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Method for production of hepatic-lobule-like cell cluster from adipose-tissue-derived cell

a technology of adiposetissue and adipose tissue, which is applied in the direction of skeletal/connective tissue cells, drug compositions, peptides, etc., can solve the problems of incomplete cure medically impossible, and no report of regenerating hepatic lobule. , to achieve the effect of promoting or inhibiting the formation of hepatic lobules and promotes or inhibi

Inactive Publication Date: 2010-07-22
FOUND FOR BIOMEDICAL RES & INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for obtaining a hepatic-lobule-like cell cluster from an adipose-tissue-derived cell and a hepatic-lobule-like cell cluster obtainable by this method. Additionally, the invention provides methods for screening for substances that promote or inhibit the formation of hepatic lobule and for substances that increase or decrease the activity of hepatic lobule. The invention also provides kits for these screenings. The technical effects of the invention include the ability to obtain a hepatic-lobule-like cell cluster with a high degree of purity and the ability to screen for substances that regulate the formation of hepatic lobule.

Problems solved by technology

Meanwhile, once liver cancer occurs, a complete cure is medically impossible, leaving surgical extraction of the tumor as the number one choice.
However, in reality, since advanced hepatic cirrhosis is to be the stage where liver cancer will occur, only surgical treatments can be selected also for liver failure, and even therapies such as TAE or PEIT cannot be attempted if the case becomes serious.
However, regarding cerebrally dead donor liver transplantation, the absolute insufficiency of donors, and regarding living donor liver transplantation, ethical issues such as safety for the donor, and insertion of a surgical knife into a healthy subject exist.
However, there was no report that a hepatic lobule could be regenerated, which is the smallest functional unit of the liver, that can be used in a treatment.

Method used

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  • Method for production of hepatic-lobule-like cell cluster from adipose-tissue-derived cell
  • Method for production of hepatic-lobule-like cell cluster from adipose-tissue-derived cell
  • Method for production of hepatic-lobule-like cell cluster from adipose-tissue-derived cell

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation and Culture of Adipose Tissue-Derived Cell

[0159]A human adipose tissue was sliced into 2 to 3 mm2-large fragments and digested using collagenase I. The digestate was cultured for 24 to 36 hours in DMEM containing 10% FBS and antibiotics and treated with 0.02% EDTA to obtain adipose tissue-derived cells. The obtained adipose tissue-derived cells were amplified by 3 to 5 passage cultures in a culture medium containing 60% DMEM (low glucose), 40% MCDB201, 1×ITS (10.0 mg / L insulin, 5.5 mg / L transferrin, 6.7 ng sodium selenite), 10 ng / mL rhEGF, 1 nM dexamethasone, 0.1 mM ascorbic acid and 5% FCS (Hyclone), in a fibronectin-coated dish. A micrograph of adipose tissue-derived cells is shown in FIG. 1.

Acquisition of Undifferentiated Cell from Adipose Tissue-Derived Cells

[0160]Cells that were singularized by treating adipose tissue-derived cells treated with 0.25% trypsin / EDTA (Nacalai Tesque) to be dissociated were cultured for 2 to 3 days in knock out DMEM (GIBCO Invitrogen) cont...

example 2

Hepatocyte Gene Expression Characteristics of Hepatic-Lobule-Like Cell Cluster

1. Extraction of RNA

[0162]Extraction of RNA from the hepatic-lobule-like cell cluster was carried out using RNeasy Protect Mini Kit (QIAGEN), as follows. The hepatic-lobule-like cell cluster was recovered, and the buffer RLT containing 10 μl / ml 2-mercaptoethanol (Naacalai Tesqu[13]) was added at a proportion of 600 μl / 107 cell. Cells were homogenized by pipetting with a 20G needle and then 600 μl of 70% ethanol was added. Transferred onto an RNeay[14] Mini column inside a 2 ml collection tube were 700 μl of the obtained mixed solution, which was centrifuged at 1000 rpm for 15 seconds. Next, 350 μl of buffer RW1 was added onto the column and centrifuged at 1000 rpm for 15 seconds. Added to 70 μl of buffer RDD were 10 μl of DNase I stock solution (QIAGEN), which were tumble-mixed, added to the RNeasy silica gel membrane inside the RNeasy Mini column, and incubated at room temperature for 15 minutes. Added wa...

example 3

Production of Hepatic Protein by Hepatic-Lobule-Like Cell Cluster

A. Western Blot Analysis

1. Sample Preparation

[0165]Hepatic-lobule-like cell cluster was washed three times with PBS (Nacalai Tesque), and then M-PER (PIERCE) was added. Cells were lysed by ultrasonication, centrifuged at 14000 g for 15 minutes to eliminate insoluble cell constituents. Sample buffer (Nacalai Tesque) was added in the same amounts as the sample, boiled at 100° C. for 5 minutes and ice-cooled. The protein concentration in the obtained sample was measured using BCA Protein Assay Reagent (PIERCE).

2. SDS-PAGE

[0166]Gel mini plate for electrophoresis (PAG mini “Daiichi”; Daiichi Pure Chemicals Co.) and running buffer were used to perform SDS-PAGE. The amount of protein used was 5 μg. The electrophoresis conditions were 10 mA in the stacking gel and 40 mA in the running gel.

3. Western Blot

[0167]The electrophoresed gel above was washed in blotting buffer for 10 minutes. Next, the proteins in the gel were copied o...

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Abstract

Disclosed are: a method for producing a hepatic-lobule-like cell mass from an adipose-tissue-derived cell, which is characterized by culturing the adipose-tissue-derived cell; a hepatic-lobule-like cell mass produced by the method; a method for the screening of a substance capable of promoting or inhibiting the formation of a hepatic-lobule-like cell mass, which is characterized by culturing an adipose-tissue-derived cell to produce the hepatic-lobule-like cell mass, wherein a candidate substance is added to a culture medium; and a kit for use in the screening method.

Description

TECHNICAL FIELD[0001]The present invention relates to a method for obtaining a hepatic lobule cell population from an adipose-tissue-derived cell, a hepatic lobule cell population obtainable thereby, a method of screening for a substance that promotes or inhibits formation of hepatic lobule and for a substance that causes the activity of a hepatic lobule to increase or decrease, and a kit therefor, and the like.BACKGROUND ART[0002]Compared to other countries, Japan has a high incidence of hepatitis C. That chronic hepatitis, hepatic cirrhosis and then liver cancer occur due to infection by hepatitis C virus is a known fact. Medical therapies mainly centered on interferon are considered to be effective in chronic hepatitis and hepatic cirrhosis states. Meanwhile, once liver cancer occurs, a complete cure is medically impossible, leaving surgical extraction of the tumor as the number one choice. However, in reality, since advanced hepatic cirrhosis is to be the stage where liver cance...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/071A61K35/00A61K35/407C12Q1/02C07K14/00C12N5/0775
CPCC12N5/0667A61P1/16A61P9/00
Inventor MATSUYAMAKOMODA, HIROSHISAWA, YOSHIKI
Owner FOUND FOR BIOMEDICAL RES & INNOVATION
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