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.

1 . An in vitro fibrosis model comprising a cell cluster differentiated from mesenchymal cells, wherein the cell cluster exhibits pathological characteristics of fibrosis. 
     
     
         2 . The model of  claim 1 , wherein the mesenchymal cells are selected from the group consisting of adipose stem cells, mesenchymal stem cells, mesenchymal stromal cells, bone marrow stem cells, and fibroblasts. 
     
     
         3 . The model of  claim 1 , wherein the cell cluster is spherical and has a diameter in a range of about 300 μm to about 2,000 μm. 
     
     
         4 . The model of  claim 1 , wherein the cell cluster is differentiated by adhering mesenchymal cells to a culture container comprising a hydrophobic surface and culturing the mesenchymal cells in the culture container. 
     
     
         5 . The model of  claim 4 , wherein the mesenchymal cells are further cultured for at least 12 hours after the mesenchymal cells are differentiated into the cell cluster. 
     
     
         6 . The model of  claim 1 , wherein the pathological characteristics of fibrosis comprise at least one selected from the group consisting of:
 formation of excessive connective tissue;   deposition of collagen;   increased expression, secretion, or synthesis of a fibrosis-related molecule including at least one selected from the group consisting of transforming growth factor (TGF)-beta, Smad, laminins, and smooth muscle actin (SMA); and   increased cell death,   or a combination thereof, in the cell cluster or cells constituting the cell cluster.   
     
     
         7 . The model of  claim 1 , wherein the fibrosis comprises at least one selected from the group consisting of idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, interstitial lung disease, nonspecific interstitial pneumonia (NSIP), usual interstitial pneumonia (UIP), endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease, chronic myocardial infarction, scleroderma/systemic sclerosis, neurofibromatosis, Hermansky-Pudlak syndrome, diabetic kidney disease, renal fibrosis, hypertrophic cardiomyopathy (HCM), hypertension-related nephropathy, renal tubulointerstitial fibrosis, focal segmental glomerulosclerosis (FSGS), radiation-induced fibrosis, fibroids, alcoholic liver disease, liver steatosis, liver fibrosis, liver cirrhosis, Hepatitis C Virus (HCV) infection, chronic rejection of transplanted organ, fibrotic skin disease, keloidal scar, Dupuytren's contracture, Ehlers-Danlos syndrome, epidermolysis bullosa dystrophica, oral submucous fibrosis, and fiber proliferative disorder. 
     
     
         8 . A method of preparing an in vitro fibrosis model, the method comprising:
 forming a cell cluster by adhering mesenchymal cells to a culture container comprising a hydrophobic surface and culturing the mesenchymal cells in the culture container; and   establishing pathological characteristics of fibrosis in the cell cluster by performing additional culturing thereof for at least 12 hours.   
     
     
         9 . The method of  claim 8 , wherein the forming of the cell cluster is performed by separating the mesenchymal cells from the culture container as a density of the mesenchymal cells increases. 
     
     
         10 . The method of  claim 8 , wherein the hydrophobic surface of the culture container is selected from the group consisting of a silanized surface, a hydrocarbon-coated surface, a polymer surface, and a metal surface. 
     
     
         11 . The method of  claim 8 , wherein the mesenchymal cells are adhered to the culture container by cell-substrate interactions with the hydrophobic surface of the culture container or interactions with a growth factor having adhesiveness to the mesenchymal cells immobilized on the surface of the culture container. 
     
     
         12 . The method of  claim 11 , wherein the growth factor is immobilized in the form of a polypeptide linker-growth factor recombinant protein on the surface of the culture container by using a polypeptide linker in such a way as to fuse an amino terminus of the growth factor to a carboxyl terminus of the polypeptide linker. 
     
     
         13 . The method of  claim 12 , wherein the polypeptide linker is selected from the group consisting of a maltose-binding protein (MBP), a hydrophobin, and a hydrophobic cell penetrating peptide (CPP). 
     
     
         14 . A method of screening a therapeutic agent for fibrosis, the method comprising:
 treating the in vitro fibrosis model of  claim 1  with a test substance; and   selecting, as a candidate substance for treatment of fibrosis, the test substance which exhibits improvement or treatment of pathological characteristics of fibrosis in the cell cluster or cells thereof in the in vitro fibrosis model, as compared with an untreated control group.   
     
     
         15 . The method of  claim 14 , wherein the pathological characteristics of fibrosis comprises at least one selected from the group consisting of:
 formation of excessive connective tissue;   deposition of collagen;   increased expression, secretion, or synthesis of a fibrosis-related molecule including at least one of transforming growth factor (TGF)-beta, Smad, laminins, and smooth muscle actin (SMA); and   increased cell death,   or a combination thereof, in the cell cluster or cells constituting the cell cluster.