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Method of separating small hepatocytes by using specific antibody

A technology of hepatocytes and antibodies, applied in the field of efficient separation of proliferative hepatocytes

Inactive Publication Date: 2011-09-07
LSIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, marker proteins specific to proliferative hepatocytes (small hepatocytes) derived from mammals including humans have not yet been elucidated, so there is still room for improvement in efficient isolation methods

Method used

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  • Method of separating small hepatocytes by using specific antibody
  • Method of separating small hepatocytes by using specific antibody
  • Method of separating small hepatocytes by using specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Preparation of cell fractions rich in small hepatocytes

[0077] Mature rats (8-10 weeks old) were intraperitoneally injected with D-galactosamine (ACROSS) at a concentration of 75mg / 200μl / PBS / 100g body weight. On day 4 after injection, cells were isolated from the liver according to the method of Seglen. Hanks solution supplemented with 0.2 mM EGTA without calcium and magnesium was perfused from the portal vein. After perfusion at a flow rate of 40 ml / min for about 4 minutes, Hanks solution containing 0.02% collagenase (Yakult) was perfused at a flow rate of 20 ml / min for 10 minutes. The hepatocytes were shaken from the digested liver into a beaker according to the prescribed method. Filter the cell suspension with a 250 μm mesh filter and a 70 μm mesh filter, and centrifuge at 50×g for 1 minute. Collect the supernatant and centrifuge at 50 x g for 1 min, repeating 2 times. The supernatant was collected and centrifuged at 50 xg for 5 minutes. The precipi...

Embodiment 2

[0078] Example 2 Expression profile analysis of CD44, D6.1A and BRI3 in small hepatocytes

[0079] The expression profiles of CD44, D6.1A and BRI3 of mature hepatocytes and the mini-hepatocytes prepared in Example 1 were investigated.

[0080] 1) Time-dependent changes in the expression profiles of CD44, D6.1A and BRI3

[0081] Total RNA was prepared from freshly prepared small hepatocytes and small hepatocytes after the initiation of culture, and the expression of CD44 was studied by RT-PCR. GPDH (glycerol-3-phosphate dehydrogenase) was used as a positive control. For the culture of small hepatocytes, the following media were used.

[0082] Dulbecco's Modified Eagle Medium (GIBCO Laboratories)

[0083] +20mM HEPES (Dojindo)

[0084] +25Mm NaHCO 3 (Katayama Chemical Co.)

[0085] +30mg / l Proline (Sigma Chemical Co.)

[0086] +0.5mg / l insulin (Sigma Chemical Co.)

[0087] +10 -7 M Dexamethasone (Sigma Chemical Co.)

[0088]+10% FBS (Hyclo...

Embodiment 3

[0119] Example 3 Isolation of Proliferating Hepatocytes by Anti-CD44 Antibody

[0120] The supernatant obtained by centrifuging at 50×g for 1 minute described in the above i) Preparation method of small hepatocyte-rich cell fraction was collected and centrifuged at 150×g for 5 minutes. The pelleted cells were suspended with the following buffer (phosphate buffered saline (PBS) + 2 mM EDTA (SIGMA Chemical Co.) + 0.5% bovine serum albumin (Serologicals Proteins Inc.)).

[0121] per 1×10 7 Add 80-99 μl of buffer solution to the cells and suspend them, add 1-20 μl of mouse anti-rat CD44 antibody (PharMingene), and incubate at 4-8° C. for 5-10 minutes. Add 10-20 times the volume of buffer, and centrifuge at 300×g for 10 minutes. Suspend the centrifuged pellet with buffer and centrifuge at 300×g for 10 minutes. to each 1 x 10 7 80 μl of buffer was added to the cells to suspend the pellet, and 20 μl of MASC anti-mouse IgG magnetic antibody (Miltenyi Biotec) was added, and incubat...

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Abstract

It is intended to provide a method of efficiently separating proliferative hepatocytes (small hepatocytes) originating in mammals including humans. In particular, it is intended to provide a method of separating small hepatocytes which comprises the following steps: i) the step of contacting a cell suspension containing cells originating in mammalian liver with one or more antibodies selected from the group consisting of an antibody against CD44 (an anti-CD44 antibody), an antibody against D6.1A (an anti-D6.1A antibody) and an antibody against BRI3 (an anti-BRI3 antibody) to form immune complex(es) of the small hepatocytes contained in the cell suspension with the one or more antibodies; and ii) the step of collecting the immune complex(es) containing the small hepatocytes.

Description

technical field [0001] The present invention relates to a method for efficiently isolating proliferative hepatocytes (mini-hepatocytes) from mammals including humans. Background technique [0002] The liver and hepatocytes perform a variety of functions and are known as the chemical factories of the body. For example, more than 90% of serum protein is produced by the liver, which metabolizes harmful substances ingested or produced in the body and has a detoxification function. Therefore, various research institutes are conducting studies to cultivate hepatocytes, use their functions to detect harmful substances (biosensors), and use them to produce substances required by the human body in vitro. Since there is currently no cell line that maintains the function of differentiated hepatocytes, mature hepatocytes must be isolated in each experiment in order to supply hepatocytes for these studies. In this case, the number of cells obtained depends on the number of hepatocytes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07G01N33/48C12M1/34C12N15/09C12N5/071
CPCG01N33/56966C12N5/067
Inventor 三高俊广今纯子
Owner LSIP