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Method for isolating hepatocytes

A technology of hepatocytes and cells, applied in the field of separation of hepatocytes, can solve problems such as rejection reaction

Inactive Publication Date: 2006-11-15
NEWSOUTH INNOVATIONS PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of using xenogeneic hepatocytes in humans is the potential for rejection

Method used

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  • Method for isolating hepatocytes
  • Method for isolating hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Collection of hepatocytes after hepatectomy

[0059] Hepatocytes from 5 patients who underwent liver resection due to liver metastases. The study was approved by the Ethics Committee of St George's Hospital, New South Wales, Australia (approval number 01 / 123). Details of the localization and resection of metastatic tumors in these patients are shown in Table 1.

[0060] patient

(gender)

primary tumor 1

liver resection day

Expect

resected segment

tumor size

(cm)

1(female)

CRC 2 -Mar 01

April 2003

4

4×3×2

2(male)

CRC-Nov 00

May 2002

2, 3 & 4

(Collect 2+

3)

4×4×2

3 (female)

CRC-Nov 00

April 2002

2， 3

2×2×1.5

4(male)

CRC-Apr 01

May 2002

2, 3 & 7

(Collect 2+

3)

2×2.5×2

4.5×2.7×

2

5(male)

Pancreatic Cancer - Apr 01

May 2002

5， 6 ...

Embodiment 2

[0066] Example 2: Isolation of tumor-free hepatocytes

[0067] After obtaining viable hepatocytes (Example 1), the hepatocytes were isolated from the accompanying tumor cells. Tumor cells were isolated by the immunomagnetic separation method described by Flatmark et al. (Clinical Cancer Research 8:444-449, 2002) using 4.5 μm superparamagnetic beads (Dynabeads M450; Dynal, Oslo, Norway) coated with MOC31 monoclonal antibody. MOC31 recognizes the Ep-CAM antigen, which is present on the surface of most epithelial cells and is particularly highly expressed in colorectal cancer.

[0068] 5 million hepatocytes were mixed with 1 million HT29 colorectal cells in 1 ml of phosphate buffered saline (PBS). 200 μl of Dynabeads M450 were suspended in 1 ml of PBS, and 20 μl of MOC31 antibody was added. The suspension was incubated at 4°C for 30 minutes before adding the MOC31-coated Dynabeads mixture to the tube containing the hepatocytes plus HT29 cell mixture to a total volume of 2 ml. ...

Embodiment 3

[0069] Example 3: Validation of Isolation of Tumor-Free Hepatocytes

[0070] After tumor cells were removed using MOC31-coated immunomagnetic beads (Example 2), the prepared hepatocytes were analyzed for residual tumor cells by detecting the expression of the epithelial cell adhesion molecule (Ep-CAM) gene using RT-PCR . Ep-CAM is a useful cell surface marker expressed on the surface of most epithelial and tumor cells including HT29. The sensitivity of RT-PCR in tumor cell detection based on Ep-CAM gene expression is about 10 tumor cells / 10 7 non-tumor cells (Sakaguchi, M et al., Brit J Cancer 79:416-422, 1999).

[0071] The following primers (primer) were used for RT-PCR analysis:

[0072] Sense strand: 5'-GAACAATGATGGGCTTTATGA-3'

[0073] Nonsense strand (antisense strand): 5'-TGAGAATTCAGGTGCTTTTT-3'

[0074] Successful PCR amplification of Ep-CAM using these primers generated a 515 bp product.

[0075] Hepatocytes were obtained as described in Example 1. Then, hepato...

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Abstract

The present invention provides a method for isolating normal hepatocytes, the method comprising the steps of: recovering liver tissue from a patient during a hepatectomy; and isolating normal hepatocytes from unwanted cells present in the recovered tissue by magnetic separation. The invention further provides a method of preparing hepatocytes for transplantation, the method comprising the steps of: recovering liver tissue from a patient during a hepatectomy; and isolating normal hepatocytes from unwanted cells present in the recovered tissue by magnetic separation.

Description

technical field [0001] The present invention generally relates to methods of isolating hepatocytes suitable for use in the treatment of patients suffering from liver disease. The invention further relates to hepatocytes isolated by the method of the invention and methods of using the hepatocytes isolated by the method of the invention for the treatment of liver diseases. Background technique [0002] Orthotopic liver transplantation is currently the best treatment for various liver diseases including acute and chronic liver failure. However, a limiting factor in liver transplantation is the availability of donor tissue. There is a shortage of transplanted organs worldwide. In some cases, this has resulted in approximately 10% mortality on the liver transplant waiting list (Gibbons, RD et al., Biostatistics 4:207-222, 2003). Other factors limiting the widespread use of liver transplantation include the cost of transplantation and the potential for transplant rejection. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/12C12N5/00C12N5/071G01N33/483G01N33/50G01N33/543
CPCA61K35/12C12N5/067G01N33/54326A61P1/16A61P11/00A61P35/00A61P3/06A61P7/00C12N5/0602C12Q1/24G01N33/53
Inventor D·L·莫里斯
Owner NEWSOUTH INNOVATIONS PTY LTD
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