In vitro fibrosis model, preparing method therefor, and use thereof

Abstract

Provided are an in vitro fibrosis model, a method of preparing the in vitro model, and use of the in vitro model, the in vitro model including a cell cluster differentiated from mesenchymal cells, wherein the cell cluster exhibits pathological characteristics of fibrosis.

Claims

1. A method of screening a candidate therapeutic agent for treatment of a fibrotic disease, the method comprising: culturing adipose stem cells to form a three-dimensional (3D) cell cluster by adhering the adipose stem cells to a culture container comprising a hydrophobic surface, wherein the hydrophobic surface is coated with a growth factor immobilized to the hydrophobic surface through a polypeptide linker, and wherein the growth factor comprises vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or a heparin-binding domain (HBD), and wherein the polypeptide linker comprises a maltose-binding protein (MBP), a hydrophobin, or a hydrophobic cell penetrating peptide (CPP); culturing the 3D cell cluster for 3 days to 10 days to provide an in vitro fibrosis model comprising the 3D cell cluster; treating the in vitro fibrosis model comprising the 3D cell cluster with a test substance; and selecting, as a candidate therapeutic agent for treatment of the fibrotic disease, a test substance which exhibits improvement or treatment of pathological characteristics of fibrosis in the 3D cell cluster or cells thereof in the in vitro fibrosis model, as compared with an untreated control group.

2. The method of claim 1, wherein the 3D cell cluster is spherical and has a diameter in a range of about 300 μm to about 2,000 μm.

3. The method of claim 1, wherein the pathological characteristics of fibrosis comprise at least one selected from the group consisting of: increased deposition of collagen; increased expression, secretion, or synthesis of a fibrosis-related molecule including at least one selected from the group consisting of transforming growth factor (TGF)-beta, Smad, laminins, and smooth muscle actin (SMA); and increased cell death induced by deposition of collagen, or a combination thereof, in the 3D cell cluster or cells constituting the 3D cell cluster compared to a two-dimensional culture of the adipose stem cells.

4. The method of claim 1, wherein the fibrotic disease comprises at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease, chronic myocardial infarction, scleroderma/systemic sclerosis, neurofibromatosis, Hermansky-Pudlak syndrome, diabetic kidney disease, renal fibrosis, hypertrophic cardiomyopathy (HCM), hypertension-related nephropathy, renal tubulointerstitial fibrosis, focal segmental glomerulosclerosis (FSGS), radiation-induced fibrosis, fibroids, alcoholic liver disease, liver steatosis, liver fibrosis, liver cirrhosis, Hepatitis C Virus (HCV) infection, chronic rejection of transplanted organ, fibrotic skin disease, keloidal scar, Dupuytren's contracture, Ehlers-Danlos syndrome, epidermolysis bullosa dystrophica, oral submucous fibrosis, and fiber proliferative disorder.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows scanning electron microscope (SEM) images of a three-dimensional cell cluster according to an embodiment;

(2) FIG. 1B shows Haematoxylin and Eosin (H&E) staining results obtained from a three-dimensional cell cluster according to an embodiment;

(3) FIG. 2 is a diagram showing immunofluorescence staining results confirming a hypoxic state of a three-dimensional cell cluster according to an embodiment;

(4) FIG. 3 is a graph showing expression of TGF-beta in a three-dimensional cell cluster according to an embodiment;

(5) FIG. 4 is a graph showing expression of fibrosis-related factors in a three-dimensional cell cluster according to an embodiment;

(6) FIG. 5A is a diagram showing results for deposition of collagen in a three-dimensional cell cluster according to an embodiment, as identified by immunofluorescence staining and analysis of hydroxyproline contents;

(7) FIG. 5B is a graph showing results for deposition of collagen in a three-dimensional cell cluster according to an embodiment, as identified by immunofluorescence staining and analysis of hydroxyproline contents;

(8) FIG. 6 is a diagram showing results for deposition of collagen type I in a three-dimensional cell cluster according to an embodiment, as identified by immunofluorescence staining;

(9) FIG. 7 is a diagram showing results for deposition of collagen type I in a three-dimensional cell cluster according to an embodiment, as identified by immunofluorescence staining;

(10) FIG. 8 shows transmission electron microscope (TEM) images of a three-dimensional cell cluster according to an embodiment;

(11) FIG. 9A is a graph showing results for viability and apoptosis of cells in a three-dimensional cell cluster according to an embodiment; and

(12) FIG. 9B is a diagram showing results for viability and apoptosis of cells in a three-dimensional cell cluster according to an embodiment.

MODE OF THE INVENTION

(13) Hereinafter, the present invention is described in detail with reference to Examples. However, Examples shown and described herein are illustrative examples of the present invention and are not intended to otherwise limit the scope of the inventive concept in any way.

Examples: Preparation of In Vitro Fibrosis Model and Characterization of Fibrosis Modeling

(14) (1) Preparation of In Vitro Fibrosis Model

(15) (1.1) Separation of Human Adipose Stem Cells (hASCs)

(16) Subcutaneous adipose tissue of normal individuals was obtained from the Department of Plastic Surgery, the Catholic University of Korea. Then, the adipose tissue was washed three times with PBS containing 1% penicillin/streptomycin (PS) to remove contaminated blood therefrom, and was cut with surgical scissors. The resulting adipose tissue was soaked in a tissue lysate containing 1% BSA (w/v), 0.3% collagenase type I, and 1% PS, and then, the mixed tissue lysate was stirred by orbital shaking for 1 hour at a temperature of 37° C. Afterwards, the supernatant was removed, and the cell suspension was filtered through a 250 μm Nitex filter (Sefar America Inc.) to remove tissue debris. Then, centrifugation was performed thereon at a speed of 1,000 rpm for 5 minutes. Cells collected by centrifugation were re-suspended in DMEM/F-12 containing 10% BSA. The isolated primary cells were plated in a tissue culture flask for 24 hours in a humidified atmosphere with 5% CO.sub.2 and 95% air. Then, non-adherent cells were removed by replacement with a fresh medium having the same volume. The morphology of adherent hASCs was observed via a phase contrast microscope, and hASCs of 5 passages were used for all experiments.

(17) (1.2) Preparation of 3D Cell Cluster Derived from Adipose Stem Cells

(18) To prepare a 3D cell cluster derived from the hASCs, the hASCs were cultured in a treated 96-well non-tissue culture plate (NTCP) (NTCP made of polystyrene and having a hydrophobic surface, Falcon Company). The NTCP was a plated coated with a fused protein of maltose binding protein (MBP)-fibroblast growth factor (FGF), wherein the plate coated with the fused protein has been described in KR 10-1109125 that is incorporated herein by reference in its entirety. In detail, 1×10.sup.5 cells/cm.sup.2 of the hASCs were inoculated into each well of the well plate, and cultured in a DMEM/F-12 medium containing 10% FBS. Within 24 hours of the culture, 3D cell clusters of the hASCs were formed on each cell adhesion surface. For analysis of characteristics of a fibrosis model with respect to the formed 3D cell clusters, 3D cell clusters were collected on the 1.sup.st day (1 Day), 3.sup.rd day (3 Day), and 5.sup.th day (5 Day) of the culture. In addition, the 3D cell clusters were confirmed to have a diameter of about 500 μm or more. Hereinafter, the 3D cell cluster was represented by ‘3DCM’.

(19) In addition, as a comparative example, the hASCs were cultured in a 2D manner. In detail, 1×10.sup.5 cells/cm.sup.2 of the hASCs were inoculated into each well of a treated 96-well tissue culture plate (TCP), and cultured in a DMEM/F-12 medium containing 10% FBS. In the same manner as in the 3D cell cluster, cells were collected on the 1.sup.st day (1 Day), 3.sup.rd day (3 Day), and 5.sup.th day (5 Day) of the culture for analysis of characteristics of a fibrosis model. Hereinafter, the cells cultured in a 2D manner are represented by ‘2D’.

(20) (2) Analysis of Fibrosis Modeling Characteristics of In Vitro Fibrosis Model

(21) (2.1) Analysis of Characteristics of 3D Cell Cluster Derived from Adipose Stem Cells

(22) To analyze the morphological characteristics of a 3D cell cluster derived from adipose stem cells, the 3D cell cluster was subjected to scanning electron microscopy and H&E staining. In addition, immunostaining was performed on the 3D cell cluster to confirm a hypoxic state in the 3D cell cluster.

(23) In detail, for scanning electron microscopy, the collected 3D cell cluster was immobilized with 2.5% glutaraldehyde at a temperature of 4° C. for 2 hours, and then post-immobilized with 1% osmium tetroxide in deionized water. The immobilized 3D cell cluster was dehydrated two times with ethanol at a series of concentrations (50%, 70%, 80%, 90%, and 100%). Afterwards, the resulting 3D cell cluster was immersed in hexamethyldisilazane (HMDS) for 2 minutes, and vibration-dried for one day. To obtain an SEM image, the 3D cell cluster was attached to an adhesive carbon tape, and sputter-coating was performed with gold for 60 minutes at 10 mA. Images were then obtained at 15 kV, and the results are shown in FIG. 1A.

(24) In addition, for H&E staining, the collected 3D cell cluster was immobilized with 4% PFA at room temperature for 30 minutes, dehydrated with ethanol at a series of concentrations (50%, 70%, 80%, 90%, and 100%), and then, placed in paraffin wax. A section having a thickness of 4 μm was prepared, and then stained with haematoxylin and eosin. The section was deparaffinized, hydrated with distilled water, and washed three times with PBS. Afterwards, the resulting section was immersed in haematoxylin (Harris; Sigma-Aldrich) for 10 seconds, washed in flowing water for 10 to 15 minutes, counter-stained with eosin for 15 seconds, and then, washed again for 10 to 15 minutes. Afterwards, the resulting section was placed on a slide to be observed with a light microscope, and the results are shown in FIG. 1B.

(25) In addition, for hypoxic immunofluorescence analysis, the 3D cell cluster was incubated, before being collected at each culture time, in 10 mmol pimonidazole hydrochloride (Hypoxyprobe™-1 kit, Hypoxyprobe, USA) in 0.1 ml solution for 2 hours. Then, the incubated 3D cell cluster was collected, immobilized with 4% paraformaldehyde at a temperature of 4° C. for 30 minutes, and embedded in an optimal cutting temperature (OCT) compound (TISSUE-TEK® 4583; Sakura Finetek USA, Inc.). A frozen section having a thickness of 6 μm was washed with PBS, and to prevent nonspecific binding thereto, the 3D cell cluster was incubated in 4% BSA in PBS for 1 hour. Accordingly, pimonidazole was detected by primary mouse antibodies (hydroxy probe) and secondary goat anti-mouse Alexa 488 antibodies (Invitrogen). In addition, 4,5-diamino-2-phenylindole (DAPI) (Vector Laboratories) was used for nuclear staining. Here, a control group was subjected to experiments performed under the same conditions, except that no primary antibody was used, and was observed with a confocal microscope (Carl Zeiss). The results are shown in FIG. 2.

(26) FIG. 1A shows SEM images and H&E staining results obtained from the 3D cell cluster according to an embodiment.

(27) FIG. 2 is a diagram showing immunofluorescence staining results confirming a hypoxic state of the 3D cell cluster according to an embodiment.

(28) As shown in FIG. 1, the outer surface the 3D cell cluster of the culture at Day 1 was densely stained by H&E, and thus, it was confirmed that the cells were connected by fibrous matrices. As the culture continued, the 3D cell cluster of the culture at Day 3 showed a decreased intercellular space between the cells, and the 3D cell cluster of the culture at Day 5 showed almost no gap between the cells (see arrows).

(29) In addition, as shown in FIG. 2, it was confirmed that the DAPI-stained cells were uniformly distributed over the 3D cell cluster on Day 1 of the culture, and that more hypoxic probe-positive cells were present inside the 3D cell cluster. As the culture continued, the 3D cell cluster of Day 3 of the culture showed increased hypoxic probe-positive cells therein, and the 3D cell cluster of Day 5 of the culture also showed increased hypoxic probe-positive cells outside of the 3D cell cluster. Accordingly, it was confirmed that hypoxia was induced in the 3D cell cluster, and then, diffused to the outside of the 3D cell cluster. That is, by referring to FIG. 1, it was confirmed that the closure of the intercellular space on the outer surface of the 3D cell cluster led to the induction of hypoxia. In fibrosis, TGF-1 is an important relevant factor which is overexpressed in hypoxia. That is, as the distance between the cells narrowed, supply of oxygen to the cell cluster was restricted, and accordingly, TGF-1 was induced, thereby causing fibrosis. Therefore, based on the results above, it was confirmed that the pathological characteristics of fibrosis were modeled by the 3D cell cluster according to an embodiment.

(30) (2.2) Analysis of Fibrosis-Related Factors in 3D Cell Cluster Derived from Adipose Stem Cells

(31) TGF-beta is a major molecule in fibrosis and is induced under hypoxic conditions. To confirm whether fibrosis-related factors have been expressed or not in the 3D cell cluster derived from adipose stem cells, ELISA was performed on fibrosis-related factors including TGF-beta.

(32) In detail, to measure total contents of TGF-β1, a culture medium was prepared with normal cell concentration (NCC), 2D cells (2D), and 3D cell cluster (3DCM). To activate latent TGF-β1 in an immunoreactive form, the culture supernatant was incubated in 1N HCL and neutralized with 1.2 N NaOH/0.5 M HEPES. The assay was performed using the Quantikine ELISA human TGF-β1 kit (R&D System) according to the manufacturers instructions. Here, the absorbance was measured using a Multiskan (Thermo) at 560 nm, and the results are shown in FIG. 3.

(33) In addition, to confirm the expression of the fibrosis-related factors in the 3D cell cluster, total RNAs were extracted from the collected 3D cell cluster by using a triazole reagent (Invitrogen, USA) according to the manufacturer's instructions. The extracted RNAs were dissolved in nuclease-free water, and then, the concentration of the resulting RNAs was quantified using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific). Here, synthesis of complementary DNA was performed by using Maxime RT PreMIX (iNtROn) according to the manufacturers instructions. All target primers were purchased from Bioneer. All polymerase chain reactions were performed using ABI Prism 7500 (Applied Biosystems), and gene expression levels were quantified using SYBR Premix Ex Taq (TaKaRa). Comparative gene expression levels were calculated using the comparative Ct method, and the results are shown in FIG. 4.

(34) FIG. 3 is a graph showing the expression of TGF-beta in the 3D cell cluster according to an embodiment.

(35) FIG. 4 is a graph showing the expression of fibrosis-related factors in the 3D cell cluster according to an embodiment.

(36) As shown in FIGS. 3 and 4, it was confirmed that the 3D cell cluster derived from adipose stem cells showed increased expression of the fibrosis-related factors including TGF-beta, laminin, smooth muscle actin (SMA), collagen type I, and SMAD3.

(37) (2.3) Analysis of Collagen Deposition in 3D Cell Cluster Derived from Adipose Stem Cells

(38) To analyze collagen deposition in the 3D cell cluster derived from adipose stem cells, the 3D cell cluster was subjected to immunofluorescence staining, immunohistochemical staining, and hydroxyproline quantification, and observed with a transmission electron microscope.

(39) In detail, pretreatment was performed on the 3D cell cluster in the same manner as in H&E staining, and staining was performed thereon using Masson's trichrome (MT) staining. In the 3D cell cluster, the percentage of fibrosis was determined by counting the number of pixels of the stained collagen area in a digital image by using the ImageJ software (NIH), and the results are shown in FIG. 5A. In addition, for a hydroxyproline assay, 2D cells and 3D cell cluster were prepared by using RIPA buffer, and then, were hydrolyzed in 12N HCL at a temperature of 120° C. for 3 hours. Assays were performed using the hydroxyproline kit (Sigma-Aldrich) according to the manufacturer's instructions. Here, the absorbance was measured using a Multiskan (Thermo) at 560 nm, and the results are shown in FIG. 5B.

(40) In addition, for immunofluorescence (IF), a 3D cell cluster was immobilized in the same manner as in H&E staining above, embedded in an OCT compound (TISSUE-TEK® 4583; Sakura Finetek USA, Inc.), and then frozen at a temperature of −28° C. The resulting 3D cell cluster was cut to a thickness of 6 μm. To avoid nonspecific binding thereto, a section was incubated in 4% BSA at room temperature for 1 hour. Afterwards, the section was incubated overnight at a temperature of 4° C. with primary antibodies (Rabit, Abicam) specific for collagen type I. Then, a sample on the section was washed with PBS, and incubated for 1 hour with corresponding fluorescent conjugated secondary antibodies (Donkey anti-rabbit)(Life Technologies) in 1% BSA. In addition, DAPI (Vector Laboratories) was used for nuclear staining. Here, a control group was subjected to experiments performed under the same conditions, except that no primary antibody was used, and was observed with a confocal microscope (Carl Zeiss). The results are shown in FIG. 6.

(41) In addition, for immunohistochemical staining, pretreatment was performed on the 3D cell cluster in the same manner as in H&E staining. Here, fibronectin (FN) and laminin (LN) were each detected by using mouse monoclonal antibodies and goat polyclonal antibodies (Santa cruz Biotechnology) that are specific to FN and LN. In addition, for αSMA analysis, mouse monoclonal antibodies (Dako) were used to detect αSMA. After a section prepared therefrom was incubated overnight at a temperature of 4° C. with primary antibodies for the fibrosis-related factors, the section was incubated at room temperature for 1 hour with horseradish-labeled anti-mouse antibodies (specific to FN and αSMA) and anti-goat secondary antibodies (specific to LN) (Vector). Then, positive staining was visualized using diaminobenzidine (DAB, Vector). Here, a control group was subjected to experiments performed under the same conditions, except that no primary antibody was used. A section obtained therefrom was counter-stained with Haematoxylin and observed with a light microscope, and the results are shown in FIG. 7.

(42) In addition, for transmission electron microscopy (TEM), pretreatment was performed on a sample in the same manner as used for scanning electron microscopy. Additionally, the immobilized 3D cell cluster was infiltrated into an epoxy resin, embedded therein, and polymerized at a temperature of 60° C. for 24 hours. An ultrathin section was prepared by using an ultramicrotome (Ultra cut C, Leica CO. Ltd), and then, was stained with uranyl acetate and lead citrate. TEM images were observed by cryo-TEM (cryoTecanai F20, FEI Co. Ltd), and the results are shown in FIG. 8.

(43) FIGS. 5A and 5B show the results for the deposition of collagen in the 3D cell cluster according to an embodiment, as identified by immunofluorescence staining and analysis of hydroxyproline contents.

(44) FIG. 6 is a diagram showing the results for the deposition of collagen type I in the 3D cell cluster according to an embodiment, as identified by immunofluorescence staining.

(45) FIG. 7 is a diagram showing the results for the deposition of collagen type I in the 3D cell cluster according to an embodiment, as identified by immunofluorescence staining.

(46) FIG. 8 shows TEM images of the 3D cell cluster according to an embodiment.

(47) As shown in FIGS. 5A and 5B, it was confirmed that a majority of collagen was stained in the 3D cell cluster by MT staining and that the content of hydroxyproline was also increased in the 3D cell cluster as compared with that of hydroxyproline in 2D cells.

(48) In addition, as shown in FIG. 6, it was confirmed that collagen type I was significantly increased in the 3D cell cluster, as identified by IF.

(49) In addition, as shown in FIG. 7, it was confirmed that αSMA was significantly increased in the 3D cell cluster, as identified by immunohistochemical staining. αSMA is a traditional marker of myofibroblasts, and collagen type I is known to be synthesized from myofibroblasts in fibrosis. That is, the results above are deemed to be consistent with the results of FIG. 6.

(50) In addition, as shown in FIG. 8, by referring to the TEM images, it was confirmed that the deposition of collagen fibers and collagen gradually increased as the culture time of the 3D cell cluster increased. In detail, thick collagen fibers were observed (see arrows) on Day 5 of the culture, wherein such observation is deemed to be caused by cross-linking of collagen. In addition, on Day 5 of the culture, it was confirmed that no intact cell structure was observed inside the 3D cell cluster. As a result, collagen fibers became thicker around the cells, which caused apoptosis of the cells due to lack of transport of nutrients. Therefore, it was confirmed that the pathological characteristics of fibrosis were modeled by the 3D cell cluster according to an embodiment.

(51) (2.4) Analysis of Viability and Apoptosis of Cells in 3D Cell Cluster Derived from Adipose Stem Cell

(52) The deposition of collagen ultimately induced apoptosis of cells in fibrosis. Thus, to confirm that such characteristics appeared in a 3D cell cluster derived from adipose stem cells, an LDH assay and a live/dead assay were performed on the 3D cell cluster.

(53) In detail, for the LDH assay, among a culture medium prepared with NCC, 2D cells, and 3D cell cluster, absolute lactic dehydrogenase (LDH) release was measured. The measurement was performed using the LDH assay kit (Promega) according to the manufacturer's instructions. Here, the absorbance was measured using Multiskan (Thermo) at 560 nm, and the results are shown in FIG. 9A. In addition, for the live/dead assay, a live/dead assay kit (Molecular probes) was used according to the manufacturer's instructions. In summary, the collected 3D cell cluster was treated with 1 ml of HEPES-buffered saline (HBSS) containing 1 μl of green-fluorescent nucleic acid staining solution (SYTO 10) and 1 μl of red-fluorescent nucleic acid staining solution (ethidium homodimer-2), and then cultured in a CO.sub.2 culture medium for 30 minutes. Afterwards, the resulting 3D cell cluster was washed three times with PBS, immobilized with 4% PFA for 30 minutes, embedded in an OCT compound (TISSUE-TEK® 4583; Sakura Finetek USA, Inc.), and then frozen at a temperature of −28° C. The resulting 3D cell cluster was cut to a thickness of 10 μm. The entire 3D cell cluster was completely cut, and two slides were selected from the middle and outer portions of each sample. Here, the sections were analyzed using a confocal microscope (Carl Zeiss), and the results are shown in FIG. 9B.

(54) FIGS. 9A and 9B show the results for viability and apoptosis of cells in the 3D cell cluster according to an embodiment.

(55) As shown in FIG. 9A, according to the LDH assay, the 3D cell cluster showed increased LDH levels as compared with those in NCC and 2D cells. In addition, as shown in FIG. 9B, the apoptosis of cells was visually identified in the 3D cell cluster in the same manner as in FIG. 9A.

(56) As a result, the 3D cell cluster according to an embodiment exhibited pathological characteristics of fibrosis, and thus it was confirmed to be suitable for use as an in vitro fibrosis model.