JPS61260022A - Artificial liver-like tissue and its manufacturing method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to an artificial liver-like tissue and a method for producing the same.

(Prior art) Various studies have been carried out in the past with the aim of obtaining factors that cause hepatocytes to proliferate, but it is no exaggeration to say that the goal is to create an artificial liver using cultured hepatocytes. ,
Its acquisition is far from satisfactory.

(Problem 2 to be Solved by the Invention The present inventor has developed a method for culturing liver parenchymal cells, liver tissue I/c
The present invention was achieved through various studies aimed at obtaining an artificial liver-like tissue with similar morphology and function.

(Means for Solving the Problems) That is, the gist of the present invention is as follows: (1) Hepatocytes are obtained by culturing isolated hepatic parenchymal cells, (11) Hepatocytes are arranged in close adhesion to each other, and An artificial liver-like tissue that has a gap corresponding to a bile canalicule and secretes (1) collagen, lipid globules, and bile substances, and a method for producing the same.

The present invention will be explained in detail below.

First, the M example of liver-like tissue according to the present invention will be explained.

That is, preparation is carried out by culturing hepatocytes in a medium containing specific factors and insulin.

As the specific factor, cell growth factors derived from the intestinal mucosa or specific serum of warm-blooded animals are used.

Examples of the warm-blooded animals include rats, mice, chickens, goats, cows, rabbits, pigs, horses, and the like.

The growth factor used in the present invention can be obtained, for example, by the following method.

(1) Intestinal mucosal origin: The types of intestinal mucosa in these animals include the small intestine, cecum,
Examples include the duodenum, ileum, and large intestine, but usually, the small intestine and cecum of cows, pigs, chickens, etc. are preferably used.

First, the intestinal mucosa of the above animal is collected. Collection is usually performed by a scraping method to obtain intestinal epithelial mucosal tissue. This is treated with a monosalt solution substantially free of calcium, preferably magnesium, to obtain a homogenate. These steps are normally carried out at a temperature of about 0 S2°C.

Examples of the above-mentioned salt solution include Tyrode's solution (Ty
rotlda 5olution) (hereinafter referred to as “CM
) is used. The composition of this solution is as follows.

Na(jl za, 00 NQHPO41,/j
KO'l O, KO'l O, KO'l PO40,20 (t7t) Next, a supernatant is obtained from the homogenate by centrifugation or the like. When using centrifugation, it is usually 1.000%/2,
000 X f in more than a few minutes. Although the obtained supernatant can be used as a growth factor in the present invention, the obtained Tsuchiura is usually heated to precipitate and remove denatured high molecular weight proteins. Heating is performed at about 0 to 100° C. for 1 minute or more. The supernatant is then dialyzed against the above saline solution to remove low molecular weight components. The resulting fraction gives three bands with a molecular weight of about ul, 000-/l, 000 K in electrophoresis on a BD8 polyacrylamide gel. This fraction can be used as a factor in the present invention, but it can also be further purified and used, for example, by the following method. Namely, the above fraction (or the above heating and /
You can also use the centrifuged fraction without dialysis).
The first of the three peaks obtained by gel filtration using Sephadec x'G-71, 100 or 200, etc.
Fractionate both fractions having the peak.

Next, DEAF! When cellulose is subjected to ion exchange chromatography, it is fractionated into 6 to 7 fractions. The second fraction is the growth factor of interest.

In electrophoresis on an 8D8 polyacrylamide gel, the purified fraction gives one band with a molecular weight of approximately -10,000.

Furthermore, by treating this factor with enzymes such as catezin and pepsin, it is further purified and has a smaller molecular weight (
For example, it can be a factor of about t,ooo to 20,000.

關) When derived from a specific serum: In this case, the serum used is one obtained from the blood of a warm-blooded animal whose liver has been damaged. The above-mentioned damage can be done by, for example, (a) damaging a part of the liver tissue by resection or puncture, or (b) ligating at least a part of the main trunk of the portal vein or its branch that enters the liver lobe. It is given by necrosis of a part of the tissue.

For example, in the case of (b) above, first, at least a portion of the main trunk of the portal vein of a warm-blooded animal or its branch that enters the liver lobe is ligated.

The portal vein is a venous system that collects blood from the digestive organs and spleen and carries it to the liver, and branches into each lobe of the liver.

In this method, the entry portion, ie, the bifurcation of the portal vein that enters each lobe of the liver, or part or all of the portal vein main trunk, is ligated with silk thread or the like.

An example of partially ligating the branch is a method of ligating about 1/2 to λ/3 of the branch.

For example, when ligating 2/3, there is a method of ligating two of the three parts entering the left lobe, middle lobe, and right lobe from the portal vein main trunk.

After a predetermined period of time, for example, several hours or more, has elapsed after the ligation, the blood of the animal such as the cow from which at least a portion of the portal vein has been ligated is collected by a conventional method.

Next, serum is prepared from the collected blood.

For example, if the blood is coagulated at room temperature and then the supernatant is obtained by centrifugation or the like, the desired serum can be obtained. When using centrifugation, usually 1,000-/2
,00Of for several minutes or more.

In the present invention, the obtained serum is further heated and/or
Alternatively, it can be purified by dialysis.

Heating is usually carried out at about 40 DEG C. for 1 minute or more, and heat-denatured polymer proteins are precipitated and removed.

Further, dialysis is performed using the aforementioned saline solution that does not substantially contain calcium.

The above dialysis treatment removes low molecular weight components in serum.

Furthermore, it can be subjected to a purification treatment in the same manner as in the case (1) above.

The cell increase obtained by the method (1) or (11) above has a molecular weight of approximately t, ooo Sμ? , 000.

b) CONA (concanavalin A)-1 cephalose”
It is eluted by chromatography. That is, insulin is optimally present along with the polysaccharide or glycoprotein offspring. The insulin is not limited to 5%, and commercially available petai/surin, bovine insulin, etc. are usually used.

During culturing, for example, the supernatant or buffer solution containing the above factors has a protein content of o, about 1-totq/-, and insulin has a concentration of lO-? ~1O-4 preferably 10-'~
/'0-'MO concentration, and this solution is added to the medium usually /-y
A method of adding it to a concentration of 10vol%, preferably 3 to jool% is usually adopted.

Then, add serum such as fetal bovine serum to the medium and O-! ~/O
It is preferable to use it by adding about vow%.

Cultivation can be done by conventional methods, for example! -〇Fake-ta! In air, 37°C, pH 7.0-7. ! It is carried out for about one week or more. Culture is carried out by renewing the medium as necessary.

The hepatic parenchymal cells used are usually those obtained from the liver of a normal or liver-damaged warm-blooded animal by the collagenase perfusion method. Particularly preferably, the liver of a warm-blooded animal is damaged, and the liver is used after about 3 hours or more, preferably about 20 hours or more, and the liver is directly perfused with collagenase, and then the liver is cut into small pieces. It is further treated with a collagenase solution, followed by filtration and centrifugation.

In addition, in the above method, the most favorable results can be obtained by repeating the filtration process using nylon fibers such as nylon gauze and centrifugation multiple times, preferably 7 or more times, when filtering and centrifuging the thin sections. .

Through the above culture, the hepatocytes proliferate in the medium and reassemble, and begin to be rearranged in close contact with each other, forming a multilayered colony-like hepatocyte population.

These colonies then fuse with each other to form a larger multilayered colony, which develops and adheres tightly, exhibiting the cell arrangement typical of the liver, with gaps corresponding to bile canaliculi between the cells. is formed (bile acids, bile pigments) and other products are secreted.

(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.

Example (Experiment-7) Fractionation of heated and dialyzed rat small intestine mucosal extract by gel Fffl method l) Experimental animals and breeding conditions Birth, 2-3 months old, weight 200
Samples obtained from ~300t males were used. Rats have free access to both feed and drinking water, with solid feed (
The animals were fed Japanese compounded feed (manufactured by Nippon) and raised in an environment kept at around 23°C.

-2) Preparation of small intestine mucosal extract Rats were lightly anesthetized with ether, the jugular vein was cut, blood was exsanguinated, the entire small intestine was excised after laparotomy, and the contents were removed, and then incised vertically with ophthalmic scissors. CMF-PH8 solution (Dulbe
The mucous membrane was collected by scraping with a bottle set, homogenized with approximately twice the volume of CMF-PH8 solution, and centrifuged for /2,000 X f, j O minutes. The supernatant was collected into the small intestine. It was used as a mucosal extract.

3) Heating and dialysis of small intestine mucosal extract After heating the above-mentioned small intestine mucosal extract at 100°C for 2 minutes, /2
Centrifuge for 1,000 x f% coO for 30,000 x
Dialysis was performed for 2 hours with twice the volume of OMF-PBa solution.

The small intestine mucosal extract obtained by fractionation heating and dialysis using the Rigel P method was compressed in a collodion bag and used as a sample for fractionation.

Pharmacia File Kenkar 7t (Pharmacia Fine Chemi
cals g) was used. The column had an inner diameter of 1 and a gel height of 0. 0. / MllaCl -20mM
The gel was washed with TrilB-HOI buffer (pH 7,7) K, 21 Ll of the sample was added, and eluted with the same buffer in a descending manner at a flow rate of Jaff/hr.

The eluted buffer was collected by a fraction collector.
An elution curve was obtained by fractionating the sample and measuring the absorbance at nm. This resulted in three fractions.

(Experiment-2) Cellulose ion exchanger H) used in a fractionation column by ion exchange cellulose column chromatography was used. The column has an inner diameter of JcI and a height of Jol.
A cellulose ion exchanger was equilibrated and used in the same buffer as in Example 7.

Nine fractions obtained in the above experiment-1/ (tube 4 j J ~
! λ) was concentrated in a collodion bag and used as a sample for fractionation.

0.1M after j-loading the sample to the column.

0.2M, 0.3M%0.11M, 0.4#
%OJM Na11K] The ionic strength was increased stepwise to elute the adsorbed substances. Note that for each type of buffer solution, the amount to be flowed is calculated as follows.

Elute at a flow rate of IIL/hr, and collect the eluted buffer using a fraction collector! d preparative J
The absorbance of I Onm was measured and an elution □ curve was obtained.

Two fractions of K and J5J were obtained, and the fractions (o, o, cape protein/-, tubes 444-73) were obtained as the desired factor (molecular weight approximately -10,000).

(Disruption of hepatic parenchymal cells) l) The animals used for culture were 11 years old and weighed approximately iz.

2 8D male rats were used and reared under the same conditions as in Experiment 7. It should be noted that all separation operations and cultivation of hepatocytes were performed under aseptic conditions, so special attention was paid to sterilization.

λ　Adult rat hepatocyte isolation method All hepatocytes were separated using the following method.

273 of the rat liver was excised and 20 hours later,
The rats are anesthetized with an intraperitoneal injection of 0.2 wt. Next, the abdomen is pricked with hair, and the entire body is disinfected with a 10% benzalkonium chloride solution (all liver cell separation operations are performed aseptically in a sterile box, except for shaking and centrifugation).

The rat is fixed in a supine position on a surgical fixation table, and the abdominal skin and abdominal muscles are opened in this order using surgical scissors. Use absorbent cotton soaked with OMF-PBX solution to scrape the intestine to the surgeon's right side to fully expose the portal vein. Loop the suture over the portal vein and use the tips of ophthalmic scissors to make a cut into the portal vein. A cannula is inserted into the perfusion device previously placed in the incision, aiming at the overflowing blood while flowing preperfusion buffer.
Quickly ligate with suture. At the same time, cut the subhepatic vena cava and drain the washing fluid. 3゜txl as it is /
After approximately 200 yd of preperfusion buffer flow at a flow rate of hr, the pump is stopped. Change the perfusate to CMF-PBEI solution and wash at the same flow rate for about 100 rds, then stop the pump, change the perfusate to collagenase solution and run at the same flow rate for about 30 rds. Once the collagenase has permeated the entire liver, stop the pump, carefully remove the entire liver, and remove the entire liver.
Transfer to IIK glass Petri dish. Add about 20 d of collagenase solution, cross the razor blade with forceps, and remove the liver.
Cut into m square pieces. The shredded tissue was mixed with collagenase solution and transferred to a 1011 volume triangular W cup, sealed tightly with a butyl stopper, and shaken at a rate of 100 liters per minute in a constant temperature shaker at 37°C. Shake for a minute.

After shaking, the digested tissue was removed using a cell sieve, and the undigested tissue was washed from above with approximately 10 OMF'-PBX solution. All the P solution was collected in a 30 d centrifuge tube and sealed. rear! Centrifuge at 0'Xf/min. Discard the supernatant, add 20 ml of OMF-PBX solution for washing, gently pipette, cap tightly, and cool the centrifuge tube on ice. T.O.
'lf, centrifuge for 7 minutes and discard the supernatant.

OMF-PBB solution is added again and suspended, passed through a small piece of dielectric nylon cloth, the P solution is collected in a new centrifuge tube, sealed tightly, cooled on ice, and centrifuged at IO x f for 7 minutes. After discarding the supernatant, washing with OMF-PBX solution is repeated twice to remove non-parenchymal cells, which have a lower specific gravity than hepatic parenchymal cells, and to separate hepatic parenchymal cells.

3) Preculture and preculture medium Since hepatocytes damaged by enzymes have lost their original liver function, preculture was performed in order to open the liver.

Insulin and fetal bovine serum from hepatocytes f10-6M purified by low-speed centrifugation! - William's E medium containing (
.wm medium) and a sterilized plastic petri dish (
J z x t o m) ha! Sow at a rate of -. The culture solution contained penicillin (tooσ/1ttl), streptomycin (tooμf/ld), and amphotericin B (
0.2 jμf/ld) etc. are added. Culture is j%
CO,-ta! Adhesion is carried out at 37°C using air as the gas phase.

After culturing in the main preculture medium for 2V hours, the growth factors obtained in experiment 2 above were added, the medium (same as in the preculture) was updated with at waiting time, and j%co, -ta! Culture was carried out at 37°C using air as the gas phase. After 3 to j days had passed (the start of main culture is defined as day 0 (Figure 1)), a large number of cells began to divide, proliferate, reassemble, and form colonies (Figure 2).
. Most cells are incorporated into colonies. Furthermore, the cells within the colony continue to proliferate, and the colony grows (
Figure 3). On the 10th day, the cells of the colony showed tight connections and began to secrete collagen, lipid globules, and bile substances around the colony (Figure μ). lO~
On the 13th day, the colonies begin to fuse with each other and form a large colony. On day II, the hepatocytes of the large, fused colony showed even tighter connections with each other and an arrangement of overlapping hexagonal cells typical of the liver, with gaps corresponding to bile canaliculi seen between the cells. It secreted large amounts of collagen, fat globules, bile substances, and other products, and developed an appearance and function similar to liver tissue (Figs. 3 and 3).
figure).

These results are considered to indicate that the isolated hepatocyte group initially plated within the dish reassembled and further developed and differentiated into microscopic liver tissues.

The production of collagen, lipid globules, and bile substances was confirmed by conventional methods. In other words, collagen is determined by the presence or absence of disappearance by collagenase treatment, lipid globules are qualitatively confirmed for the presence or absence of cholesterol and neutral fats, and bile substances are determined by conventional clinical testing methods. Confirmed by law.

(Effects of the Invention) The artificial liver-like tissue according to the present invention is useful for in VitrO liver function and pharmacological tests, and can also be applied to the use of artificial liver substitutes in clinical treatment.

[Brief explanation of drawings]

FIGS. 1 and 4 are phase contrast micrographs (Hiro 00x) showing the process of producing an artificial liver-like tissue according to the present invention. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney Yo Hase - and 1 other person

Claims (2)

[Claims] (1) (i) obtained by culturing isolated hepatic parenchymal cells, (ii) hepatocytes are arranged in close adhesion to each other, with gaps corresponding to bile canaliculi between the cells, and (
iii) An artificial liver-like tissue characterized by secreting collagen, lipid globules and bile substances. (2) in a medium containing (a) cell growth factors obtained from a homogenate of the intestinal mucosa of a warm-blooded animal or serum obtained from the blood of a warm-blooded animal with liver damage; and (b) insulin; By culturing parenchymal cells isolated from warm-blooded animals, (i) hepatocytes are arranged in close adhesion to each other, with spaces between the cells corresponding to bile canaliculi;
and (ii) a method for producing an artificial liver-like tissue, which comprises obtaining an artificial liver-like tissue that secretes collagen, lipid globules, and liver juice substances.