U.S. patent application number 16/448954 was filed with the patent office on 2019-12-26 for composition for culturing brain organoid based on decellularized brain matrix and method for preparing same.
The applicant listed for this patent is INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY. Invention is credited to Ann Na Cho, Seung Woo CHO, Yoonhee Jin, Jung Seung Lee.
Application Number | 20190390165 16/448954 |
Document ID | / |
Family ID | 67139600 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190390165 |
Kind Code |
A1 |
CHO; Seung Woo ; et
al. |
December 26, 2019 |
COMPOSITION FOR CULTURING BRAIN ORGANOID BASED ON DECELLULARIZED
BRAIN MATRIX AND METHOD FOR PREPARING SAME
Abstract
Provided is a method for preparing a composition for culturing a
brain organoid, the method comprising (a) decellularizing brain
tissue; (b) drying the brain tissue; and (c) gelating the brain
tissue.
Inventors: |
CHO; Seung Woo; (Seoul,
KR) ; Cho; Ann Na; (Seoul, KR) ; Lee; Jung
Seung; (Seoul, KR) ; Jin; Yoonhee; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI
UNIVERSITY |
Seoul |
|
KR |
|
|
Family ID: |
67139600 |
Appl. No.: |
16/448954 |
Filed: |
June 21, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0062 20130101;
C12N 2533/90 20130101; C12N 5/0622 20130101; C12N 2513/00 20130101;
C12N 5/0619 20130101; C12N 2533/50 20130101; C12M 25/14 20130101;
C12N 5/0068 20130101 |
International
Class: |
C12N 5/0793 20060101
C12N005/0793; C12N 5/00 20060101 C12N005/00; C12M 1/12 20060101
C12M001/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2018 |
KR |
10-2018-0071562 |
Claims
1. A method for preparing a composition for culturing a brain
organoid, comprising: (a) decellularizing brain tissue; (b) drying
the brain tissue; and (c) gelating the brain tissue.
2. The method according to claim 1, wherein in the (c), the brain
tissue and Matrigel are mixed to obtain a hydrogel.
3. The method according to claim 2, wherein the hydrogel contains a
decellularized brain extracellular matrix (dBEM) in an amount of
0.01 to 2.0 mg/mL.
4. The method according to claim 1, wherein in the (a), the brain
tissue is stirred in a decellularizing solution.
5. The method according to claim 4, wherein the brain tissue is
stirred for 3 to 24 hours.
6. The method according to claim 4, wherein 95% or more of cells of
the brain tissue is removed by the decellularization.
7. A hydrogel composition for culturing a brain organoid,
comprising: a decellularized brain extracellular matrix (dBEM).
8. The hydrogel composition according to claim 7, further
comprising: Matrigel.
9. The hydrogel composition according to claim 7, wherein the
decellularized brain extracellular matrix (dBEM) has a
concentration of 0.01 to 2.0 mg/mL.
10. The hydrogel composition according to claim 7, which has an
elastic modulus at 1 Hz of 100 to 150 Pa.
11. A method for culturing a brain organoid in the composition of
claims 7.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for culturing
a brain organoid using a decellularized brain matrix and a method
for preparing the same.
BACKGROUND ART
[0002] Decellularization of tissues and organs has been studied as
a promising approach for preparing a functional support or scaffold
for cell culture and transplantation.
[0003] During a decellularization process, cellular components are
removed from the tissue, but the extracellular matrix and some
growth factor proteins are preserved.
[0004] Therefore, various extracellular matrix components, which
are preserved in the decellularized tissue including collagen,
fibronectin, and laminin, provide a three-dimensional
microenvironment similar to the inside of intact tissue, and thus
can improve survival, proliferation, and differentiation of
cultured cells.
[0005] Additionally, removal of cellular components alters the
decellularized matrix into a functional scaffold with minimal
immunogenicity for cell transplantation.
[0006] Recently, studies for functional tissue-engineered scaffold,
which are obtained by applying several types of decellularized
matrices derived from the brain, heart, blood vessels, heart valve,
lungs, and kidneys, have been conducted.
[0007] In particular, the brain is an organ that serves as a major
control tower for regulating an organism, and is an important organ
that functions through a close network between neurons and other
various cells. However, due to the complicated structure and
working principle of the brain, sufficient studies have not been
conducted, and studies for causes of neurological diseases and
development of new drugs are confronted with limitations.
[0008] Therefore, there is a growing need for active studies of
brain development and brain diseases through the construction of an
in vitro model system that recapitulates the brain tissue and
functions thereof, and new studies for producing miniature organs
using human stem cell-derived organoids are in the spotlight.
[0009] Nevertheless, the brain organoid produced by the current
technique of culturing an organoid has many differences from the
actual brain tissue in terms of degree of differentiation and
function, and there is a need for the development of an elementary
technique for culture of a more mature brain organoid.
DISCLOSURE
Technical Problem
[0010] The present invention has been made to solve the
above-mentioned problems of the prior art, and an object thereof is
to provide a three-dimensional culture platform for improving
immature structural development and immature differentiation of a
brain organoid according to current culture techniques and for
producing a brain organoid similar to the actual brain.
Technical Solution
[0011] According to an aspect of the present invention, there is
provided a method for preparing a composition for culturing a brain
organoid, comprising (a) decellularizing brain tissue; (b) drying
the brain tissue; and (c) gelating the brain tissue.
[0012] In an embodiment, in the (c), the brain tissue and Matrigel
may be mixed to obtain a hydrogel.
[0013] In an embodiment, the hydrogel may contain a decellularized
brain extracellular matrix (dBEM) in an amount of 0.01 to 2.0
mg/mL.
[0014] In an embodiment, in the (a), the brain tissue may be
stirred in a decellularizing solution. In an embodiment, the brain
tissue may be stirred for 3 to 24 hours.
[0015] In an embodiment, 95% or more of cells of the brain tissue
may be removed by the decellularization.
[0016] According to another aspect of the present invention, there
is provided a hydrogel composition for culturing a brain organoid,
comprising a decellularized brain extracellular matrix (dBEM).
[0017] In an embodiment, the composition may further comprise
Matrigel.
[0018] In an embodiment, the decellularized brain extracellular
matrix (dBEM) may have a concentration of 0.01 to 2.0 mg/mL.
[0019] In an embodiment, the composition may have an elastic
modulus at 1 Hz of 100 to 150 Pa.
[0020] According to still another aspect of the present invention,
there is provided a method for culturing a brain organoid in the
composition.
Advantageous Effects
[0021] Unlike conventional techniques for culturing a brain
organoid, the present invention is highly likely to be applied as
an in vitro model for realizing actual brain tissue.
[0022] The brain matrix-based hydrogel of the present invention can
promote differentiation of a brain organoid into neurons and
cortical layer neurons, and can be usefully used for the
construction of an in vitro model in a brain tissue-like form.
[0023] It should be understood that the effects of the present
invention are not limited to the above-mentioned effects and
include all effects which can be deduced from the detailed
description of the present invention or the constitution of the
invention described in the claims.
DESCRIPTION OF DRAWINGS
[0024] FIGS. 1A and 1B illustrate schematic diagrams for a method
of producing a brain organoid using a decellularized brain
tissue-based three-dimensional hydrogel.
[0025] FIGS. 2A, 2B and 2C illustrate the results of the production
and analysis of human decellularized brain tissue.
[0026] FIGS. 3A and 3B illustrate the results obtained by analyzing
components of the decellularized brain tissue.
[0027] FIG. 4 illustrates the results obtained by analyzing
mechanical properties of the decellularized brain tissue-based
hydrogel.
[0028] FIGS. 5A and 5B illustrate the results obtained by analyzing
tissue-specific effects of the decellularized brain tissue-based
hydrogel.
[0029] FIGS. 6A, 6B, 6C and 6D illustrate the results obtained by
comparatively analyzing the neuronal differentiation of a brain
organoid cultured using the decellularized brain tissue-based
hydrogel or Matrigel.
[0030] FIGS. 7A-7D, 8A-C, and 9A-9B illustrate identification of
the neuronal differentiation, structural maturation, and degree of
development of a brain organoid cultured using the decellularized
brain tissue-based hydrogel or Matrigel.
[0031] FIGS. 10A-10G illustrate the results obtained by
comparatively analyzing the functionality of a brain organoid
cultured using the decellularized brain tissue-based hydrogel or
Matrigel.
[0032] FIGS. 11A-11D illustrate the results obtained by
comparatively analyzing the differentiation and intercellular
network of a brain organoid cultured using the decellularized brain
tissue-based hydrogel or Matrigel.
[0033] FIGS. 12A-12G illustrate the results obtained by
comparatively analyzing cortical layer development in a brain
organoid cultured using the decellularized brain tissue-based
hydrogel or Matrigel.
[0034] FIGS. 13A-13B illustrate the results obtained by
comparatively analyzing cortical layer and forebrain development in
a brain organoid cultured using the decellularized brain
tissue-based hydrogel or Matrigel.
[0035] FIG. 14 illustrates the results obtained by qPCR analysis of
gene expression of a brain organoid cultured using the
decellularized brain tissue-based hydrogel.
[0036] FIGS. 15A-15I, FIG. 16 and FIG. 17 illustrate the results
obtained by gene ontology (GO) analysis of gene expression of a
brain organoid cultured using the decellularized brain tissue-based
hydrogel.
MODES OF THE INVENTION
[0037] Hereinafter, the present invention will be described with
reference to the accompanying drawings. The present invention may,
however, be embodied in many different forms, and thus should not
be limited to the embodiments set forth herein. In a case where a
certain entity "comprises" a certain constitutional element, unless
specifically stated otherwise, the case means that the entity may
further include other constitutional elements rather than excluding
the other constitutional elements.
[0038] Unless otherwise defined, practice of the present invention
involves performing conventional techniques commonly used in
molecular biology, microbiology, protein purification, protein
engineering and DNA sequencing, and the field of recombinant DNA
within the skill of those skilled in the art. The techniques are
known to those skilled in the art and are described in numerous
standardized textbooks and reference books.
[0039] Unless otherwise defined herein, all technical and
scientific terms used have the same meanings as commonly understood
by those skilled in the art.
[0040] Various scientific dictionaries that include the terms
included herein are well known and available in the art. Although
any method and material similar or equivalent to those described
herein find use in the practice or testing of the present
invention, some methods and materials are described. The present
invention is not limited to particular methodology, protocols, and
reagents, as these may vary depending upon the context to be used
by those skilled in the art. Hereinafter, the present invention
will be described in more detail.
[0041] According to an aspect of the present invention, there is
provided a method for preparing a composition for culturing a brain
organoid, comprising (a) decellularizing brain tissue; (b) drying
the brain tissue; and (c) gelating the brain tissue.
[0042] The composition contains a three-dimensional culture
hydrogel prepared on the basis of a brain matrix composition
obtained by decellularization, and can be effectively used for
culturing a brain organoid.
[0043] The decellularized brain tissue contains actual
tissue-specific extracellular matrix components, and thus can
provide the physical, mechanical, and biochemical environment of
the tissue in question, and is highly efficient in enhancing
differentiation into brain tissue cells and tissue-specific
functionality.
[0044] The "organoid" refers to an ultraminiature body organ
obtained by culturing cells derived from the tissue or pluripotent
stem cells in a 3D form to produce a form such as an artificial
organ.
[0045] The organoid is a three-dimensional tissue analog that
contains organ-specific cells which originate from stem cells and
self-organize (or self-pattern) in a similar manner to the in vivo
condition. The organoid can be developed into a specific tissue by
restricted element (for example, growth factor) patterning.
[0046] The organoid can have the original physiological
characteristics of the cells and can have an anatomical structure
that mimics the original state of a cell mixture (including all
remaining stem cells and the neighboring physiological niche as
well as limited cell types). A three-dimensional culture method
allows the organoid to be better arranged in terms of cell to cell
functions, and to have an organ-like form with functionality and a
tissue-specific function.
[0047] In the (b), the decellularized brain tissue containing the
extracellular matrix may be air-dried or lyophilized. After drying,
the decellularized brain tissue may be finally sterilized by being
exposed to ethylene oxide gas or supercritical carbon dioxide by an
electron beam or gamma radiation. The decellularized brain tissue
may be stored, packaged or dispersed in a lyophilized state.
[0048] The sterilized decellularized brain tissue may be
solubilized in an acidic solution with a protease such as pepsin or
trypsin. The resultant may be mixed with a base such as an isotonic
buffer or NaOH for neutralization so that a pH thereof is adjusted
to 7.2 to 7.8, and may be gelated at a temperature of 25.degree. C.
to 38.degree. C.
[0049] In the (c), the brain tissue and Matrigel may be mixed to
obtain a hydrogel.
[0050] The "Matrigel" is a protein complex (product name of BD
Biosciences) extracted from Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells, and may contain extracellular matrix components such
as laminin, collagen, and heparan sulfate proteoglycan, and
fibroblast growth factor (FGF), epidermal growth factor (EGF),
insulin-like growth factor (IGF), transforming growth factor-beta
(TGF-.beta.), or platelet-derived growth factor (PDGF).
[0051] The "hydrogel" is a material in which a liquid that contains
water as a dispersion medium is hardened, through the sol-gel phase
transition, to lose fluidity and to form a porous structure. The
hydrogel can be formed by causing a hydrophilic polymer that has a
three-dimensional network structure and a microcrystalline
structure to contain water and to be expanded.
[0052] The hydrogel may contain the decellularized brain tissue and
Matrigel in a predetermined ratio, and the hydrogel may contain the
decellularized brain extracellular matrix (dBEM) in an amount of
0.01 to 2.0 mg/mL.
[0053] The "extracellular matrix" refers to a natural support for
cell growth which is prepared through decellularization of the
tissue found in mammals and multicellular organisms. The
extracellular matrix may be further treated through dialysis or
crosslinking.
[0054] The extracellular matrix may be a mixture of structural or
non-structural biomolecules including, but not limited to,
collagen, elastins, laminins, glycosaminoglycans, proteoglycans,
antimicrobials, chemoattractants, cytokines, and growth
factors.
[0055] The extracellular matrix may take various forms in mammals
and contain collagen in an amount of about 90%. Extracellular
matrices derived from a variety of body tissues may be different in
total structure and composition due to their inherent role required
for each tissue. The decellularized brain extracellular matrix may
be contained at a concentration of 0.01 to 2.0 mg/mL by being mixed
with the decellularized brain tissue in an appropriate ratio.
[0056] The "derive" or "derived" refers to components obtained from
mentioned sources by useful methods.
[0057] For example, the extracellular matrix-derived gel refers to
a gel containing extracellular matrix components obtained from the
tissue by a variety of technically known methods for isolating the
extracellular matrix. Alternatively, the decellularized brain
extracellular matrix may refer to an extracellular matrix
containing components obtained from the brain tissue by a useful
method.
[0058] In the (a), the brain tissue may be stirred in a
decellularizing solution.
[0059] In an embodiment, the brain tissue may be stirred for 3 to
24 hours, and 95% or more of cells of the brain tissue may be
removed by the decellularization.
[0060] The "decellularizing solution" may contain various detergent
components to remove the brain tissue cells, and examples thereof
may include, but are not limited to, hypertonic saline, peracetic
acid, Triton-X, SDS, or other detergent components.
[0061] The decellularized extracellular matrix may be dried, and
may be, for example, lyophilized or air-dried.
[0062] The dried extracellular matrix may be comminuted by methods
including tearing, milling, cutting, grinding and shearing. The
comminuted extracellular matrix may be processed into a powder form
by a method such as grinding or milling in a frozen or lyophilized
state.
[0063] According to another aspect of the present invention, there
is provided a hydrogel composition for culturing a brain organoid,
comprising a decellularized brain extracellular matrix (dBEM).
[0064] Since the hydrogel composition contains the decellularized
brain extracellular matrix in an appropriate ratio, the elastic
modulus thereof can be maintained at a level similar to that in the
actual brain tissue environment, and the optimum environment for
culturing a brain organoid can be created at an elastic modulus at
1 Hz of 100 to 150 Pa.
[0065] According to still another aspect of the present invention,
there is provided a method for culturing a brain organoid in the
composition.
[0066] The existing Matrigel-based culture system is an extract
derived from animal cancer tissue, has a large difference between
the batches, does not simulate the actual brain environment, and
exhibits insufficient efficiency in differentiation or development
into a brain organoid. On the other hand, the hydrogel composition
can create a brain tissue-like environment, and thus is suitable
for culturing a brain organoid.
[0067] The brain organoid cultured in the brain matrix-based
hydrogel composition can promote not only differentiation into
various neurons including cortical layer neurons, but also can be
structurally developed into a form similar to the actual brain.
[0068] The culture refers to a process of maintaining and growing
cells under suitable conditions, and the suitable conditions may
refer, for example, to the temperature at which the cells are
maintained, nutrient availability, atmospheric CO.sub.2 content,
and cell density.
[0069] Appropriate culture conditions for maintaining,
proliferating, expanding, and differentiating different types of
cells are known in the art and are documented. Suitable conditions
for the formation of the organoid may be conditions that facilitate
or allow for cell differentiation and formation of a multicellular
structure.
[0070] Hereinafter, the present invention will be further described
by way of examples. However, it is apparent that the present
invention is not limited by the following examples.
Experimental Example 1: Production of Brain Organoid Using
Decellularized Brain Extracellular Matrix (dBEM)-Based
Three-Dimensional Hydrogel
[0071] Referring to FIG. 1, a three-dimensional culture platform
which is excellent in production efficiency of a brain organoid was
prepared using a hydrogel containing a decellularized brain
extracellular matrix (dBEM).
[0072] The dBEM contains tissue-specific extracellular matrix
components, and thus provides cells in culture with a
tissue-specific physical and biochemical microenvironment, so that
production of an organoid and differentiation efficacy thereof can
be enhanced.
[0073] The lyophilized dBEM was subjected to a solubilization
process through an enzyme treatment method and utilized in hydrogel
production (FIG. 1A).
[0074] A brain organoid can be formed in the dBEM-based hydrogel
and cultured for a long period, through the culture protocol of
FIG. 1B.
Experimental Example 2: Production and Analysis of Human
Decellularized Brain Extracellular Matrix (dBEM)
[0075] Human brain tissue was cut to a size of 1.times.1.times.1
cm.sup.3, and decellularized by being stirred in decellularizing
solutions in the following order.
[0076] First, stirring (60 rpm) was performed in distilled water
for 24 hours, and stirring (60 rpm, 37.degree. C.) was performed in
0.05% (v/v) trypsin/ethylenediaminetetraacetic acid (EDTA) (Thermo
Fisher Scientific, Waltham, Mass.) for 90 minutes.
[0077] Stirring was performed for 120 minutes in 3% (v/v) Triton
X-100 (Wako, Osaka, Japan) containing 0.1% (v/v) ammonium hydroxide
(Sigma, St. Louis, Mo.).
[0078] Stirring was performed in a 1M sucrose solution (Sigma) for
30 minutes, and stirring was performed in distilled water for 15
minutes.
[0079] Stirring was performed in 3% (v/w) sodium dodecyl sulfate
(Sigma) for 60 minutes, and stirring was performed in 4% (v/v)
ethanol (Sigma) for 120 minutes.
[0080] Stirring was performed in PBS (Sigma) for 15 minutes, and
stirring (60 rpm) was performed in 1% (v/v) penicillin/streptomycin
(Thermo Fisher Scientific) for 60 minutes.
[0081] Finally, stirring was performed in distilled water for 15
minutes, and stirring was performed in PBS for 15 minutes.
[0082] At the time of replacing each solution, the existing
solution was washed with distilled water. Unless otherwise
indicated, all procedures were carried out at 4.degree. C. and
decellularization was performed by stirring at 120 rpm.
[0083] Referring to FIG. 2, cells can be efficiently removed
through the decellularization process, and the decellularized brain
extracellular matrix (dBEM) in which the matrix tissue is mostly
maintained can be secured.
[0084] Histology (H&E staining, Masson's trichrome staining; MT
staining) analysis showed that the cells are removed from the
tissue after being subjected to the decellularization process,
while the extracellular matrix components simulating the
biochemical microenvironment are retained (FIG. 2A).
[0085] DNA was quantified, and as a result, most of the cells (98%
or more) were removed from the tissue (FIG. 2B).
[0086] Glycosaminoglycan (GAG) was quantified, and as a result,
most of the GAG components were retained in the tissue without loss
after the decellularization process (FIG. 2C).
Experimental Example 3: Analysis of Hydrogel Components
[0087] Immunofluorescence staining and mass spectrometry were used
to analyze brain tissue-derived extracellular matrix components
that can affect the formation of an organoid.
[0088] Referring to FIG. 3, subtypes of laminin, which is an
important factor for the development and differentiation of cells
in brain tissue (before decellularization) were analyzed through
immunostaining, and as a result, in particular, laminin subtypes
.alpha.5, .beta.1, and .gamma.1 were abundantly present (FIG.
3A).
[0089] Mass spectrometry was used to analyze protein components in
the decellularized brain tissue, and as a result, not only various
types of collagens and glycoproteins such as fibronectin, and
laminin but also proteoglycan and lipid components were abundantly
contained (FIG. 3B).
[0090] In other words, the dBEM hydrogel was evaluated to enhance
the formation and differentiation of a brain organoid based on
biochemical components that simulate a brain tissue-specific
microenvironment.
Experimental Example 4: Analysis of Mechanical Properties
[0091] Referring to FIG. 4, a dBEM hydrogel having a concentration
of 400 .mu.g/mL was produced by incorporating a dBEM component into
Matrigel which is widely used for the production of an
organoid.
[0092] The physical properties of the Matrigel hydrogel and the
dBEM hydrogel were comparatively analyzed using a rheometer. As a
result, the elastic modulus was measured to be higher than the
viscous modulus within the measurement frequency, indicating that a
stable hydrogel was formed.
[0093] As a result of comparing the elastic moduli at 1 Hz,
Matrigel had an elastic modulus of about 115 Pa and the dBEM had an
elastic modulus of about 126 Pa, indicating that there was no
significant change in physical properties of the hydrogel due to
the addition of dBEM.
Experimental Example 5: Analysis of Tissue-Specific Effects of
Hydrogel
[0094] Referring to FIG. 5, tissue-specific three-dimensional
hydrogels were produced using decellularized extracellular matrices
derived from a variety of tissues, and brain tissue-specific
functionality of the dBEM hydrogels was identified.
[0095] Three-dimensional culture of a brain organoid derived from
human induced pluripotent stem cells (iPSCs) was performed. As a
result, the brain organoid exhibited a larger size in a case of
being cultured in the dBEM hydrogel than in a case of being
cultured in Matrigel (at an initial stage of culture, on day 20),
and clear neural tube formation which is important for the
formation of the early brain organoid and a high cell density were
exhibited (FIG. 5A).
[0096] The size of the brain organoid cultured in each hydrogel was
quantified. As a result, the brain organoid grown in the dBEM
hydrogel exhibited the largest size, and it was analyzed that the
brain tissue-specific dBEM hydrogel is effective for culture of a
brain organoid (FIG. 5B).
Experimental Example 6: Identification of Neuronal Differentiation
of Brain Organoid
[0097] Referring to FIG. 6, the brain organoid was cultured for 30
days using the dBEM hydrogel, and an analysis with respect to the
degree of differentiation and development inside the organoid was
performed.
[0098] Immunofluorescence staining was used to analyze
differentiation into a neuronal lineage.
[0099] The organoid differentiated in the dBEM hydrogel exhibited
increased expression of the neural stem cell marker (Nestin) and
mature neuron markers (Tuj1, MAP2) as compared with the organoid
differentiated in Matrigel (FIGS. 6A and 6B).
[0100] The image-based analysis showed that the dBEM hydrogel
promotes not only the differentiation of neurons but also the
proliferation of neural stem cells and the development of the brain
organoid (FIGS. 6C and 6D).
[0101] In particular, the brain organoid cultured in the dBEM
hydrogel exhibited an increased size regardless of the culture
environment (spinner flask, multiwell plate) (FIG. 6D).
Experimental Example 7: Identification of Differentiation and
Development of Brain Organoid
[0102] The brain organoid was cultured for 30 days using the dBEM
hydrogel, and an analysis with respect to the degree of
differentiation and development was performed.
[0103] Immunofluorescence staining was used to analyze the
expression of neuron markers (Tuj1, NeuN, MAP2), a dorsal marker of
early brain development Pax6, a marker (FoxG1) corresponding to the
forebrain, and a stem cell marker (Sox2) (FIG. 7).
[0104] The brain organoid cultured in the dBEM hydrogel exhibited
not only increased differentiation into neurons but also increased
production of stem cells, so that the maturation of the brain
organoid was promoted and a size thereof was also significantly
increased (FIG. 7A).
[0105] In addition, histology and SEM analysis also showed
increased cell density in the brain organoid (FIG. 7B), decreased
void space in the brain organoid (FIG. 7C), and formation of a
mature organoid in the dBEM hydrogel.
[0106] In particular, the laminin layer corresponding to the basal
lamina of the human brain was more clearly formed in the brain
organoid cultured in the dBEM (FIG. 7D).
[0107] The brain organoid was cultured for 45 days using the dBEM
hydrogel, and the degree of differentiation and development thereof
was analyzed.
[0108] Referring to FIG. 8, the staining of the cortical layer
markers TBR1 and TBR2 showed that the number of cells expressing
the markers is increased and the thickness of the layer of
expressing cells is increased, as compared with the existing
Matrigel culture environment (FIGS. 8A and 8B).
[0109] In addition, it was identified that the interaction
(N-cadherin) between the neurons is promoted (FIG. 8C).
[0110] The brain organoid was cultured for 45 days using Matrigel
or the dBEM hydrogel, and the distribution and structure
development of three-dimensional neurons in the brain organoid were
identified using a tissue clearing technique.
[0111] Referring to FIGS. 9A and 9B, in the brain organoid cultured
using the dBEM hydrogel, increased differentiation into mature
neurons was exhibited, and a brain tissue-specific wrinkled
structure was developed in a form similar to the actual brain.
Experimental Example 8: Analysis of Functionality of Brain
Organoid
[0112] Fluorescent calcium imaging experiments were carried out to
identify the reactivity of the brain organoid, which had been
cultured in the hydrogel for 45 days, to the glutamate and gamma
amino butyric acid (GAB A) neurotransmitters.
[0113] Referring to FIG. 10, the brain organoid cultured in the
dBEM hydrogel was higher in the proportion of cells responsive to
glutamate and intracellular calcium influx than the brain organoid
cultured in Matrigel (FIGS. 10A and 10D).
[0114] The brain organoid cultured in the dBEM also responded to
gamma amino butyric acid, whereas no responsive cells were observed
in the brain organoid cultured in Matrigel (FIG. 10B).
[0115] On the other hand, the brain organoid cultured in the dBEM
hydrogel was observed to respond to glutamate, and exhibited a
decreased neurotransmission signal and decreased activity in a case
of being treated with the sodium channel blocker, tetrodotoxin
(TTX) (FIG. 10C).
[0116] Patch clamps were used to analyze electrophysiological
functionality of the brain organoid cultured in the dBEM hydrogel
(FIGS. 10E to 10G).
[0117] In a voltage-clamped state, current generation by the
voltage-openable sodium channel was identified (FIG. 10E), and the
sodium channel-induced current disappeared due to the treatment
with the Na+ channel blocker, tetrodotoxin (TTX) (FIG. 10G). In a
case of being measured in a current-clamped state, an action
potential was generated (FIG. 10F).
[0118] In other words, the brain organoid cultured under the dBEM
condition exhibited remarkably superior neuroelectrophysiological
functionality as compared with the brain organoid cultured under
the Matrigel condition.
Experimental Example 9: Identification of Differentiation of Brain
Organoid and Intercellular Network Thereof
[0119] After culture of the brain organoid was conducted for 75
days using Matrigel as a control and the dBEM hydrogel,
immunofluorescence staining was used to analyze the differentiation
and intercellular network formation.
[0120] Referring to FIG. 11, the brain organoid cultured in the
dBEM hydrogel exhibited remarkably high levels of expression and
distribution of N-Cadherin, which is known to play a role in
adhesion between neurons, as compared with the brain organoid
cultured in Matrigel.
[0121] The N-cadherin is known to be a protein associated with
promotion of neurite outgrowth and synaptogenesis (FIGS. 11A and
11B).
[0122] Immunofluorescence staining was used to analyze the
expression of the synapsin I protein, a synaptic vesicle marker, in
the brain organoid cultured under the dBEM condition.
[0123] Synapsin I is an important protein in the chemical signaling
process of neural synapses, and the results suggest that the brain
organoid cultured using the dBEM exhibit a high degree of
maturation (FIG. 11C).
[0124] In addition, the expression of VGLUT, a representative
marker of a glutamatergic neuron, showed a high degree of
differentiation in the brain organoid cultured in the dBEM (FIG.
11D).
Experimental Example 10: Development of Cortical Layer in Brain
Organoid
[0125] After the brain organoid was cultured for 75 days using
Matrigel or the dBEM hydrogel, a degree of development of the
cortical layer in the brain organoid was analyzed.
[0126] Referring to FIG. 12, the brain organoid cultured in the
dBEM hydrogel exhibited increased expression and thickness of TBR1,
a marker corresponding to the cortical layer IV position, and it
was identified that differentiation into a mature brain organoid
occurs (FIG. 12A).
[0127] In the brain organoid cultured in the dBEM hydrogel, the
expression of TBR2, a marker corresponding to the subventricular
zone, was overall widely distributed (FIG. 12B), and the cortical
layer and specific wrinkled structure of the brain organoid were
well developed (FIG. 12C).
[0128] In a case where a size of the formed brain organoid is
measured based on the longest diameter, the brain organoid cultured
in the dBEM hydrogel exhibited a remarkably large size as compared
with the brain organoid cultured in Matrigel (FIG. 12D), and
cortical layer markers were continuously expressed in the dBEM
hydrogel (FIG. 12E).
[0129] In addition, two-dimensional and three-dimensional
light-sheet microscopy was used to analyze the formed brain
organoid. As a result, in the dBEM hydrogel group, the wrinkled
structure of the brain organoid was developed and the total volume
was increased (FIGS. 12F and 12G).
[0130] These results suggest that a degree of maturation of brain
organoid can be increased by the dBEM hydrogel.
Experimental Example 11: Analysis of Development of Cortical Layer
and Forebrain in Brain Organoid
[0131] The brain organoid was cultured for 75 days using the dBEM
hydrogel, and fluorescence immunostaining was used to analyze the
maturation of the cortical layer and the development of the
forebrain portion.
[0132] Referring to FIG. 13, the maturation and development of the
cortical layer in the brain organoid cultured in the dBEM
environment were identified through immunostaining of TBR2, a
marker corresponding to the subventricular zone, and CTIP, a marker
for cortical layer 5 (FIG. 13A).
[0133] In addition, in a case of comparing the overall distribution
of FoxG1, a forebrain marker, the brain organoid cultured in the
dBEM hydrogel exhibited a remarkably increased expression level of
the forebrain-specific marker, and also exhibited an increased size
and degree of maturation (FIG. 13B).
Experimental Example 12: Comparison of Gene Expression in Brain
Organoid (qPCR)
[0134] The gene expression in the brain organoid which had been
cultured in the hydrogel for 75 days was identified by qPCR, in
which gene expression levels in the human fetal neural stem cell
(hNSC) and human-derived brain tissue (hTissue) as controls were
compared together.
[0135] Referring to FIG. 14, in a case of the brain organoid
cultured using the dBEM, increased expression of Tuj1, a neuron
marker, and TH, a dopaminergic neuron marker, were exhibited, and
increased expression of CDH1 that plays an important role in
intercellular adhesion and neuron survival was also exhibited, as
compared with the brain organoid cultured in Matrigel.
Experimental Example 13: Comparison of Gene Expression in Brain
Organoid (Gene Ontology)
[0136] For the brain organoid cultured in Matrigel and the brain
organoid cultured in the dBEM, transcript expression analysis was
used to comparatively analyze characteristics of both
organoids.
[0137] RNA-sequencing was used to compare gene expression patterns
between the two groups based on the gene ontology (GO) category
analysis.
[0138] The genes which had been increased in the dBEM group as
compared with the Matrigel group (filtering condition: difference
of 1.2 or more, p<0.05, FDR<0.1, n=3) were clustered, and the
gene ontology (GO) analysis was performed.
[0139] Referring to FIG. 15A, the 30 GO terms with the highest
score were mostly associated with the nervous system.
[0140] In particular, the brain organoid cultured in the dBEM
exhibited increased expression of many markers associated with
electrophysiological functions such as synapse and neurotransmitter
transmission. These results suggest that the brain organoid is
efficiently induced in a case of being cultured in the developed
dBEM as compared with a case of being cultured in the existing
Matrigel condition.
[0141] Referring to FIGS. 15B to 15I, the expression of genes
associated with pluripotency was increased, and the expression of
forebrain-associated markers and mature neuron-associated markers
was also increased.
[0142] In particular, glial-associated markers are known to be
detected following the expression of neuron-associated markers in
the culture of the brain organoid.
[0143] The associated markers were increased in the dBEM group, and
the results indicate that a degree of maturation of the brain
organoid in the dBEM group is promoted. In addition, the expression
of many genes associated with ECM interaction and cell adhesion was
increased.
[0144] Referring to FIG. 16, the gene ontology (GO) was used to
analyze the genes which had been significantly increased
(p-value<0.05, FDR<0.1) 1.2-fold or more as compared with the
Matrigel group, and as a result, the expression of many genes
associated with synaptic chemical signaling was increased.
[0145] Referring to FIG. 17, the KEG pathway was used to analyze
the genes which had been significantly increased (p-value<0.05,
FDR<0.1) 1.2-fold or more as compared with the Matrigel group,
and as a result, the expression of genes associated with neuron
synapses of various subtypes was increased.
[0146] The foregoing description of the present invention is for
illustrative purposes, and it should be understood by those of
ordinary skill in the art that various changes and modifications
may be made without departing from the technical spirit or
essential features of the present invention. It is therefore to be
understood that the above-described embodiments are illustrative in
all aspects and not restrictive. For example, each constitutional
element described as a single entity may be implemented in a
distributed manner. Likewise, constitutional elements described as
distributed may also be implemented in a combined form.
[0147] The scope of the present invention is defined by the
appended claims, and all changes or modifications deduced from the
meaning and scope of the claims and their equivalents should be
construed as being included within the scope of the present
invention.
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