JP3186880B2 - Hepatocyte isolation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an animal (excluding humans)
The same as below) .

[0002]

2. Description of the Related Art In the study of a variety of specific functions of the liver, attention has been paid to a means of using primary cultured cells of liver parenchymal cells. For cell culture, it is essential to separate hepatocytes.

[0003] Hepatocytes are mainly composed of an adhesion factor which depends on a metal ion between cells and an intercellular matrix to constitute a liver. As a method for separating hepatocytes, a method of digesting an intercellular matrix by in situ perfusion of collagenase is mainly used. In this method, animal liver is first preperfused with a buffer solution, and then perfused with collagenase to gently separate hepatocytes. Using a perfusion solution containing EGTA etc. to remove metal ion-dependent adhesion factors during preperfusion,
Combinations of chemical separations have also been performed.

However, when hepatocytes are separated by the perfusion method as described above, the survival rate of the obtained cells is not constant,
It is difficult to isolate cells with high yield and stability. This also affects the reliability of experiments using isolated cells. It is thought to be due to lot differences of commercially available collagenase, but the cause is not clear.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for separating hepatocytes to obtain hepatocytes having a high survival rate.

[0006]

That is, the present invention provides a method for perfusing a liver of an animal with a preperfusion solution containing at least a garlic extract in situ, and further perfusing a collagenase solution in situ with a liver solution. And a method for separating hepatocytes, characterized by digesting hepatic cells.

[0007] As the basic prescription of the preperfusion solution, those conventionally used, for example, in the case of rats, EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) and HEPES are added, and calcium ions and magnesium ions are added. A Hanks buffer solution or the like excluding the above is preferably used, but the present invention is not limited to this, and the type and concentration of the additive may be appropriately selected depending on the type of the animal.

In the present invention, a garlic extract is added to the above-mentioned conventional preperfusion solution. The garlic extract is obtained by crushing garlic and immersing it in water.
Alternatively, extraction may be performed with an aqueous solution having a pH of 4 to 9 similar to that of the preperfusion solution, for example, a buffer solution. For example, when extracting with water, garlic may be immersed in 1 liter of water for 20 to 100 g and extracted at room temperature for 1 hour.

The amount of garlic extract to be added depends on its concentration, but is suitably up to about 50 ml per 500 ml of pre-refluxed liquid. If cells are to be obtained aseptically, it is necessary to sterilize the filter before adding the cells to the preperfusate.

[0010] In the method of the present invention, the technique of in situ perfusion of the liver is not limited, and may be used in accordance with an animal using a known method. For example, when perfusing the liver of a rat, a small flat method (Experimental Medicine Vol.
No. 1, pages 1105 to 1113 (1988). That is, the abdomen is incised, a cannula is inserted from the portal vein, and the perfusate is perfused. At the time of preperfusion, the inferior vena cava is simultaneously cut and blood is removed. Thereafter, the heart is also cannulated, and the perfusate containing collagenase is circulated for use. It is preferable that all the liquid to be perfused is heated to about the body temperature of the animal.

[0011] All blood in the liver is removed from the inferior vena cava by preperfusion, and calcium-dependent adhesion factors are removed by EGTA to facilitate cell separation. The preperfusion varies depending on the type and size of the animal. For example, in the case of a rat weighing about 150 g, the preperfusion is performed at a flow rate of 30 ml per minute for 5 to 15 minutes, for a total of 100 to 5 minutes.
Pour 00 ml of preperfusate. If the preperfusion is successful, the liver becomes evenly white.

After the end of preperfusion, the perfusate is changed to collagenase solution to enzymatically digest the liver. Collagenase solution
Conventionally used formulations may be used. Current,
It is mainly used by adding trypsin inhibitor and calcium ion to Hanks' solution and adding 0.05%
This is a solution to which collagenase was added. Commercially available collagenase may be used, but it may be appropriately diluted and used since its activity varies somewhat depending on the lot. Further, a thiol protease inhibitor, a serine protease inhibitor, or the like may be added thereto.

[0013] Perfusion is at the same rate as preperfusion, ie a weight of 1
For a rat weighing about 50 g, the test is performed at 30 ml per minute. As the perfusion is continued for 8 to 15 minutes and the digestion by collagenase progresses, the liver loses elasticity and cannot be returned to its original state even by lightly pressing with tweezers. At this point, the perfusion is terminated, the liver is removed and transferred to a petri dish. Insufficient collagenase digestion, or excessive digestion over time, results in reduced cell viability.

When the skin of the liver transferred to the petri dish is dissected, hepatocytes are separated so as to melt. Further, when an ice-cooled cell washing solution is added and most of the hepatocytes are separated as single cells by light pipetting with a thick Komagome pipette, the filtrate obtained by filtering with gauze or the like is used as a coarsely dispersed cell suspension. The obtained crude cell suspension may be separated by a conventional method, and purified and used if necessary.

According to the present invention, hepatic parenchymal cells can be stably obtained at a higher survival rate than those obtained by conventional methods. In addition, the separation method of the present invention can be used to collect not only parenchymal cells but also non-parenchymal cells such as Kupffer cells and endothelial cells by a conventional method.

The hepatic parenchymal cells obtained by the present invention can be cultured by a conventional method or can be applied to various tests and studies as they are. In addition to testing and research, it is possible to form a hybridoma with a cell line and use it for mass production of useful substances, for example, vaccines and the like, and for drug toxicity tests. The method of the present invention can be applied to not only rats but also livers of other animals such as mice, guinea pigs, rabbits, dogs, etc., if conditions are selected.

Hereinafter, the present invention will be described in more detail with reference to examples. The embodiment is an example of the present invention, and the present invention is not limited to the embodiment.

[0018]

EXAMPLES All animal and reagent experiments were performed on rats (male, Wistar, 4-7 weeks old).
Weight (100-250 g). Examples and comparative examples were performed in triplicate. Table 1 shows the compositions of the basal preperfusate and the collagenase solution.

[0019]

[Table 1]

The collagenase used was manufactured by Wako Pure Chemical Industries, Ltd., and the trypsin inhibitor used was manufactured by Sigma.

The basal preperfusate dissolves all reagents and
The one adjusted to pH 7.2 with OH was used. The collagenase solution dissolves reagents other than collagenase,
After adjusting the pH to 7.2-7.4, the collagenase powder is added little by little with stirring to dissolve, and the mixture is further stirred for 1 hour. After sufficiently dissolving, the pH is adjusted to exactly 7.5. After sterilization by filtration, 100 ml was dispensed and stored refrigerated.

Example 1 About 65 g of garlic was put into 1 liter of ion-exchange distilled water, pulverized with a household mixer (manufactured by Sanyo Electric Co., Ltd.), and stirred at room temperature for about 1 hour. 12,000 rpm
The supernatant was used as a garlic extract. The garlic extract (50 ml) added to the basal preperfusion solution (500 ml) was used as the preperfusion solution of Example 1.

Comparative Example As a comparative example, preperfusion was performed using a basal preperfusion solution to which no garlic extract was added.

In situ perfusion Perfusion was performed by the small flat method described above. A rat anesthetized with Nembutal solution was opened, and a cannula attached to the tip of a perfusion tube set in a peristaltic pump was inserted and fixed from the portal vein. Immediately after the inferior vena cava was cut and blood was removed, the preperfused solution of Example and Comparative Example heated to 38 ° C. was perfused at a flow rate of 30 ml / min. Preperfusion was performed for 10 minutes.

When perfused, the liver is uniformly bleached white.
During this time, a large incision was made in the thoracic cavity to expose the heart, after which the inferior vena cava below the liver was stopped with forceps and the inferior vena cava immediately entering the right atrium was cannulated and fixed. After the end of the preperfusion, the pump was stopped once, and the perfusion was performed at the same flow rate by changing the perfusion solution to a collagenase solution heated to 38 ° C. After about 1 minute, when the preperfusion solution was drained from the liver and the perfusion tube, the solution coming out of the heart was returned to the collagenase solution, and the perfusion was further continued.

The perfusion with the collagenase solution was continued for about 10 minutes, and the perfusion was terminated when the liver was digested and the hepatic lobules appeared as if they had risen, and the surface was not elastic enough to return to the original state even when pressed gently with forceps. The liver was picked up with tweezers, cut off with scissors, and transferred to a Petri dish kept on ice.

The liver transferred to the petri dish was cut through the liver capsule with a scalpel, and 20 ml of ice-cold MEM (basic culture medium for animal cell culture (manufactured by Dainippon Pharmaceutical Co., Ltd.)) was added, followed by slow pipetting with a thick Komagome pipette. And fully dispersed.
This cell dispersion was passed through a double gauze, and the filtrate was filtered for 4 hours.
The mixture was transferred to a glass Spitz-type centrifuge tube, made to be 40 ml per tube with MEM, dispersed with a thick Komagome pipette, and then centrifuged under cooling at 50 × g for 1 minute. Under these conditions, intact hepatocytes are packed and damaged hepatocytes, non-parenchymal cells, red blood cells and cell debris remain in the supernatant. The supernatant is removed, MEM is newly added, pipetting is performed, and the same centrifugation is repeated. Thus, centrifugation was performed four times while collecting the cells. Finally, hepatocytes could be obtained with a purity of 96 to 98%.

Finally, 40 ml of ME per liver
M, aliquots were taken, 0.3% trypan blue was added thereto, mixed gently, and the number of live and dead cells was counted using a hemocytometer. Survival was calculated. Dead cells are stained blue by trypan blue.

The survival rate of the obtained hepatocytes was 79 ± 3.1% in the comparative example, whereas the garlic extract extracted with water in Example 1 was added to the preperfusate. It was 85 ± 2.4% (n = 3).

[0030]

The hepatocytes isolated by the method of the present invention have a high survival rate and a constant yield.

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Claims (2)

(57) [Claims] 1. A method of perfusing a liver of an animal (excluding a human) with a preperfusion solution containing at least a garlic extract in situ, and further perfusing a collagenase solution in situ to digest the liver. A method for separating hepatocytes, characterized in that: 2. The method according to claim 1, wherein the animal is a rat.