Patent Application
20240400997
The content of the electronically submitted sequence listing, file name: Q289151_sequence listing as filed .TXT; size: 6,151 bytes; and date of creation: Jun. 27, 2023, filed Jun. 28, 2023, is incorporated herein by reference in its entirety.
The present invention relates to a transformed human hepatic stellate cell line and a use thereof, and in particular, to an immortalized human hepatic stellate cell line (hereinafter, referred to as HSC) which is derived from normal human liver tissue and is capable of proliferating infinitely, and a use thereof.
In general, since human-derived hepatic cells have different drug-degrading ability according to race, sex, and individuals, securing primary human hepatic cells (PHH) has become an important issue in the development of a patient-specific disease model. Since the primary hepatic cells are difficult to be cultured in general 2D culture for a long period of time and the drug-degrading ability is deteriorated, 3D culture has recently attracted attention.
Meanwhile, hepatic stellate cells are known to have the next majority to hepatic cells among the cell members of liver tissue in a disease model, particularly, have a great effect on liver fibrosis and cirrhosis. There are several commercially available hepatic stellate cell lines, including LX-2, but these hepatic stellate cell lines have several limitations in that the hepatic stellate cells are difficult to be used in an actual patient-specific disease model.
First, when the hepatic stellate cells start to be cultured in vitro, they naturally differentiate into myofibroblasts, and the myofibroblasts cause liver fibrosis. In fact, it is known that the co-culture of hepatic stellate cells and hepatic cells slows the differentiation of hepatic stellate cells into myofibroblasts, but there is a limit to the regulation of differentiation into myofibroblasts.
Second, as described above, since the hepatic stellate cells have a high degree of differentiation into myofibroblasts during the in vitro culture, the reactivity to fibrosis-inducing factors deteriorates. Therefore, the hepatic stellate cells have an aspect that it is difficult to test a liver fibrosis-promoting or -inhibiting drug by preparing a liver fibrosis model. In addition, in the case of co-culturing the hepatic stellate cells with hepatic cells, when the proportion of the hepatic stellate cells increases, the activity of the hepatic cells itself decreases, thereby making it difficult to confirm a decrease in hepatocyte metabolism by a drug.
Third, the experimental model using rodents due to the difference between species in drug metabolism has a limitation, and the experimental model closest to the human body is a 3D co-culture model using primary human hepatic cells, but there is no optimized model consisting of primary human hepatic cells and hepatic stellate cells so far.
Therefore, there is a need for a hepatic stellate cell line which can induce liver fibrosis by the effect of a drug without inhibiting the function of hepatic cells in a 3D culture environment.
Accordingly, the present inventors have made intensive efforts to develop a hepatic stellate cell line having a higher degree of fibrosis induction by a drug than that of a control group (conventional hepatic stellate cell line LX-2) in a drug-induced liver fibrosis experiment without inhibiting the function of hepatic cells in a normal 3D culture environment, and as a result, prepared a hepatic stellate cell line satisfying the above conditions, and confirmed that a composition including the hepatic stellate cell line could be utilized as a composition for maintaining or enhancing hepatic metabolism under normal conditions, and a composition for a drug-induced liver fibrosis model, thereby completing the present invention.
In order to solve the above problem, one aspect of the present invention provides a transformed human hepatic stellate cell line having increased liver fibrosis activity.
Another aspect of the present invention provides an immortalized human hepatic stellate cell line derived from a human liver tissue.
Still another aspect of the present invention provides a cell composition for producing an artificial liver construct, the cell composition including the transformed human hepatic stellate cell line and hepatic cells.
One aspect of the present invention provides a liver spheroid prepared by culturing the cell composition.
Another aspect of the present invention provides an artificial liver construct produced by culturing the cell composition with a scaffold.
Yet another aspect of the present invention provides a 3D culture model including the human hepatic stellate cell line and a disease model using the 3D culture model.
Still another aspect of the present invention provides a method for preparing a liver fibrosis model, the method including 3D co-culturing of the transformed hepatic stellate cell line and hepatic cells, and a liver fibrosis model prepared by the method.
Yet still another aspect of the present invention provides a method for evaluating a liver fibrosis regulator, the method including treating the liver spheroid, the liver construct, or the liver fibrosis model with a candidate material of regulating liver fibrosis.
The transformed human hepatic stellate cell line according to the present invention can induce liver fibrosis by a drug without showing any inhibition of the function of hepatic cells during co-culturing with hepatic cells, and thus can be useful as a model for liver diseases such as liver fibrosis and fatty liver.
According to the present invention, the human hepatic stellate cell line can be used as single or co-cultured constituent cells in various in vitro cultures, and can also be used as a cell line for in vivo transplantation.
Hereinafter, the present invention will be described in more detail.
One aspect of the present invention provides a transformed human hepatic stellate cell line into which a human telomerase reverse transcriptase (hTERT) gene and a SV40 large T-antigen gene, or a gene encoding a polypeptide of the hTERT or SV40 large T-antigen are introduced and which shows positive for the expression of a platelet-derived growth factor receptor beta (PDGFR-0) marker.
Meanwhile, the information on the gene sequences of the hTERT and SV40 large T-antigen of the present invention or the polypeptide amino acid sequence of the hTERT or SV40 large T-antigen may be obtained from a known database (e.g., GenBank of NCBI). In addition, the nucleotide sequences of the hTERT and SV40 large T-antigen or the polypeptide amino acid sequence of the hTERT or SV40 large T-antigen may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleotide sequence or amino acid sequence information which may be obtained from the known database.
In addition, the transformed human hepatic stellate cell line may be an immortalized human hepatic stellate cell line capable of proliferating indefinitely. According to an embodiment of the present invention, it was confirmed that the transformed human hepatic stellate cell line can be subcultured at least 19 times, at least 31 times, or up to 62 times.
The present inventors have found that the hTERT gene and the SV40 large T-antigen gene are introduced to the hepatic stellate cells isolated from a human, and thus the human hepatic stellate cell lines are transformed, and the transformed human hepatic stellate cell lines have an increase in liver fibrosis activity, and does not inhibit the activity of hepatic cells when co-cultured with the hepatic cells, and thus have completed the present invention.
In the present invention, the introduction of the hTERT gene and the SV40 large T-antigen gene may be performed by any method known in the art. Specifically, the transformation may be performed with an expression vector including a nucleotide encoding hTERT and a nucleotide encoding a SV40 large T-antigen. The nucleotides may be delivered to a single expression vector or to different expression vectors, respectively. The expression vector may be a viral vector, for example, a lentiviral vector.
In the present invention, PDGFR-0 is one of the receptors that bind to PDGF, which is the most potent division and proliferation-promoting cytokine of the hepatic stellate cells, and may be a marker showing liver fibrosis activity of the hepatic stellate cells.
The transformed human hepatic stellate cell line may include at least 80%, at least 90%, at least 95%, or at least 98% of cells that are positive for the PDGFR-0 marker. According to an embodiment of the present invention, the transformed human hepatic stellate cell line has a purity of about 98.3% of PDGFRB+ cells.
The transformed human hepatic stellate cell line may have overexpressed smooth muscle actin alpha (α-SMA).
The transformed human hepatic stellate cell line may exhibit increased expression of a hepatic stellate cell marker. Specifically, the expression of one or more hepatic stellate cell markers selected from the group consisting of α-SMA, lysyl oxidase (LOX), tissue inhibitor of matrix metalloproteinase 1 (TIMP1), and PDGFR-0 may be increased.
In addition, the transformed human hepatic stellate cell line may exhibit increased expression of an extracellular matrix marker. Specifically, the expression of one or more extracellular matrix markers selected from the group consisting of collagen type IV alpha 1 (Col-4A1), collagen type IV alpha 3 (Col-4A3), laminin subunit beta 1 (LamB1), fibronectin (FN), integrin subunit alpha 2 (ITGA2), and integrin subunit beta 1 (ITGB1) may be increased.
According to an embodiment of the present invention, the transformed human hepatic stellate cell line may have the expression of the hepatic stellate cell and/or extracellular matrix marker of at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times, compared to an LX-2 cell line, which is a known human hepatic stellate cell line. In addition, the expression of the hepatic stellate cells and/or the extracellular matrix marker may be up to 25 times compared to the LX-2 cell line, which is a known human hepatic stellate cell line. In particular, the transformed human hepatic stellate cell line may have an increased expression of α-SMA by 10 times or more, as compared to the LX-2 cell line. In this case, the cell line may be cultured in a DMEM medium or a Williams E medium.
The transformed human hepatic stellate cell line may exhibit liver fibrosis activity. The expression “exhibiting liver fibrosis activity” as used herein means that the hepatic stellate cells induce liver fibrosis, such as cell proliferation, contraction, inflammatory cytokine secretion (chemoattraction), migration, and secretion of extracellular matrix.
Another aspect of the present invention provides a cell composition for producing an artificial liver construct, the cell composition including the transformed human hepatic stellate cell line and hepatic cells.
The term “artificial liver construct” as used herein refers to a 3D cell mass that simulates human liver functions. The artificial liver construct may be a liver spheroid formed by direct binding of hepatic stellate cells and hepatic cells. In addition, the artificial liver construct may be a 3D liver analog formed by 3D culture of hepatic stellate cells and hepatic cells with a scaffold (support).
The transformed human hepatic stellate cell line is the same as described above.
The hepatic cells may be primary human hepatic cells, immortalized hepatic cell lines, or stem cell-derived hepatic cells, but are not limited thereto.
The cell composition may further include a medium for maintaining or culturing the hepatic stellate cell lines and hepatic cells.
Yet another aspect of the present invention provides a liver spheroid prepared by culturing the cell composition.
The liver spheroid may simulate a biological environment in a liver disease model such as liver fibrosis, liver disease associated with liver fibrosis, or fatty liver. Therefore, the liver spheroid may be utilized for drug evaluation such as efficacy, safety, and toxicity of candidate drugs in a liver disease model such as liver fibrosis, liver disease associated therewith, or fatty liver.
According to an embodiment of the present invention, even when the transformed human hepatic stellate cell line was in direct contact with the hepatic cells during the culturing of the cell composition, the liver spheroid exhibited increased or similar expression of HNF4α, albumin, and CYP3A4 enzyme, which are markers for the differentiation into hepatic cells, compared to those of the culture of hepatic cells alone, and high expression of all of HNF4α, albumin and CYP3A4 enzyme, which are markers for the differentiation into hepatic cells, and CK-19, which is a marker for the differentiation into cholangiocytes, compared to the spheroid co-cultured with the LX-2, and thus the liver spheroid was confirmed to exhibit excellent hepatocellular function. In addition, the liver spheroid exhibited initial liver fibrosis activity because the expression levels of Col-1A1, α-SMA, TIMP1, and LOX, which are markers for the fibrosis progress of the hepatic stellate cells, are similar to those of the spheroid co-cultured with the LX-2.
Still another aspect of the present invention provides an artificial liver construct produced by culturing the cell composition with a scaffold.
The scaffold may be a 3D matrix based on natural materials or artificial materials. Specifically, the scaffold may be a polyethylene glycol-containing hydrogel, a fibrin gel, or biosilk, but is not limited thereto.
The scaffold-based culture may be performed by crosslinking or coating an RGD (Arg-Gly-Asp) peptide, fibrin, or laminin in the scaffold.
The artificial liver construct may simulate a biological environment in a liver disease model such as liver fibrosis, liver disease associated with liver fibrosis, or fatty liver. Thus, the artificial liver construct may be utilized in drug evaluation such as efficacy, safety, and toxicity of candidate drugs in a liver disease model, such as liver fibrosis, liver disease associated with liver fibrosis, or fatty liver.
According to an embodiment of the present invention, the artificial liver construct is confirmed to exhibit high albumin secretion and CYP3A4 enzyme activity in the hepatic cells as compared to the artificial liver construct obtained by co-culturing with the LX-2 cell line, thereby exhibiting excellent drug metabolizing ability and hepatic cell activity.
When compared with the LX-2 cell line, which is a conventional hepatic stellate cell line, it was confirmed that α-SMA involved in tissue contraction was overexpressed (
Also, in an embodiment of the present invention, the gene sequence or amino acid sequence information of the hepatocyte nuclear factor 4 alpha (HNF4u), albumin, vimentin, smooth muscle actin alpha (α-SMA), type I collagen (Col-1A1), lysyl oxidase (LOX), tissue inhibitor of matrix metalloproteinase 1 (TIMP1), platelet-derived growth factor beta (PDGFR-0), collagen type IV alpha 1 (Col-4A1), collagen type IV alpha 3 (Col-4A3), laminin subunit beta 1 (LamB1), fibronectin (FN), integrin subunit alpha 2 (ITGA2), integrin subunit beta 1 (ITGB1), cytochrome 3A4 (CYP3A4), CK-19, GFAP, desmin, and nestin may be obtained from a known database (e.g., GenBank of NCBI). In addition, the gene sequences or amino acid sequences of the hepatocyte nuclear factor 4 alpha (HNF4u), albumin, vimentin, smooth muscle actin alpha (α-SMA), type I collagen (Col-1A1), lysyl oxidase (LOX), tissue inhibitor of matrix metalloproteinase 1 (TIMP1), platelet-derived growth factor beta (PDGFR-0), collagen type IV alpha 1 (Col-4A1), collagen type IV alpha 3 (Col-4A3), laminin subunit beta 1 (LamB1), fibronectin (FN), integrin subunit alpha 2 (ITGA2), integrin subunit beta 1 (ITGB1), cytochrome 3A4 (CYP3A4), CK-19, GFAP, desmin, and nestin may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the nucleotide sequence or amino acid sequence information which may be obtained from the known database.
Yet still another aspect of the present invention provides a method for preparing a liver fibrosis model including 3D co-culturing the transformed human hepatic stellate cell line and hepatic cells or a liver fibrosis model obtained by the method.
The transformed human hepatic stellate cell line and hepatic cells are the same as described above.
The 3D co-culture may be performed without a scaffold or with a scaffold, and the scaffold or scaffold-based culture is the same as described above. In addition, the 3D co-culture may be performed by adding a macromer to a culture medium in order to simulate the stiffness of liver fibrosis. The macromer may be at least 4%, at least 5%, at least 6%, at least 7%, or at least 8%, and 4% to 8% macromer may be used, but is not limited thereto. The liver fibrosis model may be a liver spheroid or an artificial liver construct, and the liver spheroid and the artificial liver construct are the same as described above. According to an embodiment of the present invention, it is confirmed that the liver fibrosis model exhibits a constant degree of hepatocellular function and a liver injury simulation regardless of the type of adhesion protein during the scaffold-based culturing.
According to an embodiment of the present invention, it is confirmed that the liver fibrosis model is capable of simulating the stiffness of liver fibrosis or liver fibrosis-associated diseases through the contraction of the culture during the scaffold-based culturing.
According to an embodiment of the present invention, the liver fibrosis or fatty liver model prepared using the transformed human hepatic stellate cell line has a higher sensitivity to TGF-β1, which is a liver fibrosis-inducing factor, than the liver fibrosis or fatty liver model prepared using a conventionally used human hepatic stellate cell line. In addition, since the transformed human hepatic stellate cell line does not have a direct effect on the differentiation or albumin production of hepatic cells even after long-term culture, the liver fibrosis or fatty liver model prepared using the transformed human hepatic stellate cell line is characterized by having improved sensitivity to fibrosis and metabolic degradation of hepatic cells caused by drugs or growth factors, and thus is possible to closely simulate the living environment and to secure a more sensitive response in disease modeling (see
Another aspect of the present invention provides a method for evaluating a liver fibrosis regulator, the method including treating the liver fibrosis model including the liver spheroid or the artificial liver construct with a candidate material of regulating liver fibrosis.
The term “liver fibrosis regulation” herein may mean inhibiting or activating liver fibrosis, and the material that inhibits liver fibrosis may be a material that prevents or treats liver diseases such as liver fibrosis, liver disease associated with liver fibrosis, or fatty liver. The liver disease associated with liver fibrosis may include liver fibrosis, liver cirrhosis, cirrhosis, and liver cancer, but is not limited thereto.
Evaluating the liver fibrosis regulator may include evaluating the efficacy, safety, and toxicity of a candidate material that inhibits or activates liver fibrosis, or screening therapeutic agents of liver diseases such as liver fibrosis, liver disease associated with liver fibrosis, or fatty liver.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for describing the present invention in more detail, and the scope of the present invention is not limited to these examples.
For RT-PCR, the total RNA of the cell line was isolated using the TRIzol (Thermo Fisher; 15596018) reagent, and then synthesized into cDNA using TOPScript™ RT DryMIX (Ezynomics; RT200). RT-PCR was performed using synthesized cDNA as a template, the FAST SYBR® Green Master Mix (Applied Biosystems; 4385614) reagent and gene-specific primers. The used primers are shown in Table 2 below.
For immunofluorescence staining, the cell line was fixed for 15 minutes using 4 paraformaldehyde 3-4 days after the culturing. Thereafter, the cell membrane of the cell line was permeabilized for 15 minutes using 0.25 Triton X-100. The primary antibodies were diluted in a buffer containing 400 bovine albumin and reacted at 4° C. for 16 hours, and then the secondary antibodies specific to the primary antibodies and attached with fluorescent materials were diluted and reacted at room temperature for 1 hour. An image corresponding to the marker was obtained using a fluorescence microscope. In this case, the primary antibodies used for immunofluorescence staining are as follows.
Human hepatic stellate cells were isolated from the liver tissues from patients (Chungnam National University Human Resources Bank) by treatment with 2 mg/mL collagenase I (Worthington, USA) at 37° C. for 30 minutes, and were recovered as single cells by filtration with a mesh of 100 μm pore size. The cells thus obtained were cultured for 16-18 hours in a primary hepatocyte maintenance media CM4000 (ThermoFisher) to which 10% FBS and 1% penicillin/streptomycin were added, and then pLenti-SV40 and pLenti-hTERT viruses (abm, US) were cultured together with 4 μg/mL polybrene (Sigma) for 16-18 hours, thereby being immortalized. Then, the cells were cultured in a hepatocyte maintenance medium.
Single-cell colonies were cultured in hepatic stellate cell line medium (DMEM/F12 (Thermofisher 11330), 10% fetal bovine serum, and 1% penicillin/streptomycin) to secure immortalized human hepatic stellate cell lines (HSCs) that are capable of proliferating infinitely. The hepatic stellate cell lines were established from the single-cell colonies. Morphological characteristics of the cell line were confirmed through a microscope, and the expression of hTERT and SV40 large T-antigen was confirmed by PCR (
In addition, the expression of the hepatic stellate cell markers (GFAP, desmin, endoglin, and α-SMA) of the prepared hepatic stellate cell line was confirmed by immunofluorescence staining, and the expression markers of the hepatic stellate cell line and the hepatic cell line HepG2 (ATCC) were confirmed by RT-PCR analysis.
As a result, the prepared hepatic stellate cell lines expressed weakly albumin and PEPCK, which are hepatocyte markers, hardly expressed HNF4u and CK18, and expressed GFAP, desmin, endoglin, and α-SMA, which are hepatic stellate cell line markers (
To confirm whether the hepatic stellate cell lines maintain hepatic stellate cell characteristics even after subculturing, the subculturing was performed in a hepatic stellate cell line medium (DMEM/F12 (Thermofisher 11330), 10% fetal bovine serum, 1% penicillin/streptomycin) up to 19 times, and immunofluorescence staining was performed. As a result, it was confirmed that the cell line expressed GFAP, desmin, endoglin, nestin, α-SMA, type I collagen, and PDGFRB, which are hepatic stellate cell markers, even after subculturing (
In order to isolate cells exhibiting the same characteristics of the hepatic stellate cells among the subcultured cells and to utilize the isolated cells as a disease model, PDGFRB+ (CD140b+) cells were isolated using a magnetic-activated cell sorting (MACS), and the isolated cells were identified using a fluorescence-activated cell sorting (FACS), and the purity of the PDGFRB+ cells was about 98.3% (
As a result of confirming, with immunofluorescence staining, the expression levels of GFAP, α-SMA, desmin, and PDGFRB, which are hepatic stellate cell markers, in the K-HSC cells, the hepatic stellate cell markers were well expressed (
For convenience of use in the future, K-HSC-GFP was prepared using retroviruses containing green fluorescence protein (GFP) genes. 1×105 cells of the hepatic stellate cell lines prepared in Example 1 were seeded in a 6-well culture dish, and the next day, 1 hour before virus infection, the cells were pretreated with 8 ng/mL polybrene reagents (Santa Cruz Biotechnology, USA) in order to increase virus infection efficiency. Then, the cells were treated with GFP-retrovirus at multiplicity of infection (MOI) of 5 and cultured in a 5% CO2 incubator at 37° C. for one day. The next day, after the medium was replaced with a fresh culture medium, the culture was continued, and after 48 hours, a strong GFP expression signal was confirmed to secure K-HSC-GFP hepatic stellate cell lines (
The hepatic stellate cell lines (K-HSC) prepared in Example 1 and the commercially available hepatic stellate cell line LX-2 (Millipore, USA) were stored in liquid nitrogen, and then the same lot was thawed and subcultured 5 times in Williams E medium (Sigma, USA) containing 2% FBS and DMEM medium (Sigma, USA), respectively. Thereafter, hepatic stellate cell-specific markers were compared through RT-PCR analysis. When the LX-2 was cultured in the DMEM medium, the expression of hepatic stellate cell-specific markers was set as 1, and relative mRNA expression was measured.
In the K-HSC according to the present invention, it was confirmed that α-SMA, Col-IA1, LOX, and TIMP1, which are liver fibrosis markers, and nestin and PDGFR-0, which are hepatic stellate cell markers, were more activated in Williams E medium, and in particular, the activity of α-SMA, which is a myofibroblast marker involved in tissue contraction, was increased by 10 times or more compared to the LX-2 (
The expression levels of markers (Col-4A3, Col-4A1, LamB1, FN, ITGA2, and ITGB1) for various materials constituting the extracellular matrix were confirmed by cell fluorescence staining and RT-PCR. As a result, the K-HSC of the present invention showed increased expression compared to the LX-2 (
The expression levels of hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1), which is a fibrosis-inducing factor, were confirmed through RT-PCR. As a result, the K-HSC of the present invention and the existing LX-2 showed significantly lower expression than the undifferentiated hepatic cell line HepaRG, but in the case of TGF-β1, the LX-2 showed increased expression level compared to K-HSC (
In order to confirm the effect of the K-HSC hepatic stellate cell line prepared in Example 1 on hepatic cells, co-culture of hepatic stellate cell lines and hepatic cells was performed.
The commercially available hepatic cell line HepaRG (Biopredic, France) and the K-HSC prepared in Example 1 were applied at a ratio of hepatic cells/hepatic stellate cells of 4:6 and 8:2 onto the surface of a 48-well plate coated with 1.5% agarose using a Williams E medium-based serum-free medium containing hepatocyte growth factor (HGF, 10 ng/mL) and epidermal growth factor (EGF, 2 ng/mL), respectively, and 3D cultured while inducing self-association through the rotary shaking (70 rpm) without medium exchange for 7 days to form a spheroid. In this case, the total cell concentration was 3×105 cells/mL. For comparison, only the hepatic cell line HepaRG was cultured in the same manner without hepatic stellate cell lines and set as a reference for the degree of hepatic cell differentiation. As a control group, the commercially available hepatic stellate cell line LX-2 was co-cultured with the hepatic cell line HepaRG in the same manner.
The effect of direct contact with the hepatic stellate cell line without a material-based matrix in the liver spheroid prepared in Example 3 on the differentiation degree of hepatic cells was confirmed by RT-PCR. As a result, it was confirmed that the K-HSC prepared in Example 1 did not inhibit the differentiation of hepatic cells because the K-HSC exhibited improved expression of HNF4α, albumin, and CYP3A4 enzyme, which are markers for the differentiation into hepatic cells (when the ratio of hepatic cells and hepatic stellate cell lines is 4:6) or exhibited similar expression levels thereof (when the ratio of hepatic cells and hepatic stellate cell lines is 8:2), compared to a spheroid composed of only hepatic cell line (HepaRG) when the K-HSC was co-cultured with the hepatic cell line (
Meanwhile, Col-1A1, α-SMA, TIMP1, and LOX, which are markers for the fibrosis progress of the hepatic stellate cells, exhibited the degree of initial liver fibrosis similar to that of LX-2 (
The above results show that the spheroid using the K-HSC prepared in Example 1 in a disease model such as liver fibrosis or fatty liver may simulate a biological environment more closely and secure more favorable initial conditions in disease modeling through the 3D culture. In addition, the results show that the K-HSC prepared in Example 1, unlike LX-2, improves the activity of the hepatic cell line and improves the sensitivity to fibrosis and metabolic degradation caused by drugs or growth factors.
The K-HSC prepared in Example 1 was encapsulated in a scaffold together with the hepatic cells, and then 3D co-culture was performed. In the same manner as in Example 3, HepaRG, which is an immortalized differentiated hepatic cell line, was used as the hepatocyte, and the LX-2, which is a commercially available hepatic stellate cell line, was used as a control group. After long-term culture in a 3D culture matrix, the network formation of cells was confirmed through a confocal microscope through actin-phalloidin fluorescence staining, and the activity of cells was confirmed through ATP analysis. In addition, drug metabolism, which is a major function of hepatic cells, was confirmed by measuring the activity of cytochrome p450 3A4 enzyme induced by rifampicin, and albumin production was confirmed through ELISA. In addition, the effect of direct contact between cell lines on the degree of differentiation of the hepatic cell line using a spheroid culture, which is a cell-based 3D culture model, was confirmed by RT-PCR. As a result, it was confirmed that the hepatic stellate cell line improves the activity of the hepatic cell line, unlike LX-2.
When the hepatic stellate cell line K-HSC prepared in Example 1 and the LX-2 were subjected to immunofluorescence staining with PDGFR-beta, which is a hepatic stellate cell marker, and collagen, which is a marker for myofibroblasts differentiated from hepatic stellate cells, and was then compared, it was found that the markers for hepatic stellate cells and the differentiated myofibroblasts were all activated in the K-HSC unlike the LX-2 (
Meanwhile, when the hepatic stellate cell line K-HSC prepared in Example 1 was 3D cultured in a fibrin gel scaffold, whether the cell adhesion ability was improved and the network formation ability were confirmed.
Specifically, liver analogs were prepared by using, as a 3D culture matrix, a fibrin gel based on a natural material and a hybrid hydrogel containing a polyethylene glycol based on an artificial material/hyaluronic acid/RGD peptide as a control group.
The hepatic cell line HepaRG maintained in a Williams E medium containing 10% fetal bovine serum, antibiotics, insulin, hydrocortisone and the hepatic stellate cell line K-HSC prepared in Example 1 maintained in a medium of the same component except containing 2% fetal bovine serum were treated with trypsin. Thereafter, the HepaRG and the K-HSC were added to a solution of PBS and thrombin, and mixed with a solution of fibrinogen (GreenPlast Q®, Green Cross, Korea) of the same volume (diluted with PBS to ½, ⅙, and ⅛, respectively) to finally make 40 μL gel having a concentration of 2×107 cells/mL and a ratio of hepatic cells/hepatic stellate cells of 5:5. Subsequently, the resulting gel was put into an incubator for 15 minutes to perform gelation.
Meanwhile, a hydrogel macromer based on polyethylene glycol (Sigma, USA) having 3 or 4 alkyl group spacers (Sigma, USA) was synthesized at a concentration of 6% in the trypsin-treated hepatic stellate cell line and hepatic cell line, and the gel was mixed with 0.36% of 1.5 MDa hyaluronic acid (Lifecore Biomedical, USA), and a crosslinkable 1 μM/mL RGD peptide (Bachem, USA, JenKem, China) to perform gelation using photopolymerization through ultraviolet irradiation. In this case, Irgacure® 2959 (Sigma, USA) as a photoinitiator was dissolved in a 70% ethanol solution, and then finally added thereto at a concentration of 0.1%, and the gelation was performed. Two slide cover glasses were fixed at a 1-mm interval and 30 μL of gel-cell-polymer solution was added in-between and the both sides were irradiated with ultraviolet rays for 4 minutes and 30 seconds, respectively, and then the hydrogel-based 3D liver analogs were polymerized. The gel thus obtained was transferred to a 24-well dish and cultured for 16 days (confocal microscope imaging,
The K-HSC prepared in Example 1 showed excellent adhesion and network formation ability in the fibrin gel, but showed poor cell adhesion ability in the hybrid hydrogel compared to the LX-2 (
In Example 4, the cells encapsulated in the fibrin gel were divided into two experimental groups while the medium was replaced the next day, and one group was cultured for 14 days in a hepatocyte differentiation medium containing 1.7% DMSO and the other group was cultured for 14 days in a normal medium containing 10% fetal bovine serum, and then in both groups, the media were replaced with normal media without DMSO, and the cells were cultured for three more days.
On the last culture day, the supernatant was collected, and albumin was quantified using the ELISA kit manufactured by Bethyl Laboratories, Inc., the medium was replaced with a medium containing 20 μM rifampicin, and the activity of the CYP3A4 enzyme was induced for 48 hours. The activity of the enzyme was confirmed by measuring the light intensity using a kit (P450-Glo CYP3A4 assay kit, V9002) manufactured by Promega Corporation.
Fibrin gel samples were dissolved using the CellTiter Glo kit manufactured by Promega Corporation, and then the ATP amount of the encapsulated cells was measured, and the affinity of the cells, the activity of the CYP3A4 enzyme, and the albumin production amount were normalized with the measured ATP amount and compared.
In addition, in the ATP analysis showing the cell activity, the hepatic stellate cell line K-HSC prepared in Example 1 did not show a difference from the LX-2, and thus, the fibrin gel affinity of the cells was similar (
The K-HSC prepared in Example 1 was subjected to 3D co-culture in another natural material-based laminin-coated biosilk foam. The hepatic cell line HepaRG and the hepatic stellate cell line were mixed at a ratio of 1:1 at a final concentration of 2×107 cells/mL with a biosilk solution (Biosilk®521, BioLamina, Norway), then applied to wells of 24-well plate in a volume of about 40 μL, and gelated in a 37° C. cell incubator for 20 minutes, a medium was added thereto, and the medium was replaced every 2 days. As a result, the K-HSC exhibited excellent adhesion ability (
In Example 5, for a 3D co-cultured hepatic cell line in the laminin-coated biosilk foam, the cell affinity, CYP3A4 enzyme activity, and albumin secretion ability were measured in the same manner as in Experimental Example 2.
The hepatic cell line co-cultured in the laminin-coated biosilk foam was confirmed to have the affinity of the laminin-coated biosilk foam similar to the LX-2 like in the fibrin gel (
The above results showed that the hepatic stellate cell line of the present invention is a cell line applicable to the hepatocyte co-culture experiment.
To determine whether the K-HSC prepared in Example 1 is applicable as a drug-induced liver fibrosis model, 4%, 5% and 6% macromer concentrations were included to have various initial stiffness. Specifically, a hydrogel precursor based on polyethylene glycol (Sigma, USA) including 3 or 4 alkyl group spacers (Sigma, USA) and 1.5 MDa hyaluronic acid (Lifecore Biomedical, USA) were added at a ratio of 0.24% (4% macromer), 0.3% (5% macromer), and 0.36% (6% macromer), respectively, and finally mixed with a crosslinkable 1 μM/mL RGD peptide (Bachem, USA, JenKem, China), and then Irgacure®2959 (Sigma, USA) dissolved in a 70% ethanol solution was added at a concentration of 0.1% to perform polymerization. Two slide cover glasses were fixed at a 1-mm interval and 30 μL of gel-cell-polymer solution was added in-between and the both sides were irradiated with ultraviolet rays for 4 minutes and 30 seconds, respectively, and then the hydrogel-based 3D liver analogs were polymerized. The K-HSC and the hepatic cell line HepaRG were co-cultured at a ratio of 6:4. As a control group, the LX-2 was co-cultured in the same manner.
Specifically, hepatic stellate cells were encapsulated in the hybrid hydrogel at a concentration of 2×107 cells/mL and a volume of about 30 μL. The hepatic cell line was cultured and differentiated in a DMSO environment for 14 days before the encapsulation, and the differentiated cells were collected and used. Primary human hepatic cells were collected from the liquid nitrogen in a frozen state and used immediately after thawing. After 24 hours of culture, the medium was converted to a serum-free medium. Thereafter, the culture was performed for 3 days, and a cycle of 24-hour drug administration-24-hour serum-free medium was performed 7 times. Experiments were performed in four groups: a control group (cont) to which a drug was not administered; experimental groups to which acetaminophen (apap) and methotrexate (MTX) were administered, respectively; and a positive control group to which TGF-β1 inducing fibrosis was administered. Thereafter, the cells were collected and the expression levels of α-SMA, Col-IA1, LOX, and TIMP-1, which are fibrosis markers, were measured by RT-PCR to confirm the degree of fibrosis progress of the hepatic stellate cells. As a result, referring to
In addition, the expression levels of HNF4α and albumin, which are hepatocyte markers, and the expression level of fibronectin, which is a cell adhesion protein, were quantitatively analyzed by RT-PCR. As a result, the K-HSC prepared in Example 1 was confirmed to be sensitive to drug reaction due to a large difference between the expression of HNF4α indicating the degree of differentiation of hepatic cells and the expression of albumin indicating the function of hepatic cells according to the drug administration, and was confirmed not to have a direct effect on the differentiation of hepatic cells or the albumin production ability (
It was confirmed that the hepatic stellate cell line prepared in Example 1 exhibited a larger cell condensation than the LX-2 cell group, thereby having excellent fibrosis activation (
The K-HSC prepared in Example 1 and the hepatic cell line HepaRG were encapsulated at a ratio of 1:1 in a hybrid hydrogel system simulating the stiffness of normal liver tissue (4.5% macromer) or fibrosis disease liver tissue (8% macromer), and changes in initial fibrosis markers according to whether the hepatic stellate cell line reacts to the change in the stiffness of the support and whether the hepatocyte growth factor (HGF) is administered were confirmed. The LX-2 was used as a control group. The hepatic cell line HepaRG and the hepatic stellate cell line or the LX-2 were mixed at a ratio of 1:1 in hydrogels with different concentrations of each macromer, were fixed with hydrogels at a total cell concentration of 2×107 cells/mL, and was mixed with 1 mM/mL RGD and 0.1% Irgacure® 2959 initiator, followed by photopolymerization for 9 minutes to prepare 3D liver analogs. The 3D liver analogs were cultured in media containing 10% bovine serum for one day, then the media were replaced with a serum-free medium to which 10 ng/mL of HGF was added (control medium) and a medium to which the HGF was not added, and then the replacement was performed once every two days, and the 3D liver analogs were cultured for 8 more days (total 9 days: initial conditions for constructing liver mimics). The 3D liver analogs were recovered and then treated with Trizol (ThermoFisher, USA), followed by RT-PCR analysis.
Referring to
In order to compare liver injury simulation according to the 3D culture of the K-HSC prepared in Example 1, for one 3D culture, the K-HSC and hepatic cell line HepaRG were encapsulated and cultured in a hybrid hydrogel having a concentration of 5% macromer, and for the other, the K-HSC and hepatic cell line HepaRG were cultured in the form of a spheroid using self-association, and as a comparative group, the K-HSC and hepatic cell line HepaRG were cultured in a biodegradable polylactide nanoparticle-hydrogel (NP-hydrogel). As a control group, the LX-2 was co-cultured in the same manner as described above.
Thereafter, methotrexate (MTX) was added for the purpose of simulating liver injury, or TGF-β1 was added for the purpose of inducing liver fibrosis, and then the activity and average expression amount (activity rate of enzyme) of the CYP3A4 enzyme, and albumin production ability were confirmed in the same manner as in Experimental Example 2.
As a result, it was confirmed that both the K-HSC and the LX-2 exhibited low activity of the CYP3A4 enzyme and low albumin production ability when cultured in the form of a spheroid using self-association, and simulated a deterioration in the activity and function according to drug and induction effects when cultured in a hydrogel or a nanoparticle-hydrogel (
In order to compare the affinity with various adhesion proteins in a 3D hydrogel environment, the hydrogel containing the RGD peptide and the LX-2 cell line were used as a control group, the surface adhesion was observed through an optical microscope after the 12-hour 2D-culture in a 5% macromer hybrid hydrogel in which fibronectin and laminin were encapsulated together, and the K-HSC and the LX-2 each were 3D cultured with the hepatic cell line HepaRG for 5 days in a hydrogel in which an adhesion protein was encapsulated, and then observed through a confocal microscope, thereby comparing the network formation of cells.
Referring to
Meanwhile, in order to determine the effect on the function of the hepatocyte according to the adhesion proteins, the hepatic stellate cell line (LX-2 or K-HSC) and the differentiated HepaRG were encapsulated at a ratio of 1:1 in a hydrogel and cultured, thereby comparing the formation of the CYP3A4 enzyme and albumin according to the adhesion proteins. In order to determine the effect on the liver fibrosis simulation according to the adhesion proteins, the expression levels of smooth muscle actin alpha (α-SMA) and type I collagen (Col-1A1) were compared, but all of the liver fibrosis simulation, the hepatocellular function, and the liver injury simulation did not show statistically significant differences even though the adhesion protein types were different (
This shows that the hepatocellular function and liver injury simulation properties of the K-HSC according to the present invention are constant regardless of the type of adhesion protein.
The K-HSC prepared in Example 1 was encapsulated with primary human hepatic cells (PHHs, Corning, USA) at a ratio of 1:1 in 4.5% polyethylene glycol/hyaluronic acid/RGD hydrogel using photopolymerization as described in Example 4. The cells frozen with nitrogen were thawed using recovery media (ThermoFisher, USA) and centrifuged, and then the number of cells were measured, and the cells were encapsulated with the K-HSC in a hydrogel, and then cultured for one day in a primary human hepatocyte plating medium (ThermoFisher, USA) containing 10% bovine serum. The medium was converted to a dedicated serum-free medium for primary human hepatic cells (Corning, USA), after 5 days, TGF-β1 inducing fibrosis, was added for 24 hours at a concentration of 10 ng/mL, and then converted back to a serum-free medium to which the TGF-β1 was not added in the following day, 3 cycles were performed, and the appearance change of the hydrogel was observed after the 24-day culture, and albumin production ability and expression levels of fibrosis markers were measured by RT-PCR.
As a result, the nanoparticle-hydrogel and the hydrogel in which the K-HSC and the primary human hepatic cells were co-cultured did not show any appearance changes in the control groups including or not including TGF-β1 (
The K-HSC prepared in Example 1 and the differentiated hepatic cell line HepaRG were applied at a ratio of hepatic cells/hepatic stellate cells of 6:4 onto the surface of a 48-well plate coated with 1.5% agarose using a serum-free medium based on Williams E medium, and were self-associated in the presence of physical stimulus through the rotary shaking (70 rpm) for a total of 7 days in Williams E medium containing 2% serum for 3 days to form spheroids in the form of 3D culture. In this case, the total cell concentration was 3×105 cells/spheroid. Thereafter, the medium was replaced with a serum-free Williams E medium on day 3 of culture, culture was performed for 4 days, and then TGF-β1, which is a liver fibrosis-inducing factor, was added thereto for 24 hours, and then the cells were exposed again to the medium without growth factor. This was set as one cycle, and the cycle was repeated 5 times (5 cycles), and then the cells were collected, and quantitatively analyzed with RT-PCR. The differentiated hepatic cell line HepaRG was cultured and differentiated in a long-term DMSO environment for 14 days or more before the formation of a 3D spheroid, and the differentiated cells were collected and used, and as a control group, the LX-2 was co-cultured with the differentiated hepatic cell line HepaRG in the same manner.
As a result, the co-culture spheroid composed of the K-HSC and the hepatic cell line HepaRG showed an increased expression level difference according to TGF-β1 introduction in the expression of smooth muscle actin alpha (α-SMA), type I collagen (Col-1A1), TIMP-1, TIMP-2, and fibronectin, which are liver fibrosis markers, compared to the co-culture spheroid composed of the LX-2 and the hepatic cell line HepaRG (
In addition, it was confirmed that the expression of HNF4α, albumin, CYP2B6, and CYP3A4 enzyme, which are markers for the differentiation into hepatic cells, was maintained in a relatively high state in the control groups to which TGF- β1 was not added, and it was confirmed that CK19, which is a marker for the differentiation into cholangiocytes, also showed a high expression level (
Thus, it was confirmed that the K-HSC may simulate the biological environment more closely, and secure more sensitive reactivity in disease modeling through 3D culture.
Meanwhile, the K-HSC was encapsulated with the hepatic cell line HepaRG at a ratio of 1:1 in a 4% hydrogel and then 3D cultured, and the groups were divided into experimental groups to which TGF-β1, which is a fibrosis-inducing material, and inhibitor A thereof were added individually or simultaneously, and a control group to which TGF-β1 and inhibitor A were not added. The groups were cultured for 18 days, and then the cell network formation and expression differences of fibrosis markers in the liver analogs were confirmed.
As a result, there was a significant difference in the formation of the network in the hydrogel after 8 days of culture (
From the above description, those of ordinary skill in the art of the present invention will be understood that the present invention can be carried out in other specific forms without changing the technical idea or essential features. Therefore, the above-disclosed embodiments are to be understood in all aspects as illustrative and not restrictive. Accordingly, the scope of the present invention is defined by the following claims rather than by the detailed description. It shall be understood that all modifications or changes in forms conceived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0186058 | Dec 2020 | KR | national |
This application is a National Stage of International Application No. PCT/KR2021/020054 filed on Dec. 28, 2021, claiming priority based on Korean Patent Application No. 10-2020-0186058 filed on Dec. 29, 2020, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/020054 | 12/28/2021 | WO |
