Isolation and culture of porcine hepatocytes

• US 6,017,760 A
• Filed: 10/10/1995
• Issued: 01/25/2000
• Est. Priority Date: 10/10/1995
• Status: Expired due to Term

• Current Assignee: Rhode Island Hospital
• Original Assignee: Rhode Island Hospital
• Inventors: Trenkler, Donna M., Naik, Sharda, Santangini, Henry, Jauregui, Hugo O.
• Primary Examiner(s): Smith, Lynette F.
• Assistant Examiner(s): Zeman, Mary K
• Application Number:
  US08/541,462
• Time in Patent Office: 1,568 Days
• Field of Search: 435/240, 435/240.2, 435/240.31, 435/240.242, 435/240.241, 435/240.243, 435/240.23, 435/1.1, 435/29, 435/174, 435/325, 435/378, 435/379, 435/384 , 435/389, 435/394, 435/395, 435/399, 435/400, 435/401, 435/402
• US Class Current: 435/378
• 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: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2503/04 Screening or testing on artificial tissues
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

A perfusion device such as a liver assist device containing a housing defining a perfusion inlet and a perfusion outlet, a porous membrane structure mounted within said housing to define a perfusion compartment and an adjacent hepatocyte compartment, and porcine hepatocytes isolated from a porcine liver by retrograde perfusion.

Claims

1. An isolated sample of primary porcine hepatocytes, comprising at least 9% pericentral cells, wherein said cells retain metabolic activity for at least 24 hours in vitro.

2. The sample of claim 1, wherein said sample comprises at least 10% pericentral cells.

3. The sample of claim 1, wherein said sample is characterized by:

(a) diazepam metabolic activity of at least 3 μ

g/ml total diazempam metabolites per hour;

(b) 7-ethoxycoumarin metabolic activity of at least 0.25 μ

g/ml 7-hydroxycoumarin per hour;

(c) acetaminophen metabolic activity of at least 10 μ

g/ml acetaminophen glucuronide per hour;

(d) NH₃ metabolism of at least 45 μ

g/ml NH₃ per hour;

or

(e) proliferative activity for at least 24 hours of in vitro culture.

4. The sample of claim 3, wherein said sample is characterized by:

(a) diazepam metabolic activity of at least 8 μ

g/ml total diazempam metabolites per hour;

(b) 7-ethoxycoumarin metabolic activity of at least 1.5 μ

g/ml 7-hydroxycoumarin per hour;

(c) acetaminophen metabolic activity of at least 25 μ

g/ml acetaminophen glucuronide per hour;

(d) NH₃ metabolism of at 60 μ

g/ml of NH₃ per hour;

or

(e) proliferative activity for at least 10 days of in vitro culture.

23. The sample of claim 1, wherein said cells retain metabolic activity for at least 3 days in vitro.

24. The sample of claim 23, wherein said cells retain metabolic activity for at least 4 days in vitro.

25. The sample of claim 24, wherein said cells retain metabolic activity for at least 6 days in vitro.

26. The sample of claim 25, wherein said cells retain metabolic activity for at least 10 days in vitro.

5. A method of isolating a sample of primary porcine hepatocytes enriched for pericentral cells, comprising

(a) providing a pig;

(b) perfusing a liver from said pig in a retrograde manner with a solution that is free of digitonin;

(c) removing said liver from said pig;

(d) retrieving said hepatocytes from said liver; and

(e) separating said hepatocytes from cellular debris and non-parenchymal cells to yield said sample of hepatocytes, said sample comprising at least 9% pericentral cells, wherein said hepatocytes retain metabolic activity for at least 24 hours in vitro.

6. The method of claim 5, wherein said porcine liver is perfused with a buffered collagenase solution.

7. The method of claim 5, wherein step (b) is carried out in situ.

8. The method of claim 5, wherein said pig weighs at least 15 pounds.

9. The method of claim 8, wherein said pig weighs between 20-35 pounds.

10. The method of claim 5, wherein said pig is at least 6 weeks of age.

11. The method of claim 10, wherein said pig is between 6-8 weeks of age.

12. A method of enhancing viability and metabolic activity of porcine hepatocytes in vitro comprising:

(a) providing a sample of porcine hepatocytes in a suspension of particles; and

(b) culturing said suspension in a roller bottle, wherein said hepatocytes and said particles form aggregates which adhere to the wall of said roller bottle and wherein said hepatocytes proliferate for at least 24 hours of in vitro culture and are at least 2 times more metabolically active than a second sample of porcine hepatocytes cultured in a monolayer.

13. The method of claim 12, wherein said particles are microcarrier beads.

14. The method of claim 12, wherein said hepatocytes proliferate for at least 10 days of in vitro culture and metabolize at least 20 times more of a toxic compound than a second sample of porcine hepatocytes cultured in a monolayer.

15. The method of claim 14, wherein said compound is diazepam.

16. The method of claim 12, wherein said suspension comprises a medium comprising at least 5% fetal bovine serum.

17. The method of claim 12, wherein said suspension comprises a serum-free medium.

18. The method of claim 12, wherein said suspension comprises a medium comprising at least 1% dimethyl sulfoxide.

19. The method of claim 18, wherein said medium comprises insulin.

20. The method of claim 18, wherein said medium comprises transferrin.

21. The method of claim 18, wherein said medium comprises selenium.

22. The method of claim 18, wherein said medium comprises insulin, transferrin, selenium, and at least 2% DMSO.