JP3186881B2 - Hepatocyte isolation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to animals (excluding humans,
The same applies to the method for separating hepatocytes.

[0002]

2. Description of the Related Art In the study of a variety of specific functions of a mammalian liver, attention has been paid to a means of using primary culture cells of hepatocytes. 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 in situ.
The present invention relates to a method for separating hepatocytes, which comprises perfusing a perfusion solution containing at least collagenase and a thiol protease inhibitor in situ.

[0007] As the preperfusion solution, a conventionally used solution may be used. For example, in the case of a rat, EGTA is used.
(A Hanks buffer solution to which ethylene glycol bis (2-aminoethyl ether) tetraacetic acid and HEPES are added and calcium ions and magnesium ions are removed is preferably used, but the present invention is not limited thereto. The type and concentration of the additive may be appropriately selected depending on the type of the additive.

[0008] In the method of the present invention, the technique of in situ perfusion of the liver is not limited, and may be used according to the animal using a known method. For example, when perfusing the liver of a rat, a small flat method (Experimental Medicine Vol.
No. 1, pages 1105-1113 (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 lower vena cava may be cut and blood removed at the same time. 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.

[0009] 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, 3 per minute is used.
Perform at a flow rate of 0 ml for 5 to 15 minutes, for a total of 100 to
Pour 500 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. The collagenase solution used in the method for separating hepatocytes of the present invention is obtained by adding a thiol protease inhibitor to a conventionally used collagenase solution. The collagenase solution currently mainly used is a solution obtained by adding a trypsin inhibitor and calcium ions to a Hanks solution, and further adding collagenase to 0.05%. Commercially available collagenase may be used, but it may be appropriately diluted and used since its activity varies somewhat depending on the lot.

The thiol protease inhibitors to be added to the collagenase solution include halogenated lower carboxylic acids,
For example, NEM (N-ethylmaleimide), antipine, leupeptin, etc., such as iodoacetic acid, are known.
Particularly, iodoacetic acid is preferably used. The addition amount of the thiol protease inhibitor varies depending on the activity of each inhibitor. For example, in the case of iodoacetic acid, it is 1 to 500 ppm, preferably 10 to 100 ppm. If the amount of the thiol protease inhibitor is too large, the growth after hepatocyte isolation is hindered, and if the amount is too small, the effects of the present invention cannot be obtained.

[0012] Perfusion is at the same rate as preperfusion, ie 1 body weight.
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 cut off, the hepatocytes are melted and separated. Further, when an ice-cooled cell washing solution is added and most of the hepatocytes are liberated 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 into parenchymal cells and non-parenchymal cells 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 method of the present invention can be used to sort 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 primary-cultured or subcultured by conventional methods and applied to various tests and studies. 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 not only to rats but also to other animals such as mice, guinea pigs, rabbits, dogs, etc., if conditions are selected.

Hereinafter, the present invention will be described in more detail by way of examples. The embodiment is merely an example of the present invention, and the present invention is not limited thereto.

[0017]

EXAMPLES All animal and reagent experiments were performed using rat male Wistar breeds, 4-7 weeks old, weighing 100-250 g). Table 1 shows the composition of the preperfusate, the examples and the collagenase solution.

[0018]

[Table 1]

Collagenase was manufactured by Wako Pure Chemical Industries, Ltd., and trypsin inhibitor was manufactured by Sigma. The preperfusion solution was prepared by dissolving all reagents, adjusting the pH to 7.2 with NaOH, dispensing 100 ml each, and sterilizing by autoclave. The collagenase solution was prepared by dissolving reagents other than collagenase and adjusting the pH to 7.2 to 7.4 with NaOH.
Collagenase powder was added little by little with stirring to dissolve, and further stirred for 1 hour to fully dissolve, adjusted to pH 7.5, sterilized by filtration, dispensed in 100 ml portions, and refrigerated. Was used.

In situ perfusion Perfusion was performed by the small flat method described above. The experiments were performed in triplicate. 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 cut the inferior vena cava to remove blood,
The heated preperfusion solution was flowed into the vessel and preperfusion was performed at 30 ml / min. Preperfusion was performed for 10 minutes.

Upon perfusion, the liver is bled uniformly 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. Thereafter, the pump was once stopped, and the perfusate was changed to a collagenase solution heated to 38 ° C., and perfusion was performed at the same flow rate. 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, added with 20 ml of ice-cold MEM (basal medium for animal cell culture (manufactured by Dainippon Pharmaceutical Co., Ltd.)), and slowly pipetted with a thick Komagome pipette. After the cell dispersion was passed through a double gauze, the filtrate was filtered for 4 hours.
After transferring to a glass Spitz-type centrifuge tube and dispersing with a thick Komagome pipette to 40 ml per tube with MEM,
Centrifuged at 50 xg for 1 minute under cooling. 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. In this manner, centrifugation was performed four times while collecting the cells, and finally, liver parenchymal cells were 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 hepatic parenchymal cells was 79 ± 3.1% in the comparative example, whereas the survival rate of the liver perfused with the collagenase solution containing the thiol protease of the example was 85 ± 3.1%. 1.0%.

[0026]

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

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Claims (3)

(57) [Claims] 1. A method of perfusing a liver of an animal (excluding a human) with a preperfusion solution in situ, and then perfusing a collagenase solution containing at least a collagenase and a thiol protease inhibitor in situ to digest the liver A method for separating hepatocytes, comprising: 2. The method according to claim 1, wherein the thiol protease inhibitor is a halogenated lower carboxylic acid. 3. The method according to claim 1, wherein the animal is a rat.