# US 7,745,120 B2 · Cell culture and method for screening for a compound useful in the treatment or prevention of hepatic cirrhosis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,120 B2 - Document ID: US7745120B2 - Status: Granted - Kind: B2 - Application number: 11/298,586 - Filed: 2005-12-12 - Published: 2006-08-03 - Granted: 2010-06-29 - Earliest claimed: 2004-12-28 - Assignees: Industrial Technology Research Institute - Inventors: Wen-Tyng Li; Rung-Jiun Gau; Yu-Shih Weng; Cheng-Ta Hsieh; Pei-Shan Li - Web page: https://patentgrep.com/patent/US7745120B2 ## CPC - G01N 33/5067: {Liver cells} - G01N 33/5023: {on expression patterns} - G01N 2800/085: Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention provides a cell-culturing model and a method for screening compounds which can be applied in treating or preventing hepatic cirrhosis. The cell culturing model comprises a population of hepatocytes and hepatic stellate cells (HSCs) derived from co-culturing, and at least one population of the cells comprises a nucleotide sequence fragment of a reporter gene and a cell specific regulatory sequence. The cell-culturing model of the present invention can be applied in high throughput screening for effective compounds of medication, and also in understanding the functional mechanism of the effective compounds. ## Claims ### Claim 1 A cell culturing model comprising co-cultured human hepatocytes and human hepatic stellate cells (HSCs) having a cell specific regulatory sequence operatively linked to a heterologous reporter gene nucleotide sequence, and wherein said cell specific regulatory sequence is a human hepatic stellate cell specific promoter comprising a collagen type 1 promoter having the nucleotide sequences selected from the group consisting of SEQ ID NO. 14 and SEQ ID NO. 15. ### Claim 2 (depends on 1) The cell culturing model of claim 1, wherein said human hepatocytes and human HSCs are co-cultured with a culture medium and a culture material which is coated with an extracellular matrix. ### Claim 3 (depends on 2) The cell culturing model of claim 2, wherein said extracellular matrix is collagen or Matrigel. ### Claim 4 (depends on 1) The cell culturing model of claim 1, wherein a ratio of initial concentration of said human hepatocytes and human HSCs is 1:10. ### Claim 5 (depends on 1) The cell culturing model of claim 1, wherein said co-cultured human hepatocytes and human HSCs are mixed co-cultured or separated co-cultured. ### Claim 6 (depends on 1) The cell culturing model of claim 1, wherein the nucleotide sequence is SEQ ID NO. 14. ### Claim 7 (depends on 1) The cell culturing model of claim 1, wherein the nucleotide sequence is SEQ ID NO. 15. ### Claim 8 (depends on 1) The cell culturing model of claim 1, wherein said reporter gene encodes β-galactosidase, luciferase or a fluorescence protein. ### Claim 9 (depends on 1) The cell culturing model of claim 1, wherein said reporter gene encodes a fluorescence protein. ### Claim 10 (depends on 9) The cell culturing model of claim 9, wherein said fluorescence protein is enhanced green fluorescence protein (EGFP). ### Claim 11 (depends on 9) The cell culturing model of claim 9, wherein said fluorescence protein is Discosoma sp. red fluorescent protein (DsRed). ### Claim 12 (depends on 1) The cell culturing model of claim 1, wherein the cells having a cell specific regulatory sequence operatively linked to a heterologous reporter gene nucleotide sequence further comprise a nucleotide sequence fragment of an antibiotic resistance gene. ### Claim 13 (depends on 12) The cell culturing model of claim 12, wherein said antibiotic resistance gene is a hygromycin resistance gene or neomycin resistance gene. ### Claim 14 (depends on 1) The cell culturing model of claim 1, wherein said cell specific regulatory sequence and said reporter gene are delivered into said cells by a liposome-mediated method. ### Claim 15 (depends on 1) The cell culturing model of claim 1, wherein the cells having a cell specific regulatory sequence operatively linked to a heterologous reporter gene nucleotide sequence further comprise a nucleotide sequence of immortalization gene to immortalize the cells. ### Claim 16 (depends on 15) The cell culturing model of claim 15, wherein said immortalization gene is Human telomerase reverse transcriptase catalytic subunit (hTERT). ### Claim 17 (depends on 15) The cell culturing model of claim 15, wherein said nucleotide sequence of immortalization gene is delivered to said cells by a liposome-mediated method. ### Claim 18 A cell culturing model comprising co-cultured human hepatocytes and human hepatic stellate cells (HSCS) a cell specific regulatory sequence and a nucleotide sequence fragment of a reporter gene, wherein the cell specific regulatory sequence is a hepatocyte specific promoter or a hepatic stellate cell specific promoter having a sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15 and the nucleotide fragment of the reporter gene is a nucleotide sequence that encodes for a product selected from the group consisting of β-galactosidase, chloramphenicol acetyl transferase (CAT), luciferase and a fluorescence protein. ### Claim 19 (depends on 18) The cell culturing model according to claim 18, wherein the nucleotide sequence of said promoter is SEQ ID NO. 14. ### Claim 20 (depends on 18) The cell culturing model according to claim 18, wherein the nucleotide sequence of said promoter is SEQ ID NO. 15.