KR20200115343A - Artificial tissue model comprising hydrogel structure with microchannel network - Google Patents

Abstract

The present invention relates to an artificial tissue model using a hydrogel structure having a microchannel network formed therein, and a disease model using the same, wherein a fluid can tubularly pass through the microchannel network. The artificial tissue model can three-dimensionally culture cells similarly to an in vivo environment. Also, when a component which induces a target disease tubularly passes through the hydrogel structure, the artificial tissue model can also be useful as an artificial disease model.

Description

Artificial tissue model including a hydrogel structure in which a microchannel network is formed {ARTIFICIAL TISSUE MODEL COMPRISING HYDROGEL STRUCTURE WITH MICROCHANNEL NETWORK}

The present invention relates to an artificial tissue model using a hydrogel structure in which a microchannel network through which fluid can flow through is formed.

Recently, in the biotechnology field, in the process of developing cosmetics or drugs, there is a growing movement to use an animal replacement test method instead of an experimental animal. As part of this movement, in Europe and Japan, toxicity tests related to skin such as cosmetics are standardized through cell tests, and in Korea, the Ministry of Food and Drug Safety and the National Institute of Toxicology are providing guidelines for alternative methods of animal testing.

An artificial tissue and an artificial organ model can be used as one of the animal replacement test methods. Artificial organ models have been developed extensively for disease modeling and drug screening, and have the potential to minimize or minimize the use of expensive laboratory animals. In particular, the artificial liver model is expected to have a variety of uses, such as drug screening, liver-related disease research such as non-alcoholic fatty liver disease (NAFLD), and clinical evaluation, as it is possible to model drug metabolism. Similar to models, an in vitro liver system that reproduces and predicts pathological events has not yet been developed. In order to develop an in vitro liver system, it is necessary to mimic the complex structure of the vascular network and reconstruct the lobe development from liver shoots, but this is difficult (S. Reardon, in Nat News, DOI: 10.1038/nature.2017.21818 , SPRINGER NATURE, 2017).

In this situation, the present inventors completed the present invention by devising a three-dimensional artificial tissue model that can be used for disease state modeling and drug screening by forming a three-dimensional microchannel network.

An object of the present invention is to provide an artificial tissue model using a hydrogel structure having a microchannel network and an artificial disease model using the artificial tissue model.

In order to achieve the above object, one aspect of the present invention

A hydrogel structure made of a biocompatible polymer and including a three-dimensional network formed by connecting a plurality of microchannels to each other; And

It provides an artificial tissue model including cells encapsulated in the hydrogel structure.

As used herein, the term "artificial tissue" refers to artificially manufactured tissue and organ analogs that perform functions similar to human tissues and organs. Artificial tissues include artificial joints, artificial bones, artificial skin, artificial tendons, artificial nerves, and artificial tumors, and artificial organs include artificial liver, artificial kidney, artificial heart, intestine, skin, and blood vessels. In the present invention, the term "artificial tissue" may be used as a term encompassing artificial tissues and artificial organs.

As a result of trying to develop a model capable of studying drug screening and disease pathogenesis, the present inventors have created an artificial tissue model capable of culturing encapsulated cells because a microchannel network similar to a functional vascular structure in a living body is formed inside. Devised.

Specifically, the artificial tissue model includes a hydrogel structure and a cell enclosed therein, and the cells include cells, organoids, and spheroids. The plurality of microchannels formed inside the hydrogel structure form a three-dimensional network, and these three-dimensional networks exist within 200 μm of each other in the three-dimensional (x-axis, y-axis, and z-axis) direction as each microchannel is connected to each other. Means to do. The distance takes into account the diffusion limit of blood vessels in a living body, and reflects the distance at which blood cells, nutrients, etc. can escape from the blood vessel and be transmitted to surrounding cells and tissues. Due to this limiting distance, the artificial tissue model of the present invention can culture cells, organoids, and/or spheroids contained within the hydrogel structure.

As used herein, the term "biocompatible polymer" refers to a polymer having tissue compatibility and anticoagulability that does not destroy tissue or coagulate blood in contact with living tissue or blood. .

According to one embodiment of the present invention, the biocompatible polymer is gelatin, alginate, polyethylene glycol (PEG), polydimethylsiloxane (PDMS), collagen, chitin, chitosan, keratin, cellulose, fibronectin, elastin, Fibrinogen, fibromodulin, laminin, tenacin, vitronectin, hyaluronic acid, heparin, collagen, chondroitin sulfate, pectin, dextran, silk protein, silk fibroin and their derivatives, agarose, polylac Polylactic acid (PLA), polyglycolic acid (PGA), a copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PCL), poly(poly(ethylene oxide) terephthalate- co-butylene terephthalate) (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester (poly(ortho ester, POE) and poly(propylene fumarate) diacrylate (PPF-DA), preferably alginate, gelatin, polydimethylsiloxane, and polyethylene glycol. I can.

In the present invention, the cells that can be enclosed in the hydrogel structure are neural stem cells (NSC), adult stem cells (ASC), embryonic stem cells (ESC), induced pluripotent stem cells. Cells (induced pluripotent stem cells, iPSCs), neurons, keratinocytes, tumor cells, fibroblasts, hepatocytes, hepatic astrocytes and endothelial cells can be selected from the group consisting of, and these cells vary depending on the artificial tissue to be manufactured. Can be used. The cells may be used alone or as a mixture of two or more types of cells, and may be enclosed in a spheroid form or an organoid, and sufficiently cultivated in a culture medium before encapsulation in a hydrogel structure or encapsulated, and then the medium is perfused through a microchannel. Can be cultured by

In the present invention, the cells encapsulated in the hydrogel structure may vary depending on the tissue model. For example, the artificial liver tissue model may include liver organoids, and the liver organoids may be derived from adult tissues or induced pluripotent stem cells. In addition, the artificial heart model may include cardiac organoids, and the cardiac organoids differentiate the induced pluripotent stem cells into cardiomyocytes and cardiac fibroblasts, respectively, and then convert them into microparticles that induce aggregation of cells. It can be produced by culturing together in a device such as a well. The artificial kidney model may include renal organoids and/or renal tubuloids (a type of organoid consisting of renal tubules), and renal organoids are cultured in three dimensions after stepwise differentiation of induced pluripotent stem cells. Can be produced by Renal tubuloids can be produced by differentiating adult stem cells obtained from kidney tissue or urine.

According to one embodiment of the present invention, the artificial tissue model may be an artificial liver tissue model, and the artificial liver tissue may include hepatocytes, hepatic stellate cells, and endothelial cells as cells. . For example, the artificial liver tissue contains hepatocytes, hepatic astrocytes, and endothelial cells in a ratio of 5 to 50:1 to 5:1 to 5, preferably 5 to 30:1 to 3:1 to 3 It may include a spheroid mixed in a ratio of, more preferably 5 to 15:1 to 2:1 to 2.

According to one embodiment of the present invention, the hydrogel structure may have an elastic modulus in a range similar to that of an in vivo tissue to be simulated. For example, the artificial liver model may have an elastic modulus in the range of 200 to 1000 Pa, and preferably may have an elastic modulus in the range of 300 to 800 Pa. The elastic modulus range is a range similar to that of the liver tissue in vivo.

In addition, the microchannel may have a diameter in the range of 5 to 50 µm, preferably 10 to 50 µm, more preferably 15 to 30 µm. In the present invention, the hydrogel structure may further include an inlet channel and an outlet channel (FIG. 1), and the inlet channel and the outlet channel may be connected to a microchannel to form a circulation system through which a fluid flows.

According to an embodiment of the present invention, the artificial tissue model may be manufactured by a method including the following steps.

(a) supplying a cell and hydrogel solution to be enclosed in a frame including a three-dimensional fiber bundle;

(b) crosslinking the hydrogel solution; And

(c) removing the three-dimensional fiber bundles from the crosslinked hydrogel.

In the present invention, the three-dimensional fiber bundle can be produced by spraying a stimulation-sensitive polymer solution with a rotary spraying device, and the diameter of the fiber can be adjusted by changing the rotational speed, and as a result, the diameter of the microchannel formed in the hydrogel And the density can be controlled.

The cells to be encapsulated in (a) may be suspended in a culture medium and supplied directly to a frame including a three-dimensional fiber bundle, or may be supplied in a form mixed with a hydrogel solution. In addition, it is possible to adjust the position of the 3D microchannel network in the hydrogel by adjusting the position of the 3D fiber bundle placed in the frame in step (a), and since the microchannels of the hydrogel are connected to each other in the 3D direction, the influent and It has the advantage of allowing closed fluid circulation without leaking between the effluents. In addition, the size and shape of the hydrogel structure can be variously modified by changing the size and shape of the frame and changing the amount of the hydrogel solution.

According to one embodiment of the present invention, the hydrogel crosslinking of step (b) may use both physical and chemical crosslinking methods, and for example, an enzymatic crosslinking agent such as transglutaminase may be used. Enzyme crosslinking agents are inexpensive and biocompatible, and are practical because cells and therapeutic agents can be easily loaded before gelation. In addition, the crosslinking time and degree of crosslinking can be controlled by adjusting the ratio of the enzyme to the hydrogel solution.

In the present invention, the step (c) may be performed by converting the three-dimensional fiber bundle into a sol, and specifically, may be performed by applying a stimulus corresponding to a stimulation-sensitive polymer. For example, a fiber made of a temperature-sensitive polymer exists in an aqueous solution at a temperature lower than LCST, and thus a fluid lower than LCST can be perfused through the hydrogel to remove it.

According to an embodiment of the present invention, the fiber made of poly(N-isopropylacrylamide)) (poly(N-isopropylacrylamide, PNIPAM; Mn=~40,000) exhibits LCST of 25 to 32° C. The PNIPAM fibers can be removed by perfusing a low fluid through the hydrogel.

The present inventors produced an artificial liver model by including a spheroid in which hepatocytes, hepatic astrocytes, and endothelial cells were mixed in the hydrogel structure, and as a result, it was found that the artificial liver model performs a function similar to that of a normal liver in vivo. . Therefore, it was confirmed that an artificial tissue model could be produced by including the cells of the tissue to be produced in the hydrogel structure.

Another aspect of the present invention comprises the steps of preparing an artificial tissue model including a target cell; And it provides a method of manufacturing an artificial disease model comprising the step of perfusing the component inducing the target disease to the artificial tissue model.

In the present invention, the disease is fatty liver, hepatitis, liver cirrhosis, heart failure, polycystic kidney disease, nephrotic syndrome, nephritis. , Acute renal failure, atopic dermatitis, inflammatory bowel disease, and atherosclerosis (arteriosclerosis).

As used herein, the term "target cell" refers to an artificial tissue model and a cell suitable for inducing a target disease, and one or a plurality of cells may be used in combination. For example, to create an artificial liver model and related disease models, hepatocytes, endothelial cells and hepatic stellate cells can be used as target cells, and to create an artificial skin model and related disease model, keratinocytes, melanocytes and fibroblasts Etc. can be used. In addition, tumor cells of various carcinomas can be used in the artificial tumor model, and these tumor cells are known in the art as well as cells isolated from tumor tissues of patients.

In the present invention, the component inducing the target disease refers to a substance that can cause a state similar to the target disease when processed in an artificial tissue model, and the type of the component inducing the target disease may vary depending on the target cell and the target disease. have.

According to an embodiment of the present invention, if the target cell is a hepatocyte and the target disease is fatty liver (salt), a fatty acid may be used as a component inducing the target disease. The fatty acid may be a saturated fatty acid, and may be selected from the group consisting of palmitic acid, stearic acid, myristic acid and lauric acid.

The present inventors confirmed that when palmitic acid or palmitic acid + TGF-β were treated together through medium perfusion in an artificial liver tissue model, a state similar to the initial inflammatory stage and the late fibrosis stage of steatohepatitis was induced, respectively, and the artificial liver tissue It was confirmed that the model can be used as an artificial steatohepatitis model.

Further, if the target cell is a kidney cell or a kidney organoid, and the target disease is acute kidney injury, cisplatin may be used as a component for inducing the target disease. If the target cell is an intestinal cell and the target disease is ulcerative colitis, dextran sodium sulfate (DSS) can be used. On the other hand, streptozotocin can induce a condition similar to diabetes when applied to pancreatic cells.

The artificial tissue model of the present invention uses a hydrogel structure in which a microchannel network through which fluid can permeate is formed, and cells can be cultured in three dimensions similar to the in vivo environment, and a target disease is induced in the hydrogel structure. Perfusion of a component can be usefully used as an artificial disease model.

1 schematically shows a manufacturing process of an artificial liver model according to an embodiment of the present invention.
FIG. 2 shows an artificial liver model including spheroid stem cells and having microchannel networks formed therein.
3 is a result of confirming whether PNIPAM fibers forming microchannels are removed in the artificial liver model according to an embodiment of the present invention.
4 is a result of confirming the cell viability after culturing hepatocytes in a two-dimensional plane in a 24-well plate.
5 is a result of confirming the size of spheroids after culturing spheroid stem cells in the artificial liver model of the present invention.
6 is a result of confirming the diameter of the microchannel by perfusing a red fluorine sphere through the liver chip of the present invention.
8 is a result of measuring the velocity of particles passing through the microchannel in the artificial liver model according to an embodiment of the present invention.
In FIG. 9, A is a result of confirming the cell viability after culturing spheroid or non-spheroidal stem cells under different conditions, and B is a spheroid and cell nucleus in an artificial liver model in which spheroid stem cells are cultured under medium perfusion conditions. And the result of staining the microchannels.
Figure 10 is a result of confirming the expression levels of cytochrome P450 4α10 (CYP4α10) and HNF4A (hepatocyte nuclear factor 4 alpha) genes after culturing spheroid or non-peroidal stem cells under different conditions (A), and CYP3A4 activity. This is the measurement result.
11 is a result of confirming the level of secretion of albumin according to the presence or absence of spheroidity (A) and the presence or absence of perfusion (B) after culturing spheroidal or non-peroidal stem cells under different conditions.
12 is a result of confirming the glucose consumption level (A) and the ratio of lactate to glucose level (B) according to the presence or absence of perfusion after culturing spheroid stem cells.
13 is a result of confirming the levels of albumin and CYP450 by immunofluorescence staining according to the presence or absence of perfusion after culturing spheroid stem cells.
In FIG. 14, A schematically shows a two-hit theory for a mechanism of progression of non-alcoholic steatohepatitis, and B schematically shows a process for inducing an initial stage of liver inflammation in the artificial liver model of the present invention.
15 is a result of confirming the cell survival rate of spheroid hepatocytes according to the presence or absence of perfusion after inducing an initial liver inflammatory stage in the artificial liver model of the present invention including spheroid stem cells.
16 is a result of confirming the degree of death of spheroid hepatocytes by immunofluorescence staining according to the presence or absence of perfusion after inducing an initial liver inflammation stage in the artificial liver model of the present invention including spheroid hepatocytes.
17 is a result of confirming the degree of lipid accumulation according to the presence or absence of perfusion and the duration of palmitic acid (PA) treatment after inducing an initial liver inflammatory stage in the artificial liver model of the present invention including spheroid hepatocytes.
In FIG. 18, A is a result of confirming the level of reactive oxygen species (ROS) after treatment of palmitic acid without perfusion in the artificial liver model of the present invention including spheroid stem cells, and B is a result of confirming the reactive oxygen species level (ROS) according to the presence or absence of perfusion. This is the result of checking the percentage of cells.
FIG. 19 is a diagram of inflammatory markers (A), ER stress markers (B), and antioxidant stress markers (C) genes depending on the presence or absence of perfusion after inducing an initial liver inflammatory stage in the artificial liver model of the present invention including spheroid hepatocytes. This is the result of checking the expression level.
Figure 20 shows intracellular lipid accumulation (A) and intracellular reactive oxygen species (B) after treatment with palmitic acid (PA) and ezetimibe (Eze) in the artificial liver model of the present invention including spheroid stem cells. ) This is the result of checking the level.
FIG. 21 is a NAFLD treated with palmitic acid (PA) and ezetimibe (Eze) together (in vitro) or methionine-choline deficiency (MCD) diet to the artificial liver model of the present invention including spheroid hepatocytes. Expression levels of triglyceride synthesis genes (A), inflammatory markers (B) and antioxidant stress markers (C) genes after treatment with ezetimibe (Eze) in a mouse model (in vivo) and protein levels of inflammatory marker genes (D) This is the result of checking.
22 is a result of confirming the intracellular lipid accumulation level after treatment with palmitic acid (PA) and ezetimibe (Eze) together without perfusion in the artificial liver model of the present invention including spheroid stem cells.
Fig. 23 is a NAFLD that is treated with palmitic acid (PA) and ezetimibe (Eze) together (in vitro) or methionine-choline deficiency (MCD) diet to the artificial liver model of the present invention including spheroid hepatocytes. This is the result of confirming the expression level of a gene involved in β-oxidation of fatty acids after treatment with ezetimibe in a mouse model (in vivo).
24 is a result of confirming the activation level of mitochondria after treatment with palmitic acid (PA) and ezetimibe (Eze) in the artificial liver model of the present invention including spheroid stem cells.
FIG. 25 is a result of confirming the level of mitochondrial activation after treatment with palmitic acid (PA) and ezetimibe (Eze) together without perfusion in the artificial liver model of the present invention including spheroid stem cells.
In Figure 26, A schematically shows the mechanism of liver cirrhosis through non-alcoholic steatohepatitis (NASH) in fatty liver, and B is a late fibrosis step by treating palmitic acid and TGF-β in the artificial liver model of the present invention. It represents the process of modeling.
FIG. 27 shows a process of culturing spheroids of hepatocytes, endothelial cells (EC), and hepatic stellate cells (HSCs) in the artificial liver model of the present invention.
28 is a result of confirming the expression levels of CYP4α10 and HNF4A genes after culturing hepatocytes alone spheroid (Monoculture) or hepatocytes/endothelial cells/astrocytic spheroids (Co-culture) in the artificial liver model of the present invention.
29 is a hepatocyte-only spheroid (Monoculture) and a hepatocyte/endothelial cell/astrocytic spheroid (Co-culture) (A), an endothelial cell alone (EC), and astrocyte alone (HSC) in the artificial liver model of the present invention. This is the result of confirming the expression level of the fibrosis marker gene according to the treatment with palmitic acid and TGF-β after culturing hepatocyte/endothelial/astrocytic spheroid (B).
30 is a result of confirming the protein expression level of αSMA after culturing hepatocytes/endothelial cells/astrocytic spheroids in the artificial liver model of the present invention.
In FIG. 31, A shows the artificial liver model of the present invention including pulverized liver tissue, and B and C are the results of confirming tissue regeneration after transplanting the artificial liver model of the present invention to a liver defect animal model.
FIG. 32 shows the concentration of alanine transaminase (ALT) after transplanting the artificial liver model of the present invention into a liver defect animal model (A), tissue staining results at the transplant site (B), tissue fibrosis levels (C and D), This is the result of confirming the tissue recovery level (E) and the hepatocyte death rate (F) at the defect site.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Experimental method

1. Hepatocyte spheroid culture

AML12, a normal mouse hepatocyte, was purchased from ATCC, and mouse endothelial cells (EC) were obtained from the technology transfer department of Emory University (Atlanta, USA). Mouse hepatic stellate cells (HSC) were purchased from Applied Biological Materials Inc. (Canada). All cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum (FBS) and 10% penicillin in 5% CO ₂ , 37° C. incubator. In the hepatocyte culture, 1% insulin transferrin selenium was additionally added.

Hepatocyte culture was performed with hepatocytes alone or two types of hepatocytes/endothelial cells/hepatic astrocytes to model early inflammation and late fibrosis stages in the process of non-alcoholic fatty liver disease (NAFLD), respectively.

Cell spheroids were prepared according to the manufacturer's protocol using non-adherent microwells of a 24-well plate (AggreWell ^TM 400 plate, STEMCELL Technologies). Briefly, microwells were pre-coated with an anti-adhesive wash solution (STEMCELL Technologies) for 30 minutes and washed with PBS. Thereafter, AML12 cells alone or AML12/EC/HSC (8:1:1 ratio based on the number of cells) were suspended in a culture medium at a concentration of 8×10 ⁵ cells/ml, and added to each well. After centrifugation at 1000 rpm for 5 minutes, the cells were incubated overnight, and confirmed with an optical microscope (EVOSTM XL Core, Invitrogen, USA) to recover cell spheroids. The diameter of the cell spheroid was confirmed with ImageJ/Fiji software.

2. Artificial liver model production

The three-dimensional microchannel network of the artificial liver model was constructed with enzyme-crosslinkable gelatin hydrogel. Non-toxic, sacrificial fibers of heat-sensitive poly(N-isopropyl acrylamide), PNIPAM; Mn=~40,000, Sigma-Aldrich, USA) are manufactured by solution-spinning technology. I did. Briefly, PNIPAM was dissolved in methanol at a concentration of 45 w/v%, and the solution was sprayed at different rpm with a custom-made circular rotating device to prepare a PNIPAM fiber bundle.

Thereafter, the PNIPAM fiber bundle was put into a polydimethylsiloxane (PDMS) frame, and a gelatin/mTG (microbial transglutaminase) solution (9:1 ratio, final concentration=5 w) containing cell spheroids (3x10 ⁶ cells/ml) /v%) was poured and the gelatin was crosslinked at 37°C for 30 minutes. Thereafter, silicone tubes were connected to both ends of the gelatin hydrogel, and PBS at room temperature was perfused to remove PNIPAM fibers through a sol-gel phase transition.

A schematic process of manufacturing an artificial liver model is illustrated in FIG. 1.

Whether the PNIPAM fibers were removed from the gelatin hydrogel was determined by high performance liquid chromatography (HPLC, Agilent Technologies Inc., USA) with water (1 ml/min) and a C18 column (120 EL-C18 InfinityLab Poroshell, Agilent Technologies Inc.). Performed and confirmed.

The channel structure was observed with a confocal microscope LSM 780 (ZEISS, Germany) after perfusion of red FluoSpheres (FluoSpheres, 20 nm; Invitrogen), and the channel size distribution was confirmed with Image J/Sebum software.

The diffusibility and perfusibility of the channel network in the gelatin hydrogel were determined by FITC-dextran (molecular weight=40,000, Sigma-Aldrich) and micro-fluorescence while perfusing the culture medium to the gel at a rate of 20 μl/min. Beads (2 μm in diameter for perfusion tracking and 20 nm in diameter for channel staining) were each perfused to confirm.

The spatial velocity of each microbead in the microchannel network in the gelatin hydrogel was analyzed with the particle tracker plug-in (MOSAIC group) of ImageJ/Sebum software, and visualized with a column map of MATLAB software (Mathworks, USA).

Cell viability was checked with LSM 780 using a confocal microscope using a Live/Dead assay kit (Life Technologies) according to the manufacturer's protocol, and images of live cells (green) and dead cells (red) were quantified with Image J/sebum software. Analyzed by.

3. In vitro non-alcoholic fatty liver model

Palmitic acid in the form of saturated fatty acids in DMEM medium containing 1% bovine serum albumin (BSA), 5% FBS and 1% penicillin to adjust the physiological ratio of bound and non-binding free fatty acids in the medium. PA) was added at a concentration of 500 μM. Subsequently, in the perfusion model, cells were cultured for 6 to 10 days while perfusing the medium, and in the non-perfusion model, cells were statically cultured with the medium.

To confirm the therapeutic effect of Ezetimibe, a non-alcoholic fatty liver target drug, ezetimibe (50 μM; Cayman chemical, USA) and palmitic acid (500 μM) were diluted in culture medium and then 20 μl/ The cells were cultured for 6 to 10 days while perfusing the hydrogel at a rate of minutes.

The late fibrosis stage of non-alcoholic fatty liver was performed by perfusion of DMEM medium containing 1% BSA, 5% FBS, 1% penicillin, palmitic acid (500 μM), and TGF-β (1.25 unit/ml) through microchannels. Culture alone or co-culture with hepatocytes/EC/HSC was performed for 6 to 10 days to induce.

4. Quantitative RT-PCR

Total RNA was isolated with TRIzol® reagent and treated with DNA degrading enzyme (DNase), and cDNA was synthesized using 1 μg of RNA, random-hexamer primer and kit (TAKARA Bio Inc., Japan). Quantitative PCR was performed with a StepOne ^™ Real-Time PCR system (Applied Biosystems, USA) using cDNA, SYBR® Green and primers (forward and reverse). The relative gene expression level was calculated by calculating a corresponding comparative Ct value based on the Ct (threshod cycle) value of GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

5. Fatty acid staining of liver

The sample was washed with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then permeated with 0.1% Triton-X solution for 30 minutes. Blocking the sample with 3% BSA, BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene; Molecular Probes, OG) and Hoechst 33342 (Life Technologies) were treated for 30 minutes to label triglycerides and nuclei, respectively. The labeled sample was confirmed with a confocal microscope LSM 780.

6. Intracellular reactive oxygen species staining

The degree of accumulation of reactive oxygen species (ROS) in the cells was confirmed with OxiSelectTM intracellular ROS assay kit (Cell biolabs, INC., USA). After washing the sample with PBS, it was reacted with DCFH-DA (2',7'-Dichlorodihydrofluorescin diacetate; green) and DAPI (4',6-Diamidino-2-phenylindole dihydrochloride; blue) for 30 minutes. Thereafter, the sample was washed with PBS, and the fluorescence image was confirmed with a confocal microscope LSM 780. Quantitative analysis was performed using sebum software to measure the ROS-positive green area and then dividing this area by the corresponding blue area (nuclei).

7. Mitochondrial transmembrane potential measurement

Mitochondrial transmembrane potential was measured with the TMRE kit. A culture medium containing TMRE (tetramethylrhodamine ethyl ester; 250 nM) was treated to the sample at 37° C. for 1 hour, and washed 3 times with PBS. Thereafter, the fluorescence image was confirmed with a confocal microscope LSM 780.

Mitochondrial activity was confirmed with cell counting kit-8 (CCK-8, Sigma-Aldrich, MO) according to the manufacturer's protocol. Briefly, the sample was washed with PBS and then treated with a serum-free medium containing 10 v/v% CCK-8 and reacted for 4 hours. Then, the absorbance was measured at 450 nm with a microplate reader (VersaMax microplate reader, Molecular Device), and the absorbance level was normalized to the total DNA concentration measured with Quant-iT ^™ PicoGreen® dsDNA kit (Invitrogen). Specifically, each sample was washed with PBS and 1 ㎖ of 1 Х RIPA buffer was treated at 4° C. overnight to lyse the cells. 10 μl of the dissolved sample solution was reacted with 190 μl of Quant-iT ^™ PicoGreen® dsDNA reagent (final concentration: 1Х), and then diluted with TE assay buffer (10 mM Tris-HCL, 1 mM EDTA, pH 7.5). Subsequently, fluorescence absorbance was measured at 480/520 nm wavelength with a fluorescent microplate reader (Varioskan ^TM LUX Multimode Microplate Reader, Thermo Fisher Scientific Inc.).

8. Biochemical experiment

The culture medium was recovered at a certain time point, and the released albumin concentration was measured at 450 nm with a microplate reader (Perkin Elmer VictorX4, Waltham, MA) using an enzyme-linked immunosorbent test (ELISA) kit (Bethyl Laboratories, USA). I did.

CYP450 activity was confirmed with a luminogenic P450-GloTM CYP350 assay kit according to the manufacturer's protocol, and fluorescence intensity was measured with a microplate Luminometer (Centro LB960 microplate Luminometer, Berthold Technologies). Fluorescence intensity was normalized to the luciferin standard.

9. Quantitative analysis of glucose consumption and lactic acid production

Glucose and lactate levels were measured by glucose-Glo (Promega) and lactate assay-Glo (Promega), respectively, and fluorescence intensity was measured by a microplate Luminometer (Centro LB960 microplate Luminometer). Fluorescence intensity was normalized to the luciferin standard.

10. Animal testing

10-week-old male C57BL/6J mice were purchased from Orient Bio (Sungnam, Korea) and randomly divided into the following 3 groups (7 to 10 mice in each group): i) Vehicle + normal diet group, ii) Vehicle +MCD (methionine-choline deficient) diet group, and iii) ezetimibe + MCD diet group. Mice were reared free-feeding in a 12-hour light/dark cycle, 60%±10% humidity, and 23±2℃ temperature conditions. In the case of the ezetimibe + MCD diet group, ezetimibe (10 mg/kg) was administered once a day for 4 weeks by oral gavage, and the vehicle + normal diet group and the vehicle + MCD diet group were the same. Volumes of PBS were administered orally for 4 weeks. During the experiment, the body weight was measured once a week, and after 4 weeks, the mice were anesthetized and sacrificed to collect blood.

The liver defect model for transplantation of the artificial liver model was a healthy male New Zealand white rabbit weighing 3.2 to 3.5 kg. The rabbit was injected intramuscularly with 5 mg/kg of xylazine and 15 mg/kg of zoletil every 15 minutes as a preliminary medicine, and after intubation into a 3.0 or 3.5 mm endotracheal tube, 2.0% isophageal to maintain inhalation anesthesia Fluran was used. All experimental animals were administered a crystalloid solution (10 ml/mg/kg/h) throughout the surgical procedure.

After anesthesia, an abdominal midline incision was performed, and the liver was removed with an 8 mm biopsy punch (Kai medical, Japan). The removed liver tissue was chopped and embedded in a channel hydrogel, and the channel hydrogel was immediately transplanted into the liver defect.

The level of alanine aminotransferase (ALT) in serum was measured with an ALT activity assay kit (FUJIFILM, Japan, 3250) according to the manufacturer's instructions. The level of ALT in the serum increases with hepatocyte damage, thus effectively indicating the degree of liver recovery from damage.

11. Organizational Analysis

After the end of the experiment, the mice were sacrificed to separate the tissues, fixed with formalin for 1 day, and embedded in paraffin. Thereafter, a tissue section was made, hematocillin & eosin staining, and Mason's trichrome staining were performed, and observed with an optical microscope. The tissue DNA fragmentation was confirmed by the TUNEL assay (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol. The fixed liver tissue was stored at 4°C using PBS containing a 0.1% polyvinyl-pyrrolidone solution. Fluorescence images were checked with an LSM 780 confocal microscope (Zeiss), and then the number of apoptotic cells was quantitatively analyzed with Image J/Sebum software.

12. Statistical Analysis

All data were analyzed by two-sided Student's t-test for comparison between the two experimental groups, or by one-way analysis of variance (ANOVA) with Turkey's honestly significant difference post-test for multiple comparisons (SPSS 21.0K for Windows, SPSS, Chicago , IL, USA). Values of *p<0.05 and ***p<0.005 were considered statistically significant.

Experiment result

1. Fabrication of liver chips and confirmation of characteristics

The present inventors include a hepatocyte spheroid that mimics the development process of a liver bud, and a hydrogel form in which microchannels having a physiologically appropriate diameter, density and complexity are formed by mimicking the complex three-dimensional capillary structure of the liver. An artificial liver model of was prepared (Fig. 2). As a result of confirming whether the PNIPAM fiber was completely removed from the prepared artificial liver model, it was found that the PNIPAM fiber was completely removed by PBS perfusion (FIG. 3). Hereinafter, the artificial liver model is referred to as “liver chip”.

Hepatocytes generally die within a few days in a two-dimensional monolayer culture (Fig. 4). Since hepatocytes are generated from the endoderm and then during development, they form hepatic buds to create a condensed tissue mass in the form of a mesenchyme. In order to mimic this process, hepatocyte spheroids were generated and cultured in liver chips while perfusion of the medium. Hepatocyte spheroids were produced with an average size of 173.79±32.09 μm using microwells with a repellent surface coating (FIG. 5).

The average G'of the gelatin hydrogel in the prepared liver chips was 652.05±58.52 Pa, which belongs to the modulus range of normal liver tissue. In addition, the interconnectivity of the microchannels in the liver chip was confirmed by perfusion of red fluorine spheres (diameter 0.2 μm), and it was confirmed that the diameter of the microchannels ranged from 5 to 36 μm, and was an average of 17.78±8.37 μm (FIG. 6). ).

In order to characterize the diffusivity and permeability of the microchannel network in the liver chip, 40 kDa FITC-dextran and fluorescent microbeads were perfused into the channel at a flow rate of 20 μl/min, resulting in dextran (green) 30 min after perfusion. It was confirmed that the microbead (red) was easily diffused from the channel to the hydrogel within the channel structure (Fig. 7). These results indicate that the stiffness and channel interconnection of the hydrogel are well controlled for perfusion of the culture medium so that nutrients and oxygen can be transported to the hydrogel body beyond the diffusion limit of 200 μm.

The flow rate of the microbeads ranged from 1 to 12 μm/sec (average 3.88 μm/sec) under the initial pressure driven flow (20 μm/min) (FIG. 8).

The diameter of the microchannel and the flow rate range of the microbead were consistent with the in vivo range.

2. Verification of normal liver function

The survival rate of hepatocyte spheroids was confirmed by culturing for 10 days under the following three conditions: i) hepatocyte spheroid + medium perfusion, ii) hepatocyte spheroid + medium nonperfusion, and iii) bispheroid hepatocyte + channelless hydrogel + Medium nonperfusion. As a result of culture, compared to the other experimental groups, it was possible to confirm that there were more live hepatocytes in the i) experimental group, which means that the microchannel network and medium perfusion can increase the cell viability (FIG. 9A). In addition, since the hepatocyte spheroid is surrounded by a channel network within 200 μm, it is expected that the cell viability increased by overcoming the diffusion limit of oxygen, nutrients and waste products (FIG. 9B).

Cytochrome P450 (CYP) expression and albumin secretion are functional indicators of lipid metabolism and hepatic homeostasis, respectively. CYP is mainly expressed in the liver and is involved in the biosynthesis and degradation of biological substances such as steroids and cholesterol. The CYP family is known to oxidize and remove small foreign organic molecules, including toxins and drugs, from the body. The experimental groups were divided as follows, and the expression level of the gene was confirmed after proceeding as a preliminary experiment for the first 6 days: i) non-ferroidal hepatocytes + channelless hydrogel + non-perfusion, ii) nonspheroidal hepatocytes + channel Hydrogel + nonperfusion, iii) hepatocyte spheroid + channelless hydrogel + nonperfusion, iv) hepatocyte spheroid + channel hydrogel + nonperfusion and ㅊ hepatocyte spheroid + channel hydrogel + perfusion

Subsequently, as a result of checking the expression levels of cytochrome P450 4α10 (CYP4α10) and HNF4A genes, it was found that the expression level of CYP4α10 was significantly higher in the medium perfusion experimental group (v) compared to the medium non-perfusion experimental group (i-iv), and the experimental group Compared with iii) or iv), it was confirmed that the expression level of the HNF4A gene was significantly higher in the experimental group v) (Fig. 10A). These results suggest that culture of hepatocyte spheroids in a liver chip perfused with medium remarkably promotes lipid metabolism activity of hepatocytes.

In addition, as a result of measuring CYP3A4 activity after culturing the cells for a long time, it was confirmed that the CYP3A4 activity significantly increased on the 10th day of culture compared to the second day of culture in the medium perfusion experimental group (+Per). On the other hand, in the medium non-perfusion experimental group (-Per), there was almost no change in CYP3A4 activity during the culture period, and it was found that the medium-perfusion experimental group showed a lower level of metabolic activity (FIG. 10B).

In addition, the level of albumin secretion was confirmed according to the presence or absence of spheroidity of hepatocytes and the presence or absence of medium perfusion. As a result, compared to the experimental group ii), albumin secretion was significantly increased in the experimental groups vi) and v), and in the medium perfusion experimental group (+Per), the albumin secretion level was maintained higher than on the second day of culture for 10 days of culture. However, in the non-perfused group, albumin secretion continued to decrease during 10 days of culture (FIG. 11). These results imply that medium perfusion of the liver chip is required to maintain normal liver function.

In addition, it was confirmed whether or not metabolic regulation, which is a major function of the liver. As a result of checking on the 6th day of culture, the level of glucose consumption was significantly higher in the medium perfusion group (+Per) compared to the medium non-perfusion group (-Per) and the level of lactic acid production was significantly lower. In addition, the ratio of lactate to glucose level was significantly lower in the medium perfusion experimental group (+Per) compared to the medium non-perfusion experimental group (-Per), suggesting the importance of perfusion to maintain metabolic regulatory activity (Fig. 12). ).

Also through immunofluorescence staining of albumin and CYP450, it was confirmed that normal liver function was maintained more in the medium perfused group (+Per) compared to the medium non-perfused experimental group (-Per) (FIG. 13 ).

3. Modeling the early stages of liver inflammation

In general, "two hit theory" is accepted for the mechanism of progression of NAFLD. It is a theory that hepatic steatosis occurs due to premature inflammation and accumulation of fatty acids in the liver (the first hit), and then severe inflammatory damage occurs due to an increase in oxidative stress and endoplasmic reticulum stress, leading to nonalcoholic steatohepatitis ( Second hit) (Fig. 14A).

Palmitic acid is known to be the most abundant fatty acid that can induce ROS-mediated cell death (lipotoxicity) in human plasma. Therefore, after treating the liver chips with palmitic acid (PA) three times for 10 days (FIG. 14B), the cell viability was confirmed by live/dead staining.

As a result, in the medium perfusion group (+Per), the initial apoptotic cell rate, 4%, was maintained for 72 hours, whereas in the medium non-perfusion group (-Per), the apoptotic cell rate was remarkably 16% within 24 hours of palmitic acid treatment. It was found that the ratio of dead cells increased and continued to be maintained until 72 hours after palmitic acid treatment (FIG. 15). As a result of additional confirmation by staining cell spheroids, if palmitic acid was not treated, dead cells stained with EthD-1 (ethidium homodimer-1) in the medium perfusion group (+Per) and non-perfusion group (-Per) (red) However, when palmitic acid was treated, it was found that the number of dead cells significantly increased in the medium non-perfused group (-Per) compared to the medium perfused group (+Per) (FIG. 16; Representing living cells). Through the above results, it was confirmed that the medium perfusion group (+Per) can effectively protect hepatocytes from fat toxicity caused by palmitic acid treatment compared to the medium non-perfusion group (-Per).

The normal liver maintains plasma glucose levels through metabolic functions, and if the liver does not perform normal metabolism, glucose is converted into fatty acids along with inflammation, which can lead to NAFLD. In this process, citrate is converted to palmitic acid, and then to oleic acid, a major fatty acid that can be accumulated in hepatocytes.

As a result of confirming intracellular lipid accumulation after treatment with palmitic acid for 10 days by medium perfusion, the intracellular lipid level of the 10-day palmitic acid treatment group was significantly higher than that of the palmitic acid 5-day treatment group and the palmitic acid non-treatment group. It was found that it was high (Fig. 17).

In addition, palmitic acid was treated for a short time (72 hours) and the residual reactive oxygen species (ROS) level was checked. As a result, it was found that the proportion of ROS-positive cells in the hepatocyte spheroid increased in the medium non-perfusion group (-Per), but the proportion of ROS-positive cells in the medium perfusion group (+Per) was remarkably low (Fig. 18). This result did not support the two-hit theory. However, when palmitic acid was treated for a long time (10 days), intracellular ROS accumulation increased even in the medium perfusion group (+Per) (FIG. 17 ), and these results were inflammatory markers (IL-6 and TNFα), ER stress markers ( EDEM and ATF4) and antioxidant stress markers (GSTA-1 and HO-1) were further confirmed by a marked increase in expression (Fig. 19).

The above results suggest that treatment with palmitic acid for 10 days by perfusion can reliably model the inflammatory (early) stage of NAFLD, a chronic liver disease.

4. Checking the effectiveness of treatment at the early stage of inflammation

Ezetimibe used in the treatment of hyperlipidemia is known to reduce hepatic steatosis, and the present inventors have confirmed in previous studies that ezetimibe reduces fat toxicity through Nrf2 activation in vivo and in vitro ( Free Radic Biol. Med 2016, 99, 520). Therefore, ezetimibe was treated with palmitic acid on liver chips, and the results (in vitro model, in vitro) of the NAFLD mouse model subjected to a methionine-choline deficiency (MCD) diet were administered as a result of ezetimibe administration (in vivo Model, in vivo).

As a result, it was confirmed that intracellular accumulation of lipids and ROS decreased in the medium perfusion group of liver chips by ezetimibe treatment (FIG. 20).

Palmitic acid is unsaturated by stearoyl-CoA desaturase-1 (SCD-1) and long-chain fatty acid elongase (LCE) to produce oleic acid used in the synthesis of triglycerides. To form. As a result of checking the expression levels of the genes, it was confirmed that the gene expression of SCD-1 and LCE was increased in the palmitic acid-only treatment group (PA), which was consistent with the above fact, and the increased gene expression was Ezetimie. It was found that it decreased in the B treatment group (PA+Eze) (Fig. 21A).

In addition, in the ezetimibe-treated group (PA+Eze and MCD+Eze), the expression level of the inflammatory marker genes (IL-6 and TNFα) increased by palmitic acid alone treatment decreased significantly in both in vitro and in vivo models. (FIG. 21B and D). However, the expression of the antioxidant target genes GSTA-1 and HO-1 was significantly increased in the ezetimibe-treated group (PA+Eze and MCD+Eze) compared to other experimental groups (FIG. 21C).

In contrast to the above results, in the medium non-perfused group, lipid accumulation increased even in the ezetimibe-treated group (PA+Eze), and the effect of ezetimibe could not be confirmed (FIG. 22). These results indicate that ezetimibe treatment and perfusion are essential to restore normal liver function similar to that in vivo.

Mitochondrial dysfunction can cause NAFLD because mitochondria regulate β-oxidation and ROS production of free fatty acids. Since mitochondrial dysfunction is caused by palmitic acid treatment, the effect of ezetimibe treatment on this was investigated.

Target genes for β-oxidation of mitochondrial free fatty acids are aconitase-2 (Aco) and peroxisome proliferator-activated receptor gamma coactivator 1-α (peroxisome proliferator-activated receptor gamma coactivator 1- α, PGC1-α), and these markers indicate mitochondrial function for NAFLD injury.

As a result of the confirmation, it was found that the expression of marker genes was increased in the palmitic acid + ezetimibe treatment group (PA + Eze and MCD + Eze) compared with palmitic acid alone treatment (PA) in both in vitro and in vivo experiments. There was (Fig. 23). In addition, palmitic acid alone treatment (PA) increases mitochondrial transmembrane potential to induce palmitic acid-induced mitochondrial dysfunction, but it was found that such dysfunction was reversed by ezetimibe treatment (Fig. 24). ). The effect of ezetimibe treatment was also confirmed in the non-perfusion group (FIG. 24B and FIG. 25).

From the above results, it can be seen that the liver chip of the present invention can be used to study the pathogenicity and treatment mechanisms related to NAFLD in accordance with the animal model.

5. Late fibrotic stage modeling

Non-alcoholic steatohepatitis (NASH) progresses from early inflammation to end-stage fibrosis (FIG. 26), and endothelial cells (EC) regulate this process, hepatic stellate cells (HSC) and It affects the function of hepatocytes. Therefore, by mimicking this aspect, spheroids of hepatocytes, endothelial cells, and hepatic astrocytes were co-cultured with palmitic acid and TGF-β treatment in a gel-channel perfusion system (FIGS. 26 and 27).

First, as a result of culturing without treatment with palmitic acid and TGF-β, the expression of CYP4α10 and HNF4A genes was doubled in the spheroid co-culture group, respectively, compared with the hepatocyte spheroid monoculture group (monoculture). It was confirmed that the 8-fold increase, and albumin secretion and CYP3A4 activity increased by 40% and 60%, respectively (FIG. 28). This result means that the spheroid co-culture group can further improve normal liver function compared to the hepatocyte spheroid single culture group.

Thereafter, spheroids were cultured by treatment with palmitic acid and TGF-β. Since TGF-β induces inflammation and fibrosis in hepatic stellate cells, it was treated to model the late fibrosis stage of NAFLD.

As a result of confirmation, when TGF-β and palmitic acid were treated together in the perfusion group, fibrosis markers in co-culture spheroids compared to the hepatocyte spheroid single culture group, the corresponding non-perfusion group, and the palmitic acid & TGF-β non-treated group. It was found that the expression of the genes αSMA, TGF-β and Col1A1 was significantly increased (FIG. 29). This result could also be confirmed through the protein expression level of αSMA (FIG. 30).

The above results indicate that co-culture spheroids in the perfusion group are a reliable model that mimics the fibrosis stage of the non-alcoholic steatohepatitis (NASH) model.

6. In vivo transplantation

The implantability and reproducibility of perfused liver chips were tested in a liver defective rabbit model. Using a rabbit model has suggested a rabbit model as a reliable in vivo platform for studying liver regeneration after hepatectomy in a recent study (M. Liao, T. Zhang, H. Wang, Y. Liu, M. Lu) , J. Huang, Y. Zeng, J Surg Res 2017, 209, 242.), because that approach is most relevant to our model.

Rabbit liver was subjected to liver resection within 30%, and the resected liver tissue was crushed and embedded in the liver chip of the present invention. The liver chip was transplanted to the liver defect site and confirmed 14 days later, compared to the liver chip non-transplant group (w/o gel), the liver chip transplant group ((w/gel) showed that clear tissue regeneration occurred at the damaged site. It could be confirmed (Fig. 31).

The above results show a rapid decrease in alanine transaminase (ALT) concentration in the liver chip transplant group (w/ hydrogel) compared to the liver chip non-transplant group (w/o hydrogel) (Fig. 32A), tissue staining at the transplant site. Results (FIG. 32B), significantly lower degree of tissue fibrosis (FIG. 32C and D), tissue recovery at a level similar to that of normal liver (FIG. 32E), and hepatocyte death rate (FIG. 32F). Could

Claims (15)

Made of biocompatible polymers,
A hydrogel structure including a three-dimensional network formed by connecting a plurality of microchannels to each other; And
Artificial tissue model including cells encapsulated in the hydrogel structure. The method of claim 1, wherein the biocompatible polymer is gelatin, alginate, polyethylene glycol, polydimethylsiloxane, collagen, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin. , Vitronectin, hyaluronic acid, silk protein, silk fibroin and derivatives thereof, agarose, polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid copolymer, polycaprolactone, poly(poly(ethylene oxide) terephthalate) -Co-butylene terephthalate, polyphosphoester, polyphosphazene, polyanhydride, polyorthoester and poly(propylene fumarate) diacrylate) that is selected from the group consisting of, artificial tissue model. The artificial tissue model of claim 1, wherein the tissue is liver, kidney, heart, intestine, skin, blood vessel or nerve. The method of claim 1, wherein the cells are selected from the group consisting of neural stem cells, adult stem cells, embryonic stem cells, induced pluripotent stem cells, nerve cells, tumor cells, keratinocytes, fibroblasts, hepatocytes, hepatic astrocytes and endothelial cells. The artificial tissue model. The artificial tissue model according to claim 3, wherein the liver tissue comprises hepatocytes, hepatic astrocytes and endothelial cells. The artificial tissue model according to claim 5, wherein the liver tissue is a mixture of hepatocytes, hepatic astrocytes and endothelial cells in a ratio of 5 to 50: 1 to 5: 1 to 5 based on the number of cells. The artificial tissue model of claim 1, wherein the hydrogel structure has an elastic modulus of 200 to 1000 Pa. The artificial tissue model of claim 1, wherein the microchannels have a diameter of 5 to 50 μm. The artificial tissue model of claim 1, wherein the plurality of microchannels exist within 200 μm of each other in a three-dimensional direction. (a) producing the artificial tissue model of claim 1; And
(b) a method for producing an artificial disease model comprising the step of perfusing a component that induces a target disease to the artificial tissue model. The method of claim 10, wherein the disease is fatty liver, hepatitis, liver cirrhosis, heart failure, polycystic kidney disease, nephrotic syndrome, nephritis. nephritis), acute renal failure, atopic dermatitis, inflammatory bowel disease and atherosclerosis (arteriosclerosis). The method of claim 10, wherein the perfusion step of (b) is performed for 2 to 20 days. The method of claim 10, wherein the component inducing the target disease is selected from the group consisting of fatty acids, cisplatin, dextran sodium sulfate (DSS), and streptozotocin. The method of claim 12, wherein the fatty acid is selected from the group consisting of palmitic acid, stearic acid, myristic acid and lauric acid. The method of claim 10, wherein the manufacturing method
(a) creating an artificial liver tissue model; And
(b) perfusing fatty acids to the artificial liver tissue model; which is a method for producing a fatty liver or steatohepatitis model, including.

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