND
CHARACTERIZATION OF FIBROSIS MODELING
(1) Preparation of In Vitro Fibrosis Model
(1.1) Separation of Human Adipose Stem Cells (hASCs)
[0050] Subcutaneous adipose tissue of normal individuals was
obtained from the Department of Plastic Surgery, the Catholic
University of Korea. Then, the adipose tissue was washed three
times with PBS containing 1% penicillin/streptomycin (PS) to remove
contaminated blood therefrom, and was cut with surgical scissors.
The resulting adipose tissue was soaked in a tissue lysate
containing 1% BSA (w/v), 0.3% collagenase type I, and 1% PS, and
then, the mixed tissue lysate was stirred by orbital shaking for 1
hour at a temperature of 37.degree. C. Afterwards, the supernatant
was removed, and the cell suspension was filtered through a 250
.mu.m Nitex filter (Sefar America Inc.) to remove tissue debris.
Then, centrifugation was performed thereon at a speed of 1,000 rpm
for 5 minutes. Cells collected by centrifugation were re-suspended
in DMEM/F-12 containing 10% BSA. The isolated primary cells were
plated in a tissue culture flask for 24 hours in a humidified
atmosphere with 5% CO.sub.2 and 95% air. Then, non-adherent cells
were removed by replacement with a fresh medium having the same
volume. The morphology of adherent hASCs was observed via a phase
contrast microscope, and hASCs of 5 passages were used for all
experiments.
(1.2) Preparation of 3D Cell Cluster Derived from Adipose Stem
Cells
[0051] To prepare a 3D cell cluster derived from the hASCs, the
hASCs were cultured in a treated 96-well non-tissue culture plate
(NTCP) (NTCP made of polystyrene and having a hydrophobic surface,
Falcon Company). The NTCP was a plated coated with a fused protein
of maltose binding protein (MBP)-fibroblast growth factor (FGF),
wherein the plate coated with the fused protein has been described
in KR 10-1109125 that is incorporated herein by reference in its
entirety. In detail, 1.times.10.sup.5 cells/cm.sup.2 of the hASCs
were inoculated into each well of the well plate, and cultured in a
DMEM/F-12 medium containing 10% FBS. Within 24 hours of the
culture, 3D cell clusters of the hASCs were formed on each cell
adhesion surface. For analysis of characteristics of a fibrosis
model with respect to the formed 3D cell clusters, 3D cell clusters
were collected on the 1.sup.st day (1 Day), 3.sup.rd day (3 Day),
and 5.sup.th day (5 Day) of the culture. In addition, the 3D cell
clusters were confirmed to have a diameter of about 500 .mu.m or
more. Hereinafter, the 3D cell cluster was represented by
`3DCM`.
[0052] In addition, as a comparative example, the hASCs were
cultured in a 2D manner. In detail, 1.times.10.sup.5 cells/cm.sup.2
of the hASCs were inoculated into each well of a treated 96-well
tissue culture plate (TCP), and cultured in a DMEM/F-12 medium
containing 10% FBS. In the same manner as in the 3D cell cluster,
cells were collected on the 1.sup.st day (1 Day), 3.sup.rd day (3
Day), and 5.sup.th day (5 Day) of the culture for analysis of
characteristics of a fibrosis model. Hereinafter, the cells
cultured in a 2D manner are represented by `2D`.
(2) Analysis of Fibrosis Modeling Characteristics of In Vitro
Fibrosis Model
(2.1) Analysis of Characteristics of 3D Cell Cluster Derived from
Adipose Stem Cells
[0053] To analyze the morphological characteristics of a 3D cell
cluster derived from adipose stem cells, the 3D cell cluster was
subjected to scanning electron microscopy and H&E staining. In
addition, immunostaining was performed on the 3D cell cluster to
confirm a hypoxic state in the 3D cell cluster.
[0054] In detail, for scanning electron microscopy, the collected
3D cell cluster was immobilized with 2.5% glutaraldehyde at a
temperature of 4.degree. C. for 2 hours, and then post-immobilized
with 1% osmium tetroxide in deionized water. The immobilized 3D
cell cluster was dehydrated two times with ethanol at a series of
concentrations (50%, 70%, 80%, 90%, and 100%). Afterwards, the
resulting 3D cell cluster was immersed in hexamethyldisilazane
(HMDS) for 2 minutes, and vibration-dried for one day. To obtain an
SEM image, the 3D cell cluster was attached to an adhesive carbon
tape, and sputter-coating was performed with gold for 60 minutes at
10 mA. Images were then obtained at 15 kV, and the results are
shown in FIG. 1A.
[0055] In addition, for H&E staining, the collected 3D cell
cluster was immobilized with 4% PFA at room temperature for 30
minutes, dehydrated with ethanol at a series of concentrations
(50%, 70%, 80%, 90%, and 100%), and then, placed in paraffin wax. A
section having a thickness of 4 .mu.m was prepared, and then
stained with haematoxylin and eosin. The section was
deparaffinized, hydrated with distilled water, and washed three
times with PBS. Afterwards, the resulting section was immersed in
haematoxylin (Harris; Sigma-Aldrich) for 10 seconds, washed in
flowing water for 10 to 15 minutes, counter-stained with eosin for
15 seconds, and then, washed again for 10 to 15 minutes.
Afterwards, the resulting section was placed on a slide to be
observed with a light microscope, and the results are shown in FIG.
1B.
[0056] In addition, for hypoxic immunofluorescence analysis, the 3D
cell cluster was incubated, before being collected at each culture
time, in 10 mmol pimonidazole hydrochloride (Hypoxyprobe.TM.-1 kit,
Hypoxyprobe, USA) in 0.1 ml solution for 2 hours. Then, the
incubated 3D cell cluster was collected, immobilized with 4%
paraformaldehyde at a temperature of 4.degree. C. for 30 minutes,
and embedded in an optimal cutting temperature (OCT) compound
(TISSUE-TEK.RTM. 4583; Sakura Finetek USA, Inc.). A frozen section
having a thickness of 6 .mu.m was washed with PBS, and to prevent
nonspecific binding thereto, the 3D cell cluster was incubated in
4% BSA in PBS for 1 hour. Accordingly, pimonidazole was detected by
primary mouse antibodies (hydroxy probe) and secondary goat
anti-mouse Alexa 488 antibodies (Invitrogen). In addition,
4,5-diamino-2-phenylindole (DAPI) (Vector Laboratories) was used
for nuclear staining. Here, a control group was subjected to
experiments performed under the same conditions, except that no
primary antibody was used, and was observed with a confocal
microscope (Carl Zeiss). The results are shown in FIG. 2.
[0057] FIG. 1A shows SEM images and H&E staining results
obtained from the 3D cell cluster according to an embodiment.
[0058] FIG. 2 is a diagram showing immunofluorescence staining
results confirming a hypoxic state of the 3D cell cluster according
to an embodiment.
[0059] As shown in FIG. 1, the outer surface the 3D cell cluster of
the culture at Day 1 was densely stained by H&E, and thus, it
was confirmed that the cells were connected by fibrous matrices. As
the culture continued, the 3D cell cluster of the culture at Day 3
showed a decreased intercellular space between the cells, and the
3D cell cluster of the culture at Day 5 showed almost no gap
between the cells (see arrows).
[0060] In addition, as shown in FIG. 2, it was confirmed that the
DAPI-stained cells were uniformly distributed over the 3D cell
cluster on Day 1 of the culture, and that more hypoxic
probe-positive cells were present inside the 3D cell cluster. As
the culture continued, the 3D cell cluster of Day 3 of the culture
showed increased hypoxic probe-positive cells therein, and the 3D
cell cluster of Day 5 of the culture also showed increased hypoxic
probe-positive cells outside of the 3D cell cluster. Accordingly,
it was confirmed that hypoxia was induced in the 3D cell cluster,
and then, diffused to the outside of the 3D cell cluster. That is,
by referring to FIG. 1, it was confirmed that the closure of the
intercellular space on the outer surface of the 3D cell cluster led
to the induction of hypoxia. In fibrosis, TGF-1 is an important
relevant factor which is overexpressed in hypoxia. That is, as the
distance between the cells narrowed, supply of oxygen to the cell
cluster was restricted, and accordingly, TGF-1 was induced, thereby
causing fibrosis. Therefore, based on the results above, it was
confirmed that the pathological characteristics of fibrosis were
modeled by the 3D cell cluster according to an embodiment.
(2.2) Analysis of Fibrosis-Related Factors in 3D Cell Cluster
Derived from Adipose Stem Cells
[0061] TGF-beta is a major molecule in fibrosis and is induced
under hypoxic conditions. To confirm whether fibrosis-related
factors have been expressed or not in the 3D cell cluster derived
from adipose stem cells, ELISA was performed on fibrosis-related
factors including TGF-beta.
[0062] In detail, to measure total contents of TGF-.beta.1, a
culture medium was prepared with normal cell concentration (NCC),
2D cells (2D), and 3D cell cluster (3DCM). To activate latent
TGF-.beta.1 in an immunoreactive form, the culture supernatant was
incubated in 1N HCL and neutralized with 1.2 N NaOH/0.5 M HEPES.
The assay was performed using the Quantikine ELISA human
TGF-.beta.1 kit (R&D System) according to the manufacturer's
instructions. Here, the absorbance was measured using a Multiskan
(Thermo) at 560 nm, and the results are shown in FIG. 3.
[0063] In addition, to confirm the expression of the
fibrosis-related factors in the 3D cell cluster, total RNAs were
extracted from the collected 3D cell cluster by using a triazole
reagent (Invitrogen, USA) according to the manufacturer's
instructions. The extracted RNAs were dissolved in nuclease-free
water, and then, the concentration of the resulting RNAs was
quantified using a NanoDrop ND1000 spectrophotometer (Thermo Fisher
Scientific). Here, synthesis of complementary DNA was performed by
using Maxime RT PreMIX (iNtROn) according to the manufacturer's
instructions. All target primers were purchased from Bioneer. All
polymerase chain reactions were performed using ABI Prism 7500
(Applied Biosystems), and gene expression levels were quantified
using SYBR Premix Ex Taq (TaKaRa). Comparative gene expression
levels were calculated using the comparative Ct method, and the
results are shown in FIG. 4.
[0064] FIG. 3 is a graph showing the expression of TGF-beta in the
3D cell cluster according to an embodiment.
[0065] FIG. 4 is a graph showing the expression of fibrosis-related
factors in the 3D cell cluster according to an embodiment.
[0066] As shown in FIGS. 3 and 4, it was confirmed that the 3D cell
cluster derived from adipose stem cells showed increased expression
of the fibrosis-related factors including TGF-beta, laminin, smooth
muscle actin (SMA), collagen type I, and SMAD3.
(2.3) Analysis of Collagen Deposition in 3D Cell Cluster Derived
from Adipose Stem Cells
[0067] To analyze collagen deposition in the 3D cell cluster
derived from adipose stem cells, the 3D cell cluster was subjected
to immunofluorescence staining, immunohistochemical staining, and
hydroxyproline quantification, and observed with a transmission
electron microscope.
[0068] In detail, pretreatment was performed on the 3D cell cluster
in the same manner as in H&E staining, and staining was
performed thereon using Masson's trichrome (MT) staining. In the 3D
cell cluster, the percentage of fibrosis was determined by counting
the number of pixels of the stained collagen area in a digital
image by using the ImageJ software (NIH), and the results are shown
in FIG. 5A. In addition, for a hydroxyproline assay, 2D cells and
3D cell cluster were prepared by using RIPA buffer, and then, were
hydrolyzed in 12N HCL at a temperature of 120.degree. C. for 3
hours. Assays were performed using the hydroxyproline kit
(Sigma-Aldrich) according to the manufacturer's instructions. Here,
the absorbance was measured using a Multiskan (Thermo) at 560 nm,
and the results are shown in FIG. 5B.
[0069] In addition, for immunofluorescence (IF), a 3D cell cluster
was immobilized in the same manner as in H&E staining above,
embedded in an OCT compound (TISSUE-TEK.RTM. 4583; Sakura Finetek
USA, Inc.), and then frozen at a temperature of -28.degree. C. The
resulting 3D cell cluster was cut to a thickness of 6 .mu.m. To
avoid nonspecific binding thereto, a section was incubated in 4%
BSA at room temperature for 1 hour. Afterwards, the section was
incubated overnight at a temperature of 4.degree. C. with primary
antibodies (Rabit, Abicam) specific for collagen type I. Then, a
sample on the section was washed with PBS, and incubated for 1 hour
with corresponding fluorescent conjugated secondary antibodies
(Donkey anti-rabbit) (Life Technologies) in 1% BSA. In addition,
DAPI (Vector Laboratories) was used for nuclear staining. Here, a
control group was subjected to experiments performed under the same
conditions, except that no primary antibody was used, and was
observed with a confocal microscope (Carl Zeiss). The results are
shown in FIG. 6.
[0070] In addition, for immunohistochemical staining, pretreatment
was performed on the 3D cell cluster in the same manner as in
H&E staining. Here, fibronectin (FN) and laminin (LN) were each
detected by using mouse monoclonal antibodies and goat polyclonal
antibodies (Santa cruz Biotechnology) that are specific to FN and
LN. In addition, for .alpha.SMA analysis, mouse monoclonal
antibodies (Dako) were used to detect .alpha.SMA. After a section
prepared therefrom was incubated overnight at a temperature of
4.degree. C. with primary antibodies for the fibrosis-related
factors, the section was incubated at room temperature for 1 hour
with horseradish-labeled anti-mouse antibodies (specific to FN and
.alpha.SMA) and anti-goat secondary antibodies (specific to LN)
(Vector). Then, positive staining was visualized using
diaminobenzidine (DAB, Vector). Here, a control group was subjected
to experiments performed under the same conditions, except that no
primary antibody was used. A section obtained therefrom was
counter-stained with Haematoxylin and observed with a light
microscope, and the results are shown in FIG. 7.
[0071] In addition, for transmission electron microscopy (TEM),
pretreatment was performed on a sample in the same manner as used
for scanning electron microscopy. Additionally, the immobilized 3D
cell cluster was infiltrated into an epoxy resin, embedded therein,
and polymerized at a temperature of 60.degree. C. for 24 hours. An
ultrathin section was prepared by using an ultramicrotome (Ultra
cut C, Leica CO. Ltd), and then, was stained with uranyl acetate
and lead citrate. TEM images were observed by cryo-TEM (cryoTecanai
F20, FEI Co. Ltd), and the results are shown in FIG. 8.
[0072] FIGS. 5A and 5B show the results for the deposition of
collagen in the 3D cell cluster according to an embodiment, as
identified by immunofluorescence staining and analysis of
hydroxyproline contents.
[0073] FIG. 6 is a diagram showing the results for the deposition
of collagen type I in the 3D cell cluster according to an
embodiment, as identified by immunofluorescence staining.
[0074] FIG. 7 is a diagram showing the results for the deposition
of collagen type I in the 3D cell cluster according to an
embodiment, as identified by immunofluorescence staining.
[0075] FIG. 8 shows TEM images of the 3D cell cluster according to
an embodiment.
[0076] As shown in FIGS. 5A and 5B, it was confirmed that a
majority of collagen was stained in the 3D cell cluster by MT
staining and that the content of hydroxyproline was also increased
in the 3D cell cluster as compared with that of hydroxyproline in
2D cells.
[0077] In addition, as shown in FIG. 6, it was confirmed that
collagen type I was significantly increased in the 3D cell cluster,
as identified by IF.
[0078] In addition, as shown in FIG. 7, it was confirmed that
.alpha.SMA was significantly increased in the 3D cell cluster, as
identified by immunohistochemical staining. .alpha.SMA is a
traditional marker of myofibroblasts, and collagen type I is known
to be synthesized from myofibroblasts in fibrosis. That is, the
results above are deemed to be consistent with the results of FIG.
6.
[0079] In addition, as shown in FIG. 8, by referring to the TEM
images, it was confirmed that the deposition of collagen fibers and
collagen gradually increased as the culture time of the 3D cell
cluster increased. In detail, thick collagen fibers were observed
(see arrows) on Day 5 of the culture, wherein such observation is
deemed to be caused by cross-linking of collagen. In addition, on
Day 5 of the culture, it was confirmed that no intact cell
structure was observed inside the 3D cell cluster. As a result,
collagen fibers became thicker around the cells, which caused
apoptosis of the cells due to lack of transport of nutrients.
Therefore, it was confirmed that the pathological characteristics
of fibrosis were modeled by the 3D cell cluster according to an
embodiment.
(2.4) Analysis of Viability and Apoptosis of Cells in 3D Cell
Cluster Derived from Adipose Stem Cell
[0080] The deposition of collagen ultimately induced apoptosis of
cells in fibrosis. Thus, to confirm that such characteristics
appeared in a 3D cell cluster derived from adipose stem cells, an
LDH assay and a live/dead assay were performed on the 3D cell
cluster.
[0081] In detail, for the LDH assay, among a culture medium
prepared with NCC, 2D cells, and 3D cell cluster, absolute lactic
dehydrogenase (LDH) release was measured. The measurement was
performed using the LDH assay kit (Promega) according to the
manufacturer's instructions. Here, the absorbance was measured
using Multiskan (Thermo) at 560 nm, and the results are shown in
FIG. 9A. In addition, for the live/dead assay, a live/dead assay
kit (Molecular probes) was used according to the manufacturer's
instructions. In summary, the collected 3D cell cluster was treated
with 1 ml of HEPES-buffered saline (HBSS) containing 1 .mu.l of
green-fluorescent nucleic acid staining solution (SYTO 10) and 1
.mu.l of red-fluorescent nucleic acid staining solution (ethidium
homodimer-2), and then cultured in a CO.sub.2 culture medium for 30
minutes. Afterwards, the resulting 3D cell cluster was washed three
times with PBS, immobilized with 4% PFA for 30 minutes, embedded in
an OCT compound (TISSUE-TEK.RTM. 4583; Sakura Finetek USA, Inc.),
and then frozen at a temperature of -28.degree. C. The resulting 3D
cell cluster was cut to a thickness of 10 .mu.m. The entire 3D cell
cluster was completely cut, and two slides were selected from the
middle and outer portions of each sample. Here, the sections were
analyzed using a confocal microscope (Carl Zeiss), and the results
are shown in FIG. 9B.
[0082] FIGS. 9A and 9B show the results for viability and apoptosis
of cells in the 3D cell cluster according to an embodiment.
[0083] As shown in FIG. 9A, according to the LDH assay, the 3D cell
cluster showed increased LDH levels as compared with those in NCC
and 2D cells. In addition, as shown in FIG. 9B, the apoptosis of
cells was visually identified in the 3D cell cluster in the same
manner as in FIG. 9A.
[0084] As a result, the 3D cell cluster according to an embodiment
exhibited pathological characteristics of fibrosis, and thus it was
confirmed to be suitable for use as an in vitro fibrosis model.
* * * * *