Immortalized hepatocytes

• US 5,869,243 A
• Filed: 03/05/1996
• Issued: 02/09/1999
• Est. Priority Date: 03/05/1996
• Status: Expired due to Term

• Current Assignee: Rhode Island Hospital
• Original Assignee: Rhode Island Hospital
• Inventors: Liu, Jin, Jauregui, Hugo O.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US08/611,171
• Time in Patent Office: 1,071 Days
• Field of Search: 435/6, 435/325, 623/12
• US Class Current: 435/6.16
• CPC Class Codes: A61F 2/022 Artificial gland structures using bioreactors hemodialysis A61M1/16; hemofiltration A61M1/34; other extracorporeal blood treatment A61M1/36
• CPC Class Codes: A61M 1/3679 by absorption A61M1/3675 takes precedence
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: G01N 33/5014 for testing toxicity

Abstract

The invention features a virally-immortalized mammalian hepatocyte, which is derived from a normal liver cell, has differentiated hepatocyte-specific metabolic activity, has the ability to proliferate, and is nontumorigenic after prolonged culture.

Claims

1. A virally-immortalized porcine hepatocyte, said hepatocyte

(a) being derived from a normal liver cell;

(b) having retained differentiated hepatocyte-specific metabolic activity; and

(c) having the ability to proliferate, wherein said hepatocyte is nontumorigenic after prolonged culture.

2. The hepatocyte of claim 1, wherein said metabolic activity comprises P450 enzyme activity.

3. The hepatocyte of claim 2, wherein said P450 enzyme activity is increased by contacting said hepatocyte with a metabolic inducer.

4. The hepatocyte of claim 1, wherein said hepatocyte comprises substantially pure simian virus 40 (SV40) DNA.

5. The hepatocyte of claim 4, wherein said DNA encodes wild type SV40 large T antigen (TAg).

6. The hepatocyte of claim 5, wherein expression of said TAg is constitutive.

7. The hepatocyte of claim 6, wherein said hepatocyte further comprises a substantially pure tumor suppressor-encoding DNA.

8. The hepatocyte of claim 4, wherein said DNA encodes a temperature-sensitive TAg.

9. The hepatocyte of claim 8, wherein said hepatocyte further comprises a substantially pure tumor suppressor-encoding DNA.

10. The hepatocyte of claim 7 or 9, wherein said hepatocyte comprises a substantially pure human p53-encoding DNA.

11. The hepatocyte of claim 5, wherein expression of said TAg is inducible.

12. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing the hepatocyte of claim 1;

(b) contacting said hepatocyte with said compound; and

(c) measuring the viability of said hepatocyte, wherein a decrease in viability in the presence of said compound compared to that in the absence of said compound indicates that said compound is toxic in vivo.

13. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing the hepatocyte of claim 1;

(b) contacting said hepatocyte with said compound; and

(c) measuring the metabolic activity of said hepatocyte, wherein a decrease in metabolic activity in the presence of said compound compared to that in the absence of said compound indicates that said compound is toxic in vivo.

14. A method of neutralizing a toxic compound in a bodily fluid of a mammal, comprising contacting said fluid with the hepatocyte of claim 1 in vitro.

15. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing a metabolically-active hepatocyte;

(b) contacting said metabolically-active hepatocyte with said compound to generate a cell supernatant;

(c) removing said cell supernatant from said metabolically-active hepatocyte;

(d) providing the hepatocyte of claim 1;

(e) contacting said hepatocyte with said supernatant; and

(f) measuring the metabolic activity of said hepatocyte, wherein a decrease in metabolic activity in the presence of said supernatant compared to that in the absence of said supernatant indicates that said compound is toxic in vivo.

16. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing a metabolically-active hepatocyte;

(b) contacting said metabolically-active hepatocyte with said compound to generate a cell supernatant;

(c) removing said cell supernatant from said metabolically-active hepatocyte;

(d) providing the hepatocyte of claim 1;

(e) contacting said hepatocyte with said supernatant; and

(f) measuring the viability of said hepatocyte, wherein a decrease in viability in the presence of said supernatant compared to that in the absence of said supernatant indicates that said compound is toxic in vivo.

17. A method of neutralizing a toxic compound in a bodily fluid of a mammal, comprising contacting said fluid with the hepatocyte of claim 1, wherein said contacting step takes place in a perfusion device.

18. The method of claim 17, wherein said mammal is a human.

19. The method of claim 17, wherein said perfusion device is a liver assist device.

20. The method of claim 17, wherein said perfusion device is a bioreactor.

21. A perfusion device comprising

(a) a housing defining a perfusion inlet and a perfusion outlet;

(b) a porous membrane structure mounted within said housing to define a perfusion compartment and an adjacent hepatocyte compartment; and

(c) virally-immortalized porcine hepatocytes, said hepatocytes (1) being derived from a normal liver cell, (2) having differentiated hepatocyte-specific metabolic activity, and (3) having the ability to proliferate, wherein said hepatocyte is nontumorigenic after prolonged culture.

22. A perfusion device comprising

(a) a housing defining a perfusion inlet and a perfusion outlet;

(b) a porous membrane structure mounted within said housing to define a perfusion compartment and an adjacent hepatocyte compartment; and

(c) virally-immortalized mammalian hepatocytes, said hepatocytes (1) being derived from a normal liver cell, (2) having differentiated hepatocyte-specific metabolic activity, (3) comprising substantially pure DNA encoding wild type SV40 TAg, wherein expression of said TAg is inducible; and

(4) having the ability to proliferate, wherein said hepatocyte is nontumorigenic after prolonged culture.

26. The hepatocyte of claim 22, wherein said DNA is operably linked to an inducible promoter.

27. The hepatocyte of claim 26, wherein said promoter is not derived from SV40.

23. A virally-immortalized mammalian hepatocyte, said hepatocyte

(a) being derived from a normal liver cell;

(b) having retained differentiated hepatocyte-specific metabolic activity;

(c) comprising substantially pure DNA encoding wild type SV40 TAg, wherein expression of said TAg is inducible; and

(d) having the ability to proliferate, wherein said hepatocyte is nontumorigenic after prolonged culture.

24. The hepatocyte of claim 23, wherein said hepatocyte further comprises a substantially pure tumor suppressor-encoding DNA.

25. The hepatocyte of claim 24, wherein said DNA encodes human p53.

28. The hepatocyte of claim 23, wherein said expression is induced by dexamethasone.

29. A method of neutralizing a toxic compound in a bodily fluid of a mammal, comprising contacting said fluid with the hepatocyte of claim 23 in vitro.

30. The method of claim 29, wherein said mammal is a human.

31. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing a metabolically-active hepatocyte;

(b) contacting said metabolically-active hepatocyte with said compound to generate a cell supernatant;

(c) removing said cell supernatant from said metabolically-active hepatocyte;

(d) providing the hepatocyte of claim 23;

(e) contacting said hepatocyte with said supernatant; and

(f) measuring the viability of said hepatocyte, wherein a decrease in viability in the presence of said supernatant compared to that in the absence of said supernatant indicates that said compound is toxic in vivo.

32. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing the hepatocyte of claim 23;

(b) contacting said hepatocyte with said compound; and

(c) measuring the viability of said hepatocyte, wherein a decrease in viability in the presence of said compound compared to that in the absence of said compound indicates that said compound is toxic in vivo.

33. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing the hepatocyte of claim 23;

(b) contacting said hepatocyte with said compound; and

(c) measuring the metabolic activity of said hepatocyte, wherein a decrease in metabolic activity in the presence of said compound compared to that in the absence of said compound indicates that said compound is toxic in vivo.

34. A method of evaluating the toxicity of a compound in vitro, comprising

(a) providing a metabolically-active hepatocyte;

(b) contacting said metabolically-active hepatocyte with said compound to generate a cell supernatant;

(c) removing said cell supernatant from said metabolically-active hepatocyte;

(d) providing the hepatocyte of claim 23;

(e) contacting said hepatocyte with said supernatant; and

(f) measuring the metabolic activity of said hepatocyte, wherein a decrease in metabolic activity in the presence of said supernatant compared to that in the absence of said supernatant indicates that said compound is toxic in vivo.

35. A method of neutralizing a toxic compound in a bodily fluid of a mammal, comprising contacting said fluid with the hepatocyte of claim 23, wherein said contacting step takes place in a perfusion device.

36. The method of claim 35, wherein said perfusion device is a liver assist device.

37. The method of claim 35, wherein said mammal is a human.

38. The method of claim 35, wherein said perfusion device is a bioreactor.