KR102405421B1 - cardiomyocyte spheroid formed on cell nonadhesive PEG polymer substrate and method for preparing the same - Google Patents

Abstract

The present invention relates to a cardiomyocyte spheroid formed on a substrate and a method for manufacturing the same, wherein the cardiomyocyte spheroid can control cell interaction through patterning, has high transparency, is easy to observe, and is differentiated under general culture conditions Because it can control and promote dedifferentiation, it can be effectively used for drug screening by accurately mimicking the physiological properties of cardiomyocytes.

Description

Cardiomyocyte spheroid formed on cell nonadhesive PEG polymer substrate and method for preparing the same

The present invention relates to a cardiomyocyte spheroid formed on a cell-non-adhesive PEG polymer substrate and a method for preparing the same.

In new drug development, animal testing is performed in non-clinical/clinical testing and initial screening. In addition, for ethical reasons worldwide, as animal testing is opposed and regulations on the use of laboratory animals are imposed, attempts are being made to replace animal testing through in vitro experiments using animals or human cells, especially human The importance of evaluating the safety and efficacy of pharmaceuticals using stem cell-derived cardiomyocytes, brain, and liver cells is growing.

However, there are many difficulties in predicting the actual clinical response using the existing in vitro experimental technology. Cells change their adhesion, growth, migration, and differentiation depending on the culture environment, so it provides a culture system similar to the in vivo environment that can three-dimensionally interact with surrounding cells and extracellular matrix to preserve the original function of cells. It is important to maintain, since the two-dimensional (2D) monolayer culture substrate is in a very different environment from the actual in vivo environment, there is a dedifferentiation phenomenon in which cells rapidly lose their intrinsic properties, and there is a problem in that it is difficult to control the differentiation of stem cells. Therefore, the existing 2D culture method and the monolayer cell culture method using a general cell culture vessel have limitations in accurately simulating the physiological characteristics of cardiomyocytes and living organs.

Accordingly, three-dimensional (3D) cell culture technology is being developed to model the internal environment of a living body, and a typical example is a technology of culturing cells in the form of a spheroid, which is a 3D spherical cell aggregate with a structure similar to the interior of a living body. have. A spheroid is a three-dimensional tissue analogue in which cells are aggregated and combined, and has the property of maintaining a cell-to-cell interaction similar to that of the body even in in vitro culture, and has the advantage of increasing clinical efficiency in drug screening. Therefore, cell spheroids are attracting attention in research on cells constituting general tissues or organs, cancer cells, and stem cells, and also for the development of new drugs including cell therapeutics, drug screening system development, and differentiation using stem cells. It also plays an important role in research.

On the other hand, the technology to form these cell spheroids is basically based on preventing the cells from adhering to the outer wall. As a representative prior art for this purpose, the Hanging Drop Technique, a method using a fine cell trapping structure, a cell floating-based method through non-adhesive surface fixing (Liquid-Overlay Culture Method), a Spinner Culture Flask ), a microchip-based method, etc. are being used (Korean Patent Publication No. 10-2017-0107145).

However, commercialized culture vessels for producing cell spheroids are commonly dented in the shape of a pocket, and due to this polymer molding structure, when a specific drug is directly processed into cells using the vessel, the concentration distribution of the drug is higher than that of the Well. ·There are problems that may vary depending on the location below. In addition, there is also a problem in that the number of spheroids is less than 100, or loss of spheroids occurs in the process of recovering the formed cell spheroids.

For overcoming the above problems and for quality control of cell spheroids and various application experiments utilizing the same, a technology for controlling cell spheroids to a certain size is required, and in particular, for treatment and analysis, cells controlled to a large homogeneous size A spheroid manufacturing technique is essential. For this purpose, a substrate can be used, and the Langmuir-Blodgett (LB) method (hereinafter “LB method”), which is a technique for aligning and coating fine particles on a substrate, can be utilized, but effective control of cell spheroids can be achieved. For this, research on the patterning of the substrate is required.

Therefore, while the present inventors were studying to find a way to efficiently differentiate stem cells into cardiomyocytes, when stem cells are cultured in a cell adhesion pattern array culture dish spaced apart at regular intervals on a non-adhesive PEG polymer substrate, cardiomyocytes By confirming that the production of spheroids is possible and efficiently differentiated into cardiomyocytes, the present invention was completed.

It is an object of the present invention to provide cardiomyocyte spheroids formed on a cell-non-adhesive PEG polymer substrate.

Another object of the present invention is to provide a method for preparing the cardiomyocyte spheroid.

In order to achieve the above object,

According to one aspect of the present invention,

Cell non-adhesive PEG polymer substrate;

an array of cell adhesion patterns formed on the substrate and spaced apart from each other; and

It provides a cardiomyocyte spheroid formed on a cell non-adhesive PEG polymer substrate comprising cardiomyocyte spheroids formed on the cell adhesion pattern array.

According to another aspect of the invention,

(a) forming an array of cell adhesion patterns spaced apart at regular intervals on a cell-non-adhesive PEG polymer substrate; and

(b) seeding and culturing human induced pluripotent stem cell-derived cardiomyocytes on the cell adhesion pattern array to form cardiomyocyte spheroids on a cell-non-adhesive PEG polymer substrate provides a method for manufacturing

Cardiomyocyte spheroids formed on the cell-non-adhesive PEG polymer substrate according to the present invention can effectively control the aggregation of cardiomyocytes on large-scale cardiomyocyte spheroids.

In particular, by forming an array of cell adhesion patterns spaced apart from each other at regular intervals on the substrate, cardiomyocyte spheroids can be concentrated in the center of individual cell adhesion patterns, and a certain distance can be maintained between cardiomyocyte spheroids. , can be effectively used for drug screening.

1 is a schematic diagram for cardiomyocyte spheroids formed on a cell-non-adhesive PEG polymer substrate.
2A and 2B are optical images of silica monolayer nanoparticles formed on a cell-non-adhesive PEG polymer substrate.
Figure 2c is an image observed through a confocal laser microscopy (confocal laser microscopy) of the silica monolayer nanoparticles formed on the cell non-adhesive PEG polymer substrate.
2D is an image of silica monolayer nanoparticles formed in a circular cell adhesion pattern observed through atomic force microscopy (AFM).
Figure 3a is an image of observing the interface of the cell non-adhesive PEG polymer substrate with an atomic force microscope.
Figure 3b is a graph showing the step difference of the atomic force microscope at the interface of the cell non-adhesive PEG polymer substrate.
Figure 3c is an optical image of the cell non-adhesive PEG polymer substrate interface.
3D is a three-dimensional image of silica monolayer nanoparticles observed with an atomic force microscope.
Figure 4a is a protocol for culturing human induced pluripotent stem cells (CMC-hiPS-009) and differentiating them into cardiomyocytes, and images observed with a phase-contrast microscope for cell morphology for each culture period.
Figure 4b is an image of observing the morphology of human induced pluripotent stem cells (CMC-hiPS-009)-derived cardiomyocytes under a phase-contrast microscope on the 45th day from the day of culture.
4C is a phase contrast microscope after seeding of 5x10 ⁵ human induced pluripotent stem cells (CMC-hiPS-009)-derived cardiomyocytes on a circular cell adhesion pattern 3D spheroid substrate (6-well plate) according to the present invention and 72 hours later observed image.
5A is an image of 5×10 ⁵ human induced pluripotent stem cell (CMC-hiPS-009)-derived cardiomyocytes seeded in a 6-well plate and observed with a phase contrast microscope at 10× magnification 12 hours after seeding.
5B is an image of 5×10 ⁵ human induced pluripotent stem cell (CMC-hiPS-009)-derived cardiomyocytes seeded in a 6-well plate and observed with a phase-contrast microscope at 10× magnification after 72 hours.
5c and 5d are images of 5x10 ⁵ human induced pluripotent stem cell (CMC-hiPS-009)-derived cardiomyocytes seeded in a 6-well plate and observed with a phase contrast microscope at 20x magnification after 72 hours.
Figure 6a shows commercially available cardiomyocytes from Cellular Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5) thawed and cultured in a 96-well plate. After 4 days, circular cell adhesion It is an image observed with a phase contrast microscope in which cardiomyocyte spheroids are formed on the pattern.
Figure 6b shows commercially available cardiomyocytes from Cellular Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5) thawed and cultured in a 96-well plate. 4 days after seeding, radial cell adhesion It is an image observed with a phase contrast microscope in which cardiomyocyte spheroids are formed on the pattern.
Figure 6c shows commercially available cardiomyocytes from Cellular Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5) thawed and cultured in a 96-well plate, 5 days after seeding, circular cell adhesion This is an image of the cardiomyocyte spheroids on the pattern stained with cTnT, Connexin 43, and DAPI.
FIG. 6d shows thawed and cultured cardiomyocytes commercially available from Cellular Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5) in a 96-well plate and 5 days after seeding, radial cell adhesion This is an image of the cardiomyocyte spheroids on the pattern stained with cTnT, Connexin 43, and DAPI.

Hereinafter, the present invention will be described in detail.

While the present inventors were conducting research on the patterning of a substrate for forming cell spheroids, when an optimized cell adhesion pattern array is formed on a cell-non-adhesive PEG polymer substrate, cardiomyocyte spheroids can be uniformly formed. It was confirmed that possible, and the present invention was completed.

The present invention is a cell non-adhesive PEG polymer substrate;

an array of cell adhesion patterns formed on the substrate and spaced apart from each other; and

It provides a cardiomyocyte spheroid formed on a cell non-adhesive PEG polymer substrate comprising cardiomyocyte spheroids formed on the cell adhesion pattern array.

The cell non-adhesive PEG polymer substrate may be a PEG hydrogel, but is not limited thereto.

The cell adhesion pattern may include particles at least partially coated with a cell adhesion material. Here, the cell-adhesive material may be a cell-adhesive peptide, a cell-adhesive protein, or a cell-adhesive nucleic acid. The cell-adhesive peptide may be RGD peptide (Arg-Gly-Asp; RGD, Arginylglycylaspartic acid (RGD) peptide), poly-L-lysine (PLL, polylysine) or poly-D-lysine (PDL, polydilysine). However, the present invention is not limited thereto. The cell adhesive protein may be collagen, osteopontin, vitronectin, fibronectin, laminin, tenascin, fibrinogen, or thrombospondin. , but is not limited thereto.

In addition, the particles may be a polymer, an inorganic material, a metal, a magnetic material, a semiconductor, etc., and may be a mixture of particles having different properties, but is not limited thereto.

Examples of the polymer include polystyrene (PS), polymethyl methacrylate (PMMA), polyacrylate, polyvinyl chloride (PVC), polyalphastyrene, polybenzyl methacrylate, polyphenyl methacrylate, polydiphenyl methacrylate acrylate, polycyclohexyl methacrylate, styrene-acrylonitrile copolymer, styrene-methyl methacrylate copolymer, and the like.

The inorganic material is silicon oxide (eg, SiO ₂ ), silver phosphate (eg, Ag ₃ PO ₄ ), titanium oxide (eg, TiO ₂ ), iron oxide (eg, Fe ₂ O ₃ ), zinc oxide , cerium oxide, tin oxide, thallium oxide, barium oxide, aluminum oxide, yttrium oxide, zirconium oxide, copper oxide, nickel oxide, and the like.

Examples of the metal include gold, silver, copper, iron, platinum, aluminum, platinum, zinc, cerium, thallium, barium, yttrium, zirconium, tin, titanium, or an alloy thereof.

The semiconductor includes silicon, germanium, or a compound semiconductor (eg, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, etc.).

The particles may be SiO ₂ coated at least partially with a cell-adhesive material, where SiO ₂ may be prepared by a known method such as a stober method. And the coating can be performed by a known SiO ₂ surface modification method, for example, by introducing an amine group to SiO ₂ and modifying a maleimide group, then modifying a peptide modified with a cysteine functional group containing an RGD sequence to coat the RGD peptide SiO ₂ particles can be prepared.

The cardiomyocyte spheroid may be an aggregate of human induced pluripotent stem cell-derived cardiomyocytes.

The cell adhesion pattern array is formed on a cell-non-adhesive PEG polymer substrate, and is characterized in that it is spaced apart at a predetermined interval, specifically, the predetermined interval is preferably maintained at 600 μm to 1,000 μm, and 800 μm to 1,000 μm. It is more preferable to maintain, but is not limited thereto.

The cell adhesion pattern array means a plurality of arrangement of individual cell adhesion patterns. Specifically, the individual cell adhesion pattern is divided into a central portion and a peripheral portion based on a 1/2 point at a distance from the center to the circumference, and the central portion may have a higher adhesive force per unit area than the peripheral portion. At this time, the adhesive force per unit area can be controlled by the cell adhesion pattern density or the cell adhesion pattern material. , a cell adhesion pattern material with high cell adhesion strength can be applied.

In particular, the perimeter may include one or more guide lines formed radially from the center. In this case, the length, thickness, and number of the guide lines may be variously adjusted. More specifically, the length of the guide line is 100 μm to 500 μm, preferably 200 to 400 μm, more preferably 250 to 350 μm, and the thickness of the guide line is 1 μm to 20 μm, preferably 1 to 10 less than μm, and the number of the guide lines may be 20 to 100. Thereby, the cardiomyocyte spheroids can be centrally located in the center within the individual cell adhesion pattern. In particular, by optimizing the length, thickness and number of guide lines, even when cells are seeded and cultured once, it has the advantage of uniformly forming cardiomyocyte spheroids.

For example, the individual cell adhesion pattern may have a circular or radial shape, preferably a radial shape, and more preferably a thin radial shape with a guide line having a thickness of less than 10 μm, but is not limited thereto.

The cardiomyocyte spheroid is characterized in that it is formed on the cell adhesion pattern array, in a three-dimensional form, it is preferable that an average diameter of 100 μm to 500 μm can implement a size similar to that of human myocardial tissue. , but is not limited thereto.

In addition, the present invention comprises the steps of (a) forming a cell adhesion pattern array spaced apart at regular intervals on a cell non-adhesive PEG polymer substrate; and

(b) seeding and culturing human induced pluripotent stem cell-derived cardiomyocytes on the cell adhesion pattern array to form cardiomyocyte spheroids on a cell-non-adhesive PEG polymer substrate provides a method for manufacturing

First, the method for forming cardiomyocyte spheroids on a cell-non-adhesive PEG polymer substrate according to the present invention includes the step of forming an array of cell adhesion patterns spaced apart at regular intervals on the substrate [step (a)]. Thereby, human induced pluripotent stem cell-derived cardiomyocytes can be seeded and cultured on the cell adhesion pattern array formed on the substrate.

The substrate may be prepared by coating a plurality of silica particles by applying pressure on a commercially available adhesive polymer substrate.

Since the cell adhesion pattern array is the same as described above, a redundant description will be omitted.

Next, in the method for forming cardiomyocyte spheroids on a cell-non-adhesive PEG polymer substrate according to the present invention, human induced pluripotent stem cell-derived cardiomyocytes are seeded and cultured on the cell adhesion pattern array to form cardiomyocytes. and forming a roid [step (b)].

As the cardiomyocytes, commercially available ones may be used or those differentiated by culturing human induced pluripotent stem cells may be used.

Human induced pluripotent stem cell-derived cardiomyocytes seeded on the cell adhesion pattern array may be 2 to 1000000 cells/well, 50 to 800000 cells/well, or 100 to 600000 cells/well, and the culture is performed at 30 to 40°C. can be performed in In some cases, the seeding and culture may be repeated two or more times. In this case, the number of human induced pluripotent stem cell-derived cardiomyocytes seeded per well may be divided by the number of repetitions.

As described above, the cardiomyocyte spheroids formed on the cell-non-adhesive PEG polymer substrate according to the present invention include: the cell-non-adhesive PEG polymer substrate; an array of cell adhesion patterns formed on the substrate and spaced apart from each other; It is characterized in that it comprises cardiomyocyte spheroids formed on the cell adhesion pattern array, so that it is possible to effectively control the aggregation of cardiomyocytes on large-scale cardiomyocyte spheroids.

In particular, by forming a cell adhesion pattern array spaced apart at regular intervals on the substrate, the cardiomyocyte spheroids can be centrally located in the individual cell adhesion pattern of the cell adhesion pattern array, as well as the cardiomyocyte adhesion pattern array. It is possible to maintain a certain distance between the roids.

Hereinafter, the present invention will be described in detail through Preparation Examples and Examples.

However, Preparation Examples and Examples to be described below are merely illustrative of the present invention in detail in one aspect, and the present invention is not limited thereto.

<Preparation Example 1> Preparation of cell-non-adhesive PEG polymer substrate

(1) SiO to which RGD peptide is attached ₂ Preparation of particles

SiO ₂ (silica, silica) particles were prepared according to the Stober method. For synthesizing SiO ₂ particles with a diameter of about 300 nm, 1000 mL of absolute ethanol, 40 mL of 28 wt% ammonia solution, and 80 mL of water were added to an Erlenmeyer flask and about Mix for 10 minutes. Then, 60 mL of TEOS (Tetraethyl orthosilicate) was added and mixed at room temperature for 12 hours. TEOS was continuously added to increase the particle size, and the injection of TEOS was stopped when the particle diameter reached about 750 nm.

SiO ₂ having a particle diameter of 750 nm was purified with ethanol three times through centrifugation and stored as a solution. 750 nm SiO ₂ particles stored in ethanol were uniformly dispersed through sonication, and 3-aminopropyltriethoxysilane (APTES) of 0.1% (v/v) was injected with respect to the solution volume and reacted for 12 hours. Through the reaction, SiO ₂ particles introduced with an amine functional group were purified with ethanol three times, and dried in an oven at 80° C. for 12 hours. Silica particles introduced with an amine functional group were dispersed in a phosphate buffer having a pH of 7.4 and 0.05 M to a concentration of 50 mg/mL, and then 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3- (N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3- having a concentration of 0.1 mg/mL A maleimide group was modified on the particle surface by adding sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) reagent.

The maleimide group-modified particles were purified with distilled water three times through centrifugation and dispersed in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer at pH 6.6 and 0.05 M, and then at a concentration of 0.1 mg/ml. A peptide modified with a cysteine functional group containing the RGD sequence was added, and a reducing agent, tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 0.01 mg/mL was added and reacted for 12 hours. After the reaction was completed, the SiO ₂ particles were purified with distilled water three times through centrifugation to prepare SiO ₂ coated with RGD peptide.

(2) Preparation of a substrate on which a circular cell adhesion pattern array is formed

An adhesive polymer substrate made of polyethylene glycol (PEG) was prepared by including 10% by weight of a curing agent based on Sylgard 184 (Dow Corning, USA) product. A plurality of silica particles are placed on the adhesive polymer substrate and rubbed while applying pressure by hand using a sponge wrapped in latex film to form a recess on the surface of the adhesive polymer substrate while forming a recess on the surface of the adhesive polymer substrate with silica and polystyrene (PS) particles and adhesive polymer substrate By bonding to form a silica-coated film, a circular cell adhesion pattern array with a center-to-center spacing of 800 μm spaced apart on a substrate formed of polyethylene glycol (PEG) hydrogel as a cell non-adhesive material was formed.

At this time, the diameter of the circular cell adhesion pattern is 400 μm, and the patterning is performed using the SiO ₂ particles coated with the cell adhesion RGD peptide (Arg-Gly-Asp; RGD, Arginylglycylaspartic acid (RGD) peptide) prepared by the above method. However, it was carried out in a manner of locally exposing ultraviolet rays through a photomask.

The substrate on which the circular cell adhesion pattern array prepared according to the above method was formed was observed with an optical image, a confocal laser microscope, and an atomic force microscope, and it was confirmed that the size of the patterned silica nanoparticles was 750 nm (Figs. 2a to 2d). and FIGS. 3A-3D ).

(3) Preparation of the substrate on which the radial cell adhesion pattern array is formed

An adhesive polymer substrate made of polyethylene glycol (PEG) was prepared by including 10% by weight of a curing agent based on Sylgard 184 (Dow Corning, USA) product. A plurality of silica particles are placed on the adhesive polymer substrate and rubbed while applying pressure by hand using a sponge wrapped in latex film to form a recess on the surface of the adhesive polymer substrate while forming a recess on the surface of the adhesive polymer substrate with silica and polystyrene (PS) particles and adhesive polymer substrate By bonding to form a silica-coated film, a radial cell adhesion pattern array with a center-to-center spacing of 800 μm spaced apart on a substrate formed of polyethylene glycol (PEG) hydrogel as a cell non-adhesive material was formed.

At this time, the diameter (guide line length Х2) of the radial cell adhesion pattern was 400 µm, the guide line thickness and number were 10 µm and 72 pieces, and the patterning was performed using the cell adhesion RGD peptide (Arg-Gly-Asp; RGD, Arginylglycylaspartic). acid (RGD) peptide) coated SiO ₂ particles were used, but local exposure to ultraviolet light through a photomask was performed.

<Preparation Example 2> Preparation of differentiated cardiomyocytes by culturing human induced pluripotent stem cells

To differentiate human induced pluripotent stem cells (CMC-hiPSC-009) from the Korea Centers for Disease Control and Prevention into their own cardiomyocytes, 10 μg of Vitronectin XF (Vitronectin XF, Cat#.07180, Stem cell technologies) per well was added to coat the wells of a 6 well plate. Prepared by culturing human induced pluripotent stem cells (CMC-hiPSC-009) on a coated plate using TeSR™-E8™ medium (Cat#.05940, Stem cell technologies) at 37°C, 5% CO ₂ conditions, The medium was changed daily during the subculture period. In order to differentiate the cultured cells into cardiomyocytes, a pre-culture step was performed.

First, the Matrigel-hESC qualified Matrix (Corning, Cat#.354277) was diluted at a volume ratio of 200:1 using DMEM/F12 medium, and it was added to 200 mL per well of a 6-well plate, 37°C, 5 The wells of the plate were coated by standing for 1 hour under % CO ₂ conditions. After dispensing 5x10 ⁵ human induced pluripotent stem cells into the wells of the coated plate, mTeSR™ medium (Cat#. 5850, Stem cell technologies).

After culture, when the cell density becomes 100%, 10 μM of CHIR99021 (Selleckchem, Cat#.S2924) was added to STEMdiff™APEL™ 2 Medium medium in which Growth factor reduced form Matrigel (Corning, Cat# 354230) was diluted 200:1. added and incubated. After 24 hours, the medium was removed and replaced with new STEMdiff™APEL™ 2 Medium (STEMCELL TECHNOLOGIES, cat# 05270). After 3 days of culture, B27 supplement without insulin (Invitrogen, Cat# A1895601) was administered. 50 mL of RPMI-1640 medium (Gibco, Cat#11875) was replaced with a differentiation medium diluted at a volume of 200:1, and 5 μM of IWP4 (Stemgent, Cat# 04-0036) was added.

After 5 days from the culture day, the differentiation medium was replaced, and the new differentiation medium was replaced every 2 days to successfully differentiate into cardiomyocytes. After 8 days from the day of culture, it was possible to observe that the cardiomyocytes were beating (beating). The differentiated cardiomyocytes were continuously cultured, recovered 45 days after the culture day, and used for seeding on a substrate on which a circular cell adhesion pattern array was formed in the following example (see FIGS. 4A and 4B ).

<Preparation Example 3> Thaw and culture of cardiomyocytes

After thawing and culturing commercially available cardiomyocytes from Cellular Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5), they were used for seeding on a substrate in the following examples, and the specific procedure is as follows.

Take out the iCell Cardiomyocytes Plating Medium (iCell, M1001) and iCell Cardiomyocytes Maintenance Medium (iCell, M1003) stored at -80℃, and store at 4℃ in the refrigerator to thaw slowly for one day. In order to make commercially available cardiomyocytes from Cellular Dynamics adhere well to a 6-well plate, 1 mL of 0.1% gelatin (STEMCELL Technologies, 07903) was dispensed per well of a 6-well plate and coated for about 1 hour at room temperature, at 4°C. The stored Plating Medium was left at room temperature for 1 hour.

The cardiomyocyte tube stored in liquid nitrogen was taken out and placed in a constant temperature water bath at 37° C. for 3 to 4 minutes, and the melted cardiomyocyte tube was placed on a sterile workbench. 1 mL of Plating Medium was placed in a tube, and all the solutions in the tube were slowly placed in a separate 50 mL empty tube. Afterwards, 1 mL of Plating Medium is added to the empty tube containing cardiomyocytes again, and the solution is added dropwise at intervals of about 4 to 5 seconds, and 8 mL of Plating Medium is added dropwise at intervals of 2 to 3 seconds. After try-in, the tube was closed, turned over slowly 2-3 times, and the number of cells was checked using trypan blue.

Dispense 2 mL of Plating Medium per well of a 6-well plate, dispense so that the number of cardiomyocytes is 6x10 ⁵ in each well, and use Maintenance Medium stored at 4°C under 37°C and 5% CO ₂ conditions for 24 hours incubated during After that, each well was replaced with 2 mL of Maintenance Medium, maintained at 37°C and 5% CO ₂ , and replaced with a new 2 mL of Maintenance Medium every other day. After 2 days from the start of the culture, the beating of cardiomyocytes could be confirmed, and this was used for seeding on a substrate on which a circular cell adhesion pattern or a radial cell adhesion pattern array was formed.

<Example 1> Formation of cardiomyocyte spheroids through seeding of cardiomyocytes

On the cell-non-adhesive PEG polymer substrate prepared in Preparation Example 1, the following experiment was performed to confirm whether cardiomyocyte spheroids were formed, and the results are shown in FIGS. 4c, 5a to 5d, 6a and 6b. shown in

(1) Formation of circular cell adhesion pattern cardiomyocyte spheroids

[Culturing and seeding of cardiomyocytes self-differentiated from human induced pluripotent stem cells (CMC-hiPSC-009)]

For the formation of cardiomyocyte spheroids, the human induced pluripotent stem cells (CMC) of Preparation Example 2 in a 6-well plate patterned with nanoparticles on the substrate on which the circular cell adhesion pattern array prepared in (2) of Preparation Example 1 is formed. -hiPSC-009) self-differentiated cardiomyocytes (human pluripotent stem cell-derived cardiomyocyte, hiPSC-CM) were seeded in each well in a total volume of 100 μL, 5×10 ⁵ each.

As a result of observing the cells with a phase-contrast microscope 24 hours after seeding, it was confirmed that three-dimensional spheroids were formed in a circular pattern, did not fall off even after 72 hours after seeding, and further confirmed that the cells were adhered in an aggregated form. was confirmed (see FIGS. 4C and 5A to 5D).

[Seeding of cardiomyocytes from Cellular Dynamics]

For the formation of cardiomyocyte spheroids, the cardiomyocytes of Preparation Example 3, that is, commercially available Cellular, in a 96-well plate in which nanoparticles are patterned on the substrate on which the circular cell adhesion pattern array prepared in (2) of Preparation Example 1 is formed. Cardiomyocytes from Dynamics (iCell Cardiomyocytes2 Kit, 01434, cat# CMC-100-012-000.5) were thawed and cultured, 1×10 ⁵ each, in a total volume of 100 μL, and seeded in each well.

As a result of observing the cells with a phase-contrast microscope 12 hours after seeding, it was confirmed that the cardiomyocytes formed a three-dimensional spheroid in a circular pattern. It was confirmed that it was adhered to a spheroid in the form of a spheroid.

In both cases of culturing cardiomyocytes differentiated from human induced pluripotent stem cells (CMC-hiPSC-009) and thawing and culturing commercially available cardiomyocytes, when seeded on a substrate having a circular cell adhesion pattern array, cells aggregated As a result, a three-dimensional cardiomyocyte spheroid was formed.

(2) Cardiomyocyte spheroid formation of circular cell adhesion pattern and radial cell adhesion pattern

For cardiomyocyte spheroid formation, the cardiomyocytes of Preparation Example 3, i.e., commercially available cardiomyocytes (iCell Cardiomyocytes2 Kit) from Cellular Dynamics, were placed on a 96-well plate on each substrate prepared in (2) and (3) of Preparation Example 1. , 01434, cat# CMC-100-012-000.5) were thawed and cultured 1×10 ⁵ each was seeded.

As a result of observing the cells 24 hours after seeding, it was confirmed that cardiomyocytes were strongly adhered to both the circular cell adhesion pattern and the radial cell adhesion pattern substrate and formed a spheroid shape, and the myocardium adhered to each substrate It was confirmed that the cell spheroids were beating (Beating).

In addition, 4 days after seeding, the cardiomyocyte spheroids on the circular cell adhesion pattern and the radial cell adhesion pattern became more synchronized, and it was confirmed that the cardiomyocyte spheroids with a constant beating rate were maintained ( 6a and 6b).

<Experimental Example 1> Immunofluorescence staining for cardiomyocyte spheroids

In order to confirm the expression of cardiomyocyte markers on the cardiomyocyte spheroids according to the present invention, an immunofluorescence staining experiment was performed in the following manner, and the results are shown in FIGS. 6c and 6d . The cardiomyocyte marker cTnT antibody (invitrogen, Mouse host, cat # MA1-26935, 1:50) and the gap junction protein Connexin 43 (Cx43, also known as GJA1; Abcam, Rabbit host, cat#. ab217676, 1:50) ) When immunofluorescent staining is performed using an antibody, it can be confirmed whether the spheroid formed in (2) of Example 1 is a cardiomyocyte spheroid.

Cells 5 days after seeding on the substrate are washed 3 times with DPBS (Dulbecco's phosphate-buffered saline), 100 μL of 4% paraformaldehyde is added, and the cells are reacted at room temperature for 20 minutes to fix the cells. After washing the fixed cells twice with DPBS, add 20 μL of 1M Glycine and react at room temperature for 5 minutes to quench the remaining 4% paraformaldehyde. After washing once with DPBS, 200 μL of 0.5% Triton X-100 solution is added and reacted at room temperature for 5 minutes for permeabilization.

After washing the permeabilized cells 3 times with 0.1% Triton X-100 solution, dilute the primary antibody in 0.1% Triton X-100 solution containing 2% BSA (Bovine Serum Albumin) and put 100 μL of it. The reaction was carried out at 37°C for 1 hour. After the reaction, the cells are washed 3 times with 0.1% Triton X-100 to remove the remaining primary antibody. Secondary antibody (anti-Mouse Alexa 488 (cTnT): invitrogen, A-10680, 1:5,000; anti-Rabbit Alexa 594 (Connexin 43): invitrogen in 0.1% Triton X-100 solution with fresh 2% BSA added , A-11037, 1:500), add 100 μL, react at room temperature for 45 minutes, and wash 4 times with 0.1% Triton X-100 solution.

Thereafter, DAPI solution was added to each well to stain the nucleus, and then the degree of fluorescence expression was observed using a fluorescence microscope.

As a result of immunofluorescence staining, it was confirmed that the spheroids on the circular cell adhesion pattern and the radial cell adhesion pattern all expressed cTnT, and in particular, the expression of Connexin 43 increased in the spheroids on the radial cell adhesion pattern (Fig. 6c and 6d).

Through this, it can be seen that both the circular cell adhesion pattern and the radial cell adhesion pattern substrate can form cardiomyocyte spheroids, and in particular, it can be seen that the radial cell adhesion pattern substrate has excellent differentiation into cardiomyocytes. From the above results, it can be seen that the cardiomyocyte spheroids formed on the cell-non-adhesive PEG polymer substrate of the present invention can effectively control the aggregation of cardiomyocytes and can be effectively used for drug screening.

Claims (12)

An array for culturing cardiomyocytes spheroids, comprising a cell adhesion pattern spaced apart from a cell non-adhesive PEG polymer substrate at regular intervals, wherein the cell adhesion pattern comprises SiO2 particles coated with a cell-adhesive RGD peptide.
The array of claim 1, wherein the PEG polymer substrate is a PEG hydrogel.
The array according to claim 1, wherein the predetermined interval is 600 μm to 1,000 μm.
The array according to claim 1, wherein the individual cell adhesion pattern of the array is divided into a central portion and a peripheral portion, and the central portion has a higher adhesive force per unit area than the peripheral portion.
5. The method of claim 4, characterized in that the individual cell adhesion pattern is circular or radial. Array.
The array according to claim 4, wherein the adhesive force per unit area is controlled by a cell adhesion pattern density or a cell adhesion pattern material.
5. The array of claim 4, wherein the perimeter comprises one or more guide lines formed radially from the center.
The array according to claim 7, wherein the length of the guide line is 100 μm to 500 μm, the thickness of the guide line is 1 μm to 20 μm, and the number of guide lines is 20 to 100.
The array according to claim 1, characterized in that the average diameter of the cardiomyocyte spheroids is 100 μm to 500 μm.
delete (a) seeding cardiomyocytes on the array of claim 1; and
(b) culturing the cells to form cardiomyocyte spheroids;
A method for producing a cardiomyocyte spheroid comprising a.
The method according to claim 11, wherein the method is characterized in that the human induced pluripotent stem cell-derived cardiomyocytes are seeded at 2 to 1,000,000 cells/well.

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