Method of expanding human hepatocytes in vivo

A technology of hepatocytes and liver, applied in the field of hepatocyte expansion

Inactive Publication Date: 2012-05-16
OREGON HEALTH & SCI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The availability of high-quality human hepatocytes is further hampered by the fact that they cannot be expanded in large numbers in tissue culture (Runge et al., Biochem. Biophys. Res. Commun. 274:1-3, 2000; Cascio S.M., Artif. Organs 25:529-538, 2001)

Method used

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  • Method of expanding human hepatocytes in vivo
  • Method of expanding human hepatocytes in vivo
  • Method of expanding human hepatocytes in vivo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0284] Example 1: Fah - / - / Rag2 - / - / Il2rg - / - Generation of (FRG) mice

[0285] A number of different strategies can be applied to generate immunodeficient mice, including, for example, by administering immunosuppressive drugs or by introducing one or more genetic alterations. This example describes the generation of genetically altered immunodeficient mice.

[0286] To generate immunodeficient Fah that completely lacks T cells, B cells, and NK cells, but does not have DNA repair defects - / - mouse strain that produced the Fah - / - / Rag2 - / - / Il2rg - / - (FRG) mice. Will the male Fah - / - 129S4 mice (Grompe et al., Genes Dev. 7:2298-2307, 1993) with female Rag2 - / - / Il2rg - / - Mice (Taconic) were crossed. All animals were maintained with drinking water containing 2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (NTBC) at a concentration of 1.6 mg / L (Grompe et al ., Nat. Genet. 70:453-460, 1995). To confirm the genotype of each animal, PCR-based genotyping was...

Embodiment 2

[0288] Example 2: Histology and Engraftment Detection Analysis

[0289] Histology and Immunocytochemistry

[0290] FAH immunohistochemistry was performed as previously described (Wang et al., Am. J. Pathol. 161:565-574, 2002). Briefly, liver and kidney tissues fixed in 10% phosphate-buffered formalin at pH 7.4, dehydrated in 100% ethanol, and embedded in paraffin at 58 °C. Deparaffinized 4 μm sections were stained with hematoxylin and eosin. For immunohistochemistry, wash sections with 3% H 2 o 2 treated with methanol solution to block endogenous peroxidase. Prior to incubation with the primary antibody, avidin and biotin blocking was also performed. Sections were incubated with anti-FAH rabbit antibody or HepPar antibody (DAKO) for 2 hours at room temperature. Then incubated with HRP-conjugated secondary antibody. Signals were detected by diaminobenzidine (DAB).

[0291] FAH enzyme assay

[0292] Fumaryl acetoacetate was incubated with the cytoplasmic liver fraction ...

Embodiment 3

[0308] Example 3: Isolation and Cryopreservation of Human Hepatocytes

[0309] Human hepatocytes were isolated from donor livers not used for liver transplantation following a previously described procedure (Strom et al., Cell Transplant. 15:S 105-110, 2006). Briefly, liver tissue was perfused with Hanks' balanced salt solution (Cambrex) without calcium and magnesium supplemented with 0.5 mM EGTA (Sigma) and HEPES (Cellgro), and then treated with Eagle's minimal essential medium (Cambrex). 100mg / L collagenase XI (Sigma) and 50mg / L deoxyribonuclease I (Sigma) were digested through the existing vasculature. The cells were washed 3 times with Eagle's minimal essential medium supplemented with 7% bovine serum (Hyclone) at 50 x g for 2 minutes each. Transfer pelleted hepatocytes to cold VIASPAN TM Medium (universal active pulse wash and cold storage solution for preservation of intra-abdominal organs; also known as University of Wisconsin solution or UW).

[0310] Transport the ...

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Abstract

Described herein is a method of expanding human hepatocytes in vivo using an immunodeficient mouse which is further deficient in fumarylacetoacetate hydrolase (Fah). The method comprises transplanting human hepatocytes into the immunodeficient and Fan-deficient mice, administering an IL-IR antagonist to the mouse and allowing the hepatocytes to expand. Alternatively, the method includes transplanting human hepatocytes into the immunodeficient and Fah-deficient mice, wherein the mouse is further deficient for IL-IR and allowing the hepatocytes to expand. The method also allows serial transplantation of the human hepatocytes into secondary, tertiary, quaternary or additional mice.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 61 / 296,774, filed January 20, 2010, and U.S. Provisional Application No. 61 / 174,791, filed May 1, 2009, which are hereby incorporated by reference in their entirety incorporated into this article. technical field [0003] The present disclosure relates to a method for expanding hepatocytes, such as human hepatocytes, in particular to a method for expanding hepatocytes using immunodeficient and Fah-deficient mice. [0004] Thanks for government support [0005] This invention was made with government support under Grant No. DK051592 awarded by the National Institutes of Health. The US Government has certain rights in this invention. Background technique [0006] The liver is an important site for the metabolism of xenobiotic compounds, including medicinal drugs. Because many liver enzymes are species-specific, it is necessary ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50C12N5/071
CPCA01K2217/075A01K67/0271A01K67/0276A01K2227/105A01K2267/035
Inventor M·葛洛佩H·兰
Owner OREGON HEALTH & SCI UNIV
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