Human liver cell line

• US 20060160216A1
• Filed: 01/19/2006
• Published: 07/20/2006
• Est. Priority Date: 09/25/2000
• Status: Active Grant

• Current Assignee: United States Army (U.S. Department Of Defense)
• Original Assignee: United States Army (U.S. Department Of Defense)
• Inventors: Beerheide, Walter, Prachumsri, Jetsumon, Yimamnuaychok, Nongnuch
• Granted Patent: US 7,781,186 B2
• Time in Patent Office: Days
• US Class Current: 435/370
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/62 DMSO
• CPC Class Codes: C12N 2501/30 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C12N2501/85
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/375 Thyroid stimulating hormone [TSH]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

In this application is described the establishment and maintanence of a normal human hepatocyte cell line able to support complete development of malaria parasite development in vitro. Advantages and uses of the cell line are also described.

Claims

1. An immortalized cell line of normal human hepatocytes.

2. The cell line of claim 1 having the ability to support growth of a liver stage malaria parasite.

3. The cell line of claim 2 wherein said malaria parasite is chosen from the group P. falciparum and P. vivax.

4. The cell line of claim 1 wherein said cell line is HC-04 having ATCC accession number PTA-3441.

34. A method for measuring uptake of drugs or chemicals into liver cells, comprising

(i) incubating the cell line of claim 4 with a desired drug wherein said drug or chemical has a detectable label;

(ii) removing said cells and,

(iii) measuring amount of detectable label present in said cells wherein said amount of label is proportional to amount of drug uptake by said cells.

35. A method for measuring metabolism of a drug or chemical in a liver cell, comprising

(i) incubating at least one cell from the cell line of claim 4 with a desired drug or chemical;

(ii) lysing cell to create a homogenate;

(iii) analyzing homogenate for the presence of drug metabolites.

36. The method of claim 35 wherein said incubating in (i) is in the presence of an inhibitor of said drug.

37. The method of claim 35 wherein said incubating in (i) is in the presence of an enhancer of said drug.

38. A method for determining presence of reactive metabolites of drug or chemical, comprising,

(i) incubating at lease one cell from the cell line of claim 4 with said drug or chemical wherein said drug or chemical has a detectable label;

(ii) isolating proteins from said cell; and

,

(iii) measuring protein-bound label wherein presence of protein-bound label indicates presence of reactive metabolites.

39. A method for measuring toxicity of a drug or chemical in a liver cell, comprising

(i) incubating at least one cell from the cell line of claim 4 with said drug or chemical in culture medium;

(ii) isolating culture medium, and

(iii) measuring amount of lactate dehydrogenase in culture medium wherein level of lactate dehydrogenase indicates level of toxicity of drug or chemical on cell.

40. A method for measuring liver cell mitochondria-mediated induction of toxicity of a drug or chemical, comprising

(i) incubating at least one cell from the cell line of claim 4 with said drug or chemical in culture medium;

(ii) isolating culture medium, and

(iii) measuring amount of cytochrome c in said culture medium wherein presence of cytochrom c in culture medium indicates mitochondria-mediated induction of drug toxicity.

41. A method for measuring drug-drug interactions in a liver cell, comprising

(i) incubating at least one cell from the cell line of claim 4 with a drug A together with a drug B in culture medium;

(ii) harvesting cell and culture medium, and

(iii) measuring a decrease in drug A wherein a decrease in drug A indicates an inhibition of drug A by drug B.

42. The method of claim 41, wherein said measuring in (iii) is for an incease in metabolites of drug A, wherein a decrease in metabolites indicates inhibition of drug A by drug B.

43. The method of claim 41 wherein drug B is added after incubating cells with drug A.

5. A method of making an immortalized cell line of normal human hepatocytes comprising:

isolating a suspension of normal human hepatocytes,

and plating said hepatocytes.

6. The method of claim 5 further comprising the step of growing the cells in medium.

7. The method of claim 6 wherein said medium comprises MEM+F12 medium supplemented with 10% FBS, Insulin (10 μ

g/ml), Hydrocortizone (3.625 mg/ml.), Linoleic acid-BSA, Nicotinamin (0.122 g/ml.), TRF (3.625 mg./ml), Glucagon (34.828 mg/ml), L-EGF (10 μ

g/ml), Selenous acid (1.29 mtg/ml), 20% Lactabumin and penicillin (5 unit/ml)-streptomycin (5 ug/ml).

8. A method for maintaining an immortalized cell line of normal human hepatocytes comprising:

passaging the cells in a medium suitable for said hepatocytes, and

culturing said hepatocytes.

9. A method for developing liver stage malaria parasites in vitro, said method comprising

infecting an immortalized normal human hepatocyte cell line with sporozoites of said parasites.

11. The method of claim 9 wherein said cell line is HC-04.

14. The method of claim 9 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

15. The method of claim 11 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

10. A method for completely developing liver stage malaria parasites in vitro, said method comprising

infecting an immortalized normal human hepatocyte cell line with sporozoites of said parasites;

and

incubating the cell with the parasites in the presence of erythrocytes.

12. The method of claim 10 wherein said cell line is HC-04.

13. The method of claim 10 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

16. The method of claim 12 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

17. A method for producing cDNA of a developmental stage of malaria parasites comprising:

infecting an immortalized normal human hepatocyte cell with liver stage sporozoites of said parasites;

detecting the presence of the desired developmental stage; and

,

preparing cDNA from RNA expressed by the desired parasite stage.

18. The method according to claim 17 wherein said hepatocyte cell is HC-04.

19. The method according to claim 17 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

20. The method according to claim 18 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

21. A method for producing cDNA of an infected hepatocyte, said hepatocyte infected with a liver stage malaria parasite comprising:

infecting an immortalized normal human hepatocyte cell in vitro with sporozoites of said parasite;

detecting the presence of the desired developmental stage of the parasite in an infected liver cell; and

,

preparing cDNA from mRNA expressed by said infected liver cell.

22. The method according to claim 21 wherein said

hepatocyte is from the cell line HC-04.

23. The method of claim 22 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

24. The method of claim 21 wherein said parasite is chosen from the group consisting of P. falciparum and P. vivax.

25. A method for screening a compound affecting malaria parasite development comprising

incubating components comprising the compound and at least one immortalized normal human hepatocyte which has been infected with the parasite under conditions sufficient to allow the compound and cell to interact;

and

determining the effect of the compound on the parasite.

26. The method of claim 25 wherein said immortalized normal human hepatocyte is from HC-04.

27. The method of claim 26 wherein said malaria parasite is P. falciparum or P. vivax.

28. A method for screening a compound affecting a liver cell infected with a malaria parasite:

incubating components comprising the compound and at least one immortalized normal human hepatocyte infected with the parasite under conditions sufficient to allow the compound and cell to interact; and

determining the effect of the compound on the cell.

29. The method of claim 28 wherein said immortalized normal human hepatocyte is from HC-04.

30. The method of claim 29 wherein said malaria parasite is P. falciparum or P. vivax.

31. An immortalized normal human hepatocyte infected with a liver stage parasite.

32. The hepatocyte of claim 31 wherein said hepatocyte is from HC-04.

33. The hepatocyte of claim 32 wherein said parasite is P. falciparum or P. vivax.