JP3058438B2 - Immortalized primate hepatocytes capable of infecting human liver tissue-specific virus and methods for preparing the same - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to primates capable of infecting or persistently infecting viruses with high human hepatocyte specificity, such as hepatitis A virus, hepatitis B virus, non-A non-B hepatitis virus. And a method for preparing the immortalized hepatocytes.

2. Description of the Related Art One of the reasons that it has been difficult to identify and isolate a virus using a human host as a host is that a system for propagating the virus in addition to a human host is suitable. It did not exist.
This problem is caused by non-A non-B hepatitis (hereinafter referred to as HNANB).
It was a big close-up in conducting research targeting viruses. Conventionally, HNANB virus has been proved directly (as phenomenology) by infection of chimpanzees in the case of HNANB after blood transfusion and in the case of cynomolgus monkeys in the case of aqueous HNANB as a host system other than human as a non-human host system. However, the fact that the chimpanzee, a protected animal in the former, was the only experimental animal for virus research, severely limited the isolation of this virus and subsequent studies. The same problem was serious in the case of hepatitis B (hereinafter referred to as HB) virus.

Recently, HBs have been used in primary cultured human hepatocytes or adult hepatocytes.
Virus infection and growth were confirmed, suggesting that human hepatocyte-specific virus growth is also possible in an in vitro system (Shimizu, Y. et al., J. Med. Virol., 20 :
p.313 (1986); Ochiya, T. et al., Proc. Natl. Acad. Sci.U
SA 86: p.1875 (1989)).

On the other hand, such viruses have not been successfully propagated in hepatocellular carcinoma cell lines derived from hepatocellular carcinoma or cells derived from normal liver and accidentally acquired permanent growth. This fact implies that the primary culture method, although not proliferable, is quite in vivo in terms of maintaining liver-specific functions. On the other hand, hepatocytes that have acquired proliferative potential by chance become malignant tumors (transform,
It is conceivable that hepatocyte specificity such as expression of many receptors, for example, hepatitis virus, has been lost.

The following report (Marceau, N. et al., In.) Explains the above-mentioned phenomenon, that is, the relationship between differentiation, regeneration, and canceration of hepatocytes.
Vitro, 25: p. 336 (1989)). “Hepocytes differentiate and mature from immature bile duct cells. Liver regeneration is a mechanism in which mature hepatocytes become slightly immature, become small hepatocytes, proliferate and differentiate again. It becomes a state of formation and cannot return to mature normal cells. Therefore, many liver-specific functions do not exist in liver cancer cell lines. "In general, diploid normal cells cultured in the primary culture are permanent unless the cells are mutated. It cannot keep growing.
However, some cells maintain normal cell morphology and do not exhibit tumorigenicity even if the cells acquire permanent growth. This indicates that the canceration of the cells is a permanent growth potential (immortalizatio
It is understood by the description that it can be divided into two steps: n) and transformation.
Here, acquisition of immortalization (immortalization) means in vitro (in
In vitro), cells that have been removed (differentiated) continue to proliferate while maintaining the original specific function of the cells (differentiation).
In addition, if the immortalized cells are transformed, some of them may cause a decrease in contact inhibition, a decrease in anchorage dependence, a decrease in serum dependence, tumor formation in animals, etc., and the specificity of the cells also decreases. (Immature, dedifferentiated).

To date, many researchers have made efforts to render isolated hepatocytes permanent. However, for example, the addition of various growth factors and hormones to the culture medium only gives the primary cultured hepatocytes temporary DNA synthesis or a limited increase in the culturing time. It did not lead to the grant. (To
mita, Y. et al., Exp. Cell Res., 135: p. 363 (1981); Naka
mura, T. et al., J. Biochem., 94: p. 1029 (1983)) In the 1970's, research on hepatocytes, particularly research using primary culture systems of the hepatocytes, has progressed rapidly. Since the efficient method for separating parenchymal cells by the collagenase perfusion method was reported, many reports have been known to date. However, many reports relate to non-primate hepatocytes such as rats, and the culture period is limited to a maximum of about one month. Recently, isolation of a growth factor (HGF) specifically acting on hepatocytes and determination of the nucleotide sequence of a gene encoding the same have also been reported (Nakamura, T. et al., Na
ture. 342: p. 440 (1989)). At present, no remarkable in vitro hepatocyte proliferation promoting action has been shown.

By the way, by infecting cells with a certain tumor virus, for example, SV40, polyoma virus, or the like, or by inserting (transfection) a T antigen that is an oncogene of those viruses in the form of a gene, transfection is performed. Some types of primary cells are immortalized (immortaliz
e), and furthermore, there is a report that it becomes malignant and cancerous (transform).

For example, D encoding only the N-terminal 44% of the SV40T antigen
The NA fragment has an activity of permanently growing rat fetal primary cells (Colby, W. et al., Proc. Natl. Acad. Sci. USA,
79: p.5189 (1982)), and furthermore, a permanent growing cell line Rat-
The activity to completely transform one cell was also shown (Clayton, C. et al., Nature, 299: p. 59 (1982)). Transformation of cells by these viruses involves two types of oncogenes and factors expressed by them. One is a factor that imparts permanent growth to primary cells, and the other is a factor that imparts permanent growth. Are transformed into factors that transform transformed cells. In the case of SV40, it has been revealed that the large T antigen gene has two necessary functions (Fujinaga, K. Oncologia, 8: p. 127 (1984)).

However, immortalization of human cells is extremely difficult, and no such report has been found especially for hepatocytes of humans or higher primates such as chimpanzees. The reasons for the difficulty are, in part, the difficulty in obtaining normal liver tissue from higher primates as a material and the problem of the technology for separating hepatocytes from very small amounts of liver tissue. The problem is that no method has been found for efficiently obtaining hepatocytes whose characteristics are maintained as much as possible by the oncogene, ie, differentiated immortalized hepatocytes that are not transformed.

The latter involves, first of all, the following problems caused by the characteristics of hepatocytes themselves. First, hepatocytes have many specific functions, such as synthesis and secretion of serum proteins, synthesis of liver-specific proteins (enzymes), gluconeogenesis, urea formation, lipid synthesis, bile secretion, and detoxification. Is in vitro
Rapidly disappears when placed in culture conditions. This is presumed to be based on the fact that hepatocytes are highly regulated in vivo by many nutrients, nerves, hormones or non-hepatic cells. Furthermore, it is considered that the intercellular connection, density, polarity, and the like also change, and that the cell is placed in an environment that is significantly different from the original “field”.

The second problem is the difficulty in integrating oncogenes into hepatocytes. Infection of tumor viruses, such as monkey SV40 cells, results in the production of progeny virus in permissive cells, such as monkey-derived cells, and death of the cells. On the other hand, when cells derived from rats or mice (non-permissive cells) are infected with SV40,
No replication of the viral DNA occurs and the cells do not die. Then, only a limited number of the cells are permanently grown or transformed. This is viral DNA
Is integrated into the gene of the cell, and only the early gene of the virus is expressed, and its transforming activity is considered to be working. Human cells are semi-permissive for SV40. That is, virus particle production occurs in about 1% of cells, and replication of viral DNA occurs in 1-2% of cells. Therefore, it is hardly suitable for the purpose of immortalizing or transforming cells without the possibility of virus particle or DNA replication.

In order to solve this problem, a method of inserting the initial gene itself into cells has been attempted. However, transfection efficiency is extremely low, and particularly in primary cultured hepatocytes, cell damage is extremely large. Therefore, efficient integration of oncogenes is required, for example, by applying the technology using a vector virus or the like as shown in the present invention.

Against this background, efficient hepatocyte immortalization requires efficient hepatocyte isolation technology, establishment of a vector system for the integration of oncogenes, and maintenance and proliferation of hepatocyte functions. Three of the culture methods must be fulfilled. Moreover, their efficient combination is even more important. By utilizing the present invention, these problems can be suitably solved.

An object of the present invention is to provide a primate hepatocyte which can be stably cultured while maintaining its specific function as much as possible and can be infected with a hepatocyte-specific human virus, and a method for preparing the cell. The primary purpose. By achieving this object, it becomes possible to easily carry out culturing of a hepatocyte-specific virus and, consequently, production of a vaccine derived from the virus.

Another major object of the present invention is to provide a virus-specific antigen produced by viral infection of cells, and a detection system for detecting virus-specific antibodies in human or monkey serum infected with viral factors. To supply cells that can provide the antigen.

It is another important object of the present invention to prepare stable immortalized hepatocytes in place of primary culture cells in metabolism studies, carcinogenesis studies, and the like. Yet another object is to prepare cells as recombinants for expressing and producing hepatocyte-specific proteins.

These objects and features of the invention will become more fully apparent from the following detailed description of the invention.

Configuration of the Invention As described above (problems to be solved by the present invention),
In order to immortalize human or non-human primate hepatocytes efficiently, the following requirements must be satisfied.

A method for obtaining a sufficient amount of hepatocytes from a very small amount of human or non-human primate liver tissue.

A method for efficiently inserting an oncogene into an extremely small amount of hepatocytes.

A culture method for efficiently and sufficiently transferring a hepatocyte into which an oncogene has been incorporated to a growth phase while maintaining its specific function.

An efficient combination method to satisfy the above three factors.

In the present invention, all of these requirements can be sufficiently satisfied by the following constitutions.

(1) Separation / Purification of Hepatocytes The cells from which immortalized hepatocytes are prepared are liver parenchymal cells that can be separated from liver tissues of human or non-human higher primates. Non-human primates include human primates, such as chimpanzees, which respond similarly to humans upon infection with the virus of interest, eg, the HNANB virus. The cells are isolated from normal liver tissue or liver tissue infected with the virus of interest, eg, HNANB virus.

In general, in animals that can be killed for experiments, such as rats and dogs, the target hepatic parenchymal cells (hepatocytes) can be easily prepared by in situ collagenase perfusion as a cell separation method (Seglen). , POExp.Cell Re
s.82: 391 (1973)). However, in many primates, especially humans, it is difficult to obtain the liver as an organ fresh, so that very small amounts of liver tissue (often less than one gram of tissue) require more liver. There is a strong need for a method for separating hepatocytes. The first feature of the present invention is to provide such an efficient method for separating hepatocytes.

In the method for separating hepatocytes according to the present invention, EGTA (ethylene glycol-bis (β-aminoethyl ether)-) is first used in a tube made of a flexible material such as a cannula to remove blood from liver tissue that has been surgically resected. (N, N, N ', N'-tetraacetic acid) solution is injected into the blood vessel, then collagenase and then an enzyme that digests intercellular substances such as dispase are injected, and the tissue is finely scalpel. It has the characteristic that liver parenchymal cells can be efficiently separated without cutting. Therefore, the technique of the present invention is extremely useful for separating cells from a tissue with developed connective tissue, such as the liver of a human or non-human primate.

(2) Providing Permanent Proliferation to Isolated Hepatocytes The principle of the present invention is described as follows.

As mentioned in the background section, various oncogenes are known to immortalize or transform cells. Thus, these oncogenes can be incorporated into primary cultured cells, their expression proteins impart permanent growth to the cells, and these cells can be transformed. However, as described above, the method of inserting an oncogene itself into hepatocytes in the form of a DNA fragment or a plasmid by a physical transfer method (calcium phosphate method, DEAE dextran method, electroporation, etc.) involves damage to cells or transfection. This is extremely disadvantageous from the viewpoint of projection efficiency.

It is known that adenovirus has a very wide range of infection and can infect most cultured cells (primary cultures, cell lines). The present inventors have proposed that the adenovirus type 5
A recombinant virus incorporating the 40 large T gene was constructed, and the virus was successfully transmitted to human fibroblasts for efficient transformation (Gluzman, Y Mol. Ce).
ll. Biol. 4: 1653-1656, 1984). Further, in the present invention, by using this, an attempt was made to make permanent culture of the primary culture cells, and particularly to add permanent growth to cells that are almost impossible to grow in vitro, such as hepatocytes.

As described above, the SV40 large T antigen has both functions of permanently growing primary cells and transforming the proliferating cells. Therefore, cells artificially incorporating the SV40T antigen gene and imparting permanent growth by its expression can be obtained by mutation at various stages of immortalization → transformation. In the case of a cell line derived from a cancer cell or accidentally transformed, a specific immature cell clone has acquired high proliferation,
It is probabilistically difficult to obtain satisfactory differentiated cells.

Therefore, in order to obtain more suitable immortalized cells with a high degree of differentiation, efficient cell separation, which is a pre-stage, and more aggressive oncogene integration are required. As a result of intensive studies, the present inventor has completed the present invention that can sufficiently satisfy these two points.

The recombinant virus used in the present invention does not show a life cycle that destroys cells against hepatocytes, and expresses only the SV40T antigen. As a vector virus system for constructing a recombinant virus having a similar function, SV40, polyoma virus, retrovirus, vaccinia virus, herpes virus and the like can be considered in addition to adenovirus. Examples of oncogenes for imparting permanent proliferation to hepatocytes include, in addition to the SV40T gene, viral oncogenes such as adenovirus E1, polyomavirus T, and retrovirus v-onc, and cm-m in normal cells.
Many oncogenes can be used in addition to c-onc such as yc and cH-ras. Depending on the various combinations of vector virus-oncogene, there is a possibility that the same or better effects as the present invention can be obtained. However, they are in principle the same in all cases as the present invention, and it goes without saying that they are all included in the present invention.

In order to make full use of the features of the present invention, the following two requirements should be further noted, which are also shown by Example 3 described later.

The first relates to a method for infecting hepatocytes with a recombinant virus. In general, cultured cells are hardly damaged by normal virus adsorption methods,
Virus adsorption occurs efficiently. However, hepatocytes, especially those that have been primarily cultured in monolayers, are only infected at a very low rate. In the present invention, as a method for satisfying this, a method has been developed in which hepatocytes immediately after separation are infected (adsorbed) with a recombinant virus in a suspended (suspended) state.

Second, hepatocytes infected by the recombinant virus are cultured three-dimensionally (three-dimensionally) in an intercellular matrix such as a gel composed of collagen, fibronectin, laminin, proteoglycan, and the like. It was shown to contribute to the induction of immortalized hepatocytes much more efficiently than when cultured in monolayer (planar). These two methods have shown tremendous effects on the expression of oncogenes by the recombinant virus and subsequent induction and stabilization of hepatocyte proliferation (division).

(2) Selection of immortalized hepatocytes Prior to the examination of the infectivity of the hepatocyte-specific virus to the immortalized hepatocytes, the immortalized hepatocytes must be capable of transfecting the liver, in particular, the liver-specific protein secreted by hepatocytes. It is significant to confirm the ability to secrete.

For example, a cell with high albumin secretion ability is a hepatocyte that has been imparted with perpetual proliferative property that maintains the properties of a more natural hepatocyte. This is because it can be considered to be maintained.

Because of the close structural relationship between secreted proteins in humans and non-human primates, antibodies specific for human secreted proteins, such as human serum albumin (hereinafter referred to as HSA), are not compatible with the corresponding non-human primates. Can also be used for the analysis of secreted proteins. The presence of secreted proteins such as HSA in the medium of immortalized hepatocytes was confirmed by means such as enzyme-linked immunoassay (hereinafter, referred to as ELISA).

Generally, first, an antibody (immune animal serum) against HSA to be detected is immobilized, and then a sample (culture supernatant)
And react for a certain period of time. After washing, an antibody against HSA is reacted with a conjugate in which an enzyme (such as peroxidase) as a color developing system is bound, and the color is developed in a color indicator. Depending on the degree of color development, the ability of the immortalized hepatocytes to secrete albumin can be measured.

(4) Viral infectivity One of the important advantages provided by the present invention is that immortalized hepatocytes have previously been unable to grow by culture in human or non-human primate tissue-specific viruses, such as It can be used as a host capable of infecting hepatocyte-specific HB virus or HNANB virus. A
Hepatitis B (hereinafter referred to as HA) virus has also recently been able to be cultured in cell lines other than hepatocytes,
Much evidence has revealed that the virus is originally a hepatocyte-specific virus (Ashida, M. et al., J. Gen.
Virol., 70: p.2487 (1989)). Therefore, showing the infectivity of these three viruses in the immortalized hepatocytes supports the higher utility of the present invention.

In general, the method of viral infection involves first infecting cells with a known, active virus, and analyzing and detecting the cells for the presence or absence of virus growth during an incubation period of one to several weeks.

Typically, the cells are cultured in a monolayer, the medium is removed before inoculation of the virus, and a small amount of virus liquid enough to immerse the cells is added, and the mixture is kept warm and adsorbed on the cells. Cell changes caused by virus growth are easily and clearly detectable cell changes, for example, observed as cytopathic effects (CPE) such as cell lysis, multinucleation, aggregation. Alternatively, it is determined by the presence of virus-specific surface or intracellular or extracellular antigens expressed as a result of viral growth. Example 6 illustrates the analysis of immortalized hepatocytes infected with the HA virus. In addition, immortalized hepatocytes that can be infected by the HA virus
It became clear that HB virus and HNANB virus could also be infected.

Hereinafter, in order to clarify the features of the present invention, the present invention will be described in detail with reference to examples. However, these examples explain various specific examples of the present invention. However, the present invention is not limited to this.

Example 1. Isolation of hepatocytes From a human liver tissue removed by a surgical procedure, an ice-cooled preperfusion solution was injected from a blood vessel on a cut surface of the tissue in a short time as possible to remove blood. The composition of the preperfusate used Hanks buffer containing EGTA according to the method of Seglen. An ice-cooled culture solution of blood-deprived liver tissue (Williams E medium, Dainippon Pharmaceutical)
And carried into the laboratory. In a sterile hood, a preperfusate warmed to 37 ° C. was infused into the hepatic tissue from the cut blood vessel by cannulation using a peristaltic pump. After injection at a flow rate of 7 to 10 ml / min for about 20 minutes, the solution was digested with a 0.05% collagenase solution containing 1000 unit / ml dispase. Approximately 10 minutes later, when the viscosity of the tissue surface increased, the solution was
When the digestion was continued for another 20-30 minutes instead of the% collagenase solution, the hepatic lobules floated, the inside became muddy, and the blood vessels became unclear. So, I will break the organization with my palm,
The digested and effluent cells were received in a petri dish. According to this method, the only undigested tissue was a small amount of connective tissue, and there was no need to mince with a scalpel. After passing the digested cells through a triple gauze-treated cell filter,
In order to remove non-parenchymal cells, cell debris, damaged hepatocytes, erythrocytes, and the like, low-speed centrifugation was performed at 50 × g for 2 minutes, and the supernatant was discarded. A medium was added to the cell sediment, and centrifugation was repeated three times, and the number of cells was measured. Cell viability by trypan blue exclusion always exceeds 90% and cell yield is about 10 / g of liver.
More than ⁷ hepatocytes were obtained.

In addition to human hepatocytes, chimpanzee and cynomolgus monkey hepatocytes were isolated in a similar manner. A feature of the method for separating hepatocytes of the present invention is to provide fresh hepatocytes from liver tissues that are difficult to obtain, such as humans, without using in situ perfusion. Because of the remarkable yield and low damage, the hepatocytes obtained by this method have very good results in the next step, immortalization with recombinant virus or primary culture for other purposes. Can be given.

Example 2 Method for culturing hepatocytes stimulated by Ad5SVR4 infection and proliferation by the recombinant virus Hepatocytes separated and purified by the method of Example 1 were immortalized by the following method.

Recombinant virus Ad5SVR4 (Gluzman, Y Mol, Cell. Biol.
4: 1653-1656, 1984), the cells could be transmitted in at least two ways. In general,
First, it is started by monolayer culture of the separated hepatocytes after centrifugation. The cells are suspended in a William E medium containing 10% fetal bovine serum (more preferably, a hormone, for example, insulin or dexamethasone, and more preferably a growth factor, for example, EGF), and 2 × 10 ⁴ to 2 × 10 ^5. / cm ² and seeded on culture dishes. After cultivation, typically after 1 to 7 days, preferably about 2 days, the above-described recombinant virus has an MOI of 0.01
Inoculated at ~ 100. The virus contains 293 cells (ATCC, CRL
-1573), and about 10 ⁹ PFU of the obtained culture supernatant was diluted and used. The virus was adsorbed according to a method generally used in virology. That is, the monolayer cultured cells before virus inoculation were washed about three times with a serum-free medium,
The virus solution was added and incubated for 30-120 minutes. After removing the virus solution, the cells were further washed twice with a serum-free medium, the above-mentioned serum-containing medium was added, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ).

A second method of infection of the virus, characteristic of the invention, was started by adding the virus directly to isolated hepatocytes. The virus solution is added to cells 10 ^{6 to} 5 × 10 ⁷ after centrifugation by MOI.
The cells were suspended to be 0.01-100. After adsorbing the virus at room temperature or 37 ° C. for 30 to 120 minutes, the cells were submerged under the above-mentioned centrifugation conditions, and the supernatant, that is, the virus solution was discarded. The cells washed twice by the same method were cultured by the following three culture methods.

In the first method, the treated cells were subjected to the method described above, ie, monolayer culture. In the second and third methods, the treated cells were subjected to embedded culture on or in a collagen gel. The collagen gel was prepared using Nitta Gelatin's Cell Matrix-1A according to the company's method of use. First, 0.3% cell matrix-1A, 10-fold concentration William E medium (containing antibiotics), buffer (0.05 N NaOH 100 ml)
Then, 2.2 g of NaHCO ₃ and 4.77 g of HAPES were dissolved and over-sterilized), mixed at a ratio of 8: 1: 1 while cooling, and the fetal bovine serum containing hormones, growth factors, etc. was added to the final concentration.
It was added to 10%.

Immediately pour the pre-gelled matrix solution into a 35 mm or 60 mm diameter plastic culture dish and at 37 ° C for 10-20
Minutes standing and gelled, on cell matrix gel, treated cells i.e. the virus inoculated cells 2 × 10 ⁴ ~
Seeding was performed at a concentration of 2 × 10 ⁵ / cm ² . Alternatively, the treated cells were suspended in the matrix before gelling and overlaid on the gelled matrix. After further gelation, complete medium was added and 2-4
Every day, preferably every three days, the medium on the gel was completely exchanged and maintained.

Example 3. Isolation and passage of proliferating cell colonies In the case of virus-infected hepatocytes cultured in monolayer, 7 to
At 10 days, the adherent cells gradually began to detach, but 3-6
After a week, many colonies grown from one cell began to be observed. The grown colonies were mainly isolated by the following two methods. Silicon grease is applied to one end of a stainless steel cylinder having an inner diameter of 3 to 8 mm and surrounds a colony to be separated. The medium inside the cylinder is removed, a small amount of cell digestion solution (phosphate buffer solution containing 0.05% trypsin, 0.02% EDTA) is added, and the mixture is incubated at 37 ° C for about 5 minutes. Add complete medium, pipet lightly, and transfer to another culture dish, for example, a 24-well plate. Carefully peel the colonies using a Pasteur pipette while observing the culture dish where the growing colonies are observed with a microscope. The colonies peeled off in the form of a sheet are transferred to another culture dish.

The colonies isolated by the above two methods grew in a monolayer in the well and spread over the entire well in about 1-4 weeks. When the cells had grown sufficiently, the medium was removed, a cell digest solution was added, and the mixture was incubated at 37 ° C. for about 5 minutes to detach the cells from the culture dish. Add complete medium, pipet, add another culture dish,
For example, it was transferred to a 6-well plate. Several days later, when the cells proliferated in the entire well, the cells were subcultured in the same manner, and this operation was repeated.

In the case of virus-infected hepatocytes cultured in or on a collagen gel, no phenomenon was observed in which cells were detached as the culture progressed. However, as in the case of monolayer culture, some cells in the gel or on the gel became atrophy and became opaque from the 7th to 10th days, and it was determined that such cells had died. However, many cells have 3-6
After a week, the colonies grow to the extent that they can be seen with the naked eye, and colonies that grow in the gel often have a diameter of 0.5
It grew to a spherical shape of ~ 1 mm. However, even if it grows to a larger size, the inside of the colony will cause necrosis, so it was considered preferable to carry out the passage by this time.

One of the important features of the present invention is that, by such a culture method using a collagen gel and a method in which the virus is infected immediately after hepatocyte isolation, the hepatocytes can be imparted with permanent proliferation very efficiently. The point is that you can do it. This important feature was more pronounced in higher animal species (eg, chimpanzees than cynomolgus monkeys, and even humans).

First, Table 1 shows that Ad5SVR4 is highly useful for immortalizing hepatocytes. Monocytic cynomolgus monkey hepatocytes were infected with Ad5SVR4 at various MOIs, and the appearance of proliferative foci was observed. It showed a much higher appearance rate (10 ^-3 ) than the original SV40 virus, indicating that the SV40T gene was integrated into hepatocytes at a high rate.

Table 2 shows the difference in the immortalization efficiency of hepatocytes depending on the animal species. In the case of the normal virus adsorption method and the monolayer culture method, chimpanzee hepatocytes were given permanent proliferation only at a very low rate. Similar results were shown in the case of human hepatocytes, and a method characteristic of the present invention as described above was devised. Table 2 also shows the Ad5SVR4 infection method and SV40T
Significant differences in efficiency with the gene-only transfection method are also shown. Here, in the pX-8 is pUC118 ori ^- of
It is a pramid incorporating the SV40T gene.

Table 3 shows that chimpanzees and even human hepatocytes can be immortalized very efficiently by applying the two characteristic methods described above.

Isolation of colonies and passage of cells from collagen gel was performed by digesting collagen gel with collagenase. As a collagenase for gel digestion, a 0.05% solution for separating hepatocytes from liver tissue was used. First, the medium on the gel was removed as much as possible, and a collagenase solution equivalent to the gel was added. Transfer each gel to a separate container, such as a flask for cell digestion, at 37 ° C for 30-60
The gel was digested and the colonies were exposed when the mixture was kept warm and stirred for 1 minute. Before the cells in the colony were dissociated, the colony was aspirated with, for example, a Pasteur pipette. The colonies were transferred to a fresh medium and allowed to stand to settle the colonies, thereby washing them. After 2-3 exchanges of the medium, the colonies were transferred to another culture dish, for example, a 24-well plate, and allowed to stand in a CO ₂ incubator. 1-3
Within days, the colonies adhered to the culture dish and epithelial cells spread from the periphery. The expanded cells proliferated to form a monolayer. When the cells had grown to full wells, they were subcultured according to the monolayer culture subculture method.

Example 4 Albumin Secretion of Immortalized Hepatocytes As a goat anti-human HSA antibody specific to HSA, BIOSYS was used. At a dilution of 1: 2000, 50 μ / well was poured into a microtiter plate and left at 4 ° C. overnight. next day,
The antibody solution is aspirated and washed three times with PBS / 0.05% Tween-20.
A 50% 0.5% gelatin solution was added and left at room temperature for 1 hour. These plates were then washed three times, and 50 μl of an analytical sample (culture supernatant or standard HSA) was added and incubated at 37 ° C. for 1 hour. After washing three times, a peroxidase-conjugated goat antibody against HSA (Kappel) was added, incubated at 37 ° C. for 1 hour, and washed three times. A substrate mixture (OPD) was added, the color was developed at room temperature for 10 to 30 minutes, and the reaction was stopped with a 1N sulfuric acid solution. The solution after the reaction was measured at a difference between two wavelengths of 492 nm and 600 nm. The sensitivity of this ELISA using human purified albumin as a standard was about 0.5 ng or less. As a result of examining the albumin secretion ability of all the isolated colonies (clones), it was determined that the colony was positive in many cases, and it was confirmed that hepatocytes were immortalized with high probability.

Example 5 SV40T Antigen Expression in Immortalized Hepatocytes Cells immortalized by infection with Ad5SVR4 depend on the expression of SV40T antigen inserted into the viral gene for their growth. Therefore, confirming that the immortalized hepatocytes express the SV40T antigen can be one basis for the validity of the present invention.

Expression was confirmed by detecting the SV40T antigen in the cells by a fluorescent antibody method. Immortalized hepatocytes were detached from the culture dish with a trypsin solution during the growth phase (typically 2 days after the passage) and dissociated into isolated cells. About in complete medium
It was seeded 300 microns / WE11 to 10 ⁵ / ml to adjust 8 wells Rabutekku Chamber slides (Miles Inc.). 1 to
Two days later, when the cells formed a monolayer, they were washed five times with PBS so as not to dry the cells. After washing, all PBS was discarded by suction, the slide was dried at room temperature, the cells were fixed with acetone cooled to 4 ° C. for 15 minutes, and then dried again, and dried.
A 50-fold diluted mouse anti-SV40T antibody (Oncogene) was reacted. After reacting at 37 ° C. for 1 hour, the plate was washed gently with PBS and reacted with FITC-conjugated anti-mouse IgG (Kappel) diluted 1:50 as a secondary antibody. After one hour, the cells were washed with PBS, embedded in glycerin, covered with a cover glass, and observed with a fluorescence microscope.

Several clones were selected at random from immortalized hepatocytes obtained from humans, chimpanzees, and cynomolgus monkeys, and the expression of SV40T protein in the cells was examined. Fluorescence was observed. Furthermore, the frequency was positive in 100% of the cells, supporting that the provision of the permanent proliferation of hepatocytes according to the present invention was due to the expression of SV40T.

Example 6 Propagation of HA virus Among the immortalized hepatocytes prepared according to Example 2, clones secreting albumin were analyzed for HA virus infectivity. The immortalized hepatocytes were seeded at a rate of 2 × 10 ⁵ / well in 24 wells / plate and covered with 100 μl of the HA virus solution maintained in green monkey kidney cells. As the virus solution, a 10 ⁸ TCID ₅₀ infectious titer obtained from green monkey kidney cells was diluted 1:50 and used. After incubation at 37 ° C. for 1 hour, 500 μl of complete medium was added, and the cells were cultured at 37 ° C. in a 5% CO ₂ incubator. Almost all of the medium was changed every 3-4 days and maintained for 3 weeks. Since the HA virus is not secreted extracellularly, the extra virus inoculated by such medium exchange was completely removed. The cells were destroyed every week, and the presence of HA virus in the cells was examined.
After completely removing the medium and washing the cells three times with PBS,
Solubilization solution (1% NP-40, 0.4% deoxycholic acid, 15 mM
By adding EDTA-containing Tris-HCl buffer), the cell membrane was destroyed and the cytoplasm was drained. The lysate containing cell components is obtained by the following immunological method (ELISA)
The presence of the virus was confirmed.

First, a rabbit antiserum specific to the HA virus was obtained, and 50 μ / well at a 1: 5000 dilution was poured into a microtiter plate and left at 4 ° C. overnight. The next day, remove the liquid,
After washing three times with PBS / 0.05% Tween 20, 0.2% bovine serum albumin was added to saturate non-specific binding sites,
Stored at 4 ° C. Wash this plate if necessary,
The cell lysate and HA virus standard solution were poured at 50 μ / well and left at 4 ° C. overnight.

The next day, the plate was washed, and a peroxidase-conjugated anti-HA virus antibody was poured at a dilution of 1: 5000, and reacted at 37 ° C. for 3 hours. After washing in the same manner, add the above-mentioned substrate mixture and add
Allowed to react for minutes. The reaction was stopped by adding a 1% sulfuric acid solution, and the absorbance was measured. The HA virus was quantified by measuring HA virus standard solutions at 10, 20, and 30 ng / ml, and was calculated by a parallel line test with the sample.

As a result of examining HA virus proliferation of immortalized hepatocytes selected from humans, chimpanzees, and cynomolgus monkeys, many clones capable of secreting albumin were determined to be positive (Table 4). The viral load decreased in the order of cynomolgus monkey, human, and chimpanzee, but this was considered to be a problem on the virus side. In other words, it is considered that Urus isolated from humans had considerably adapted to kidney cells of green monkeys, which are considered to be more closely related to cynomolgus monkeys. Therefore, it was considered that the hepatocytes to which the present invention has been imparted with permanent proliferation maintain many characteristics of hepatocytes.

Example 7 Propagation of HB virus HB virus infectivity was analyzed for human immortalized hepatocytes H6Ad-1 and H8Ad-17, which were confirmed to be infected with the HA virus in Example 6. The two types of human hepatocytes are
A plastic culture flask (Falcon, 3013) was inoculated at 1-3 × 10 ⁶ / flask at the time of virus inoculation so as to be almost monolayer. According to the usual virus adsorption method, the medium was completely removed, and 0.5 ml of the HB virus solution was inoculated. . HB virus HB611 cells (HB viral genes incorporating hepatoma cell lines, are about ^{107/10 6} cells producing the Dane particles (Turimoto, T, et al., Proc.Natl.Acad.Sci.USA 84: 444- Four
48 (1987)). After adsorption at 37 ° C. for 2 hours, the virus solution was removed, and the medium was washed twice with a medium to remove excess virus. Then, a fresh medium was added and the cells were cultured at 37 ° C. 2
The medium was collected every day, and the HBs antigen and HBe antigen in the medium were measured by Abbott RIA or EIA kit. First result
Shown in the figure. HB virus-specific protein was detected on day 6 post-infection and continued to rise until day 14. This clearly indicates that the cells are producing virus particles.

Next, replication of the viral nucleic acid in the cell and virus-specific transcription were confirmed by the following method. First, extrachromosomal DNA is H
Prepared by the method of irt (J. Mol. Biol. 26: 365-369, 1967). After inoculation of the HB virus, the cells were treated with 0.5 M NaCl / 10 mM T
It was dissolved in ris-Hcl, 1 mM EDTA / 1% SDS, and left at 4 ° C. overnight. After centrifugation at 1600 xg for 1 hour, extrachromosomal DNA in the supernatant
Is reacted with pronase E at 37 ° C. for 1 hour.
It was treated with chloroform and recovered by ethanol precipitation. The obtained DNA was prepared in a TE buffer (10 mM Tris-HCl, pH 7.4 / 1 mM EDT).
The cells were dissolved in (A), electrophoresed on 1.5% agarose, transferred to a nitrocellulose membrane, and analyzed by Southern blotting. Probe using all HB viral DNA was ³² P labeled. Total RNA
Was prepared by homogenizing cells in guanidine thiocyanate and performing cesium chloride equal density gradient centrifugation. The resulting RNA was purified by electrophoresis on a 1% agarose gel containing 6.6% formaldehyde, transferred to a nitrocellulose membrane, and analyzed by Northern blotting.

Southern blot results revealed the presence of virus-specific extrachromosomal DNA. On the other hand, the results of the Northern blot confirmed that RNA at 3.6, 2.4, and 2.1 kilobases was found in the cells 14 days after infection. This is consistent with the results obtained in vivo from HB virus-infected liver, with 3.6
Kilobase mRNA stands for transcription of the entire genome of the virus. Also, the other 2.4,2.1 kilobase mRNA is large H
It implies transcription for Bs and small HBs.

Example 8 Propagation of HNANB virus The HNANB virus infectivity was analyzed for the human immortalized hepatocyte clone H6Ad-1, for which the proliferability of the HB virus was confirmed in Example 7.

The cells were seeded in a 6-well culture plate (Falcon, 3046) at a concentration of about 1 × 10 ⁶ / well, and cultured overnight in a CO ₂ incubator to form a monolayer cell sheet. To the cells were added 0.5 ml of the following two types of serum or plasma which are thought to contain a virus infectious agent and normal human serum as a negative control, and the mixture was incubated at 37 ° C. for 2 hours.

Serum of acute chimpanzee who developed HNANAB hepatitis Exchanged plasma of patients with severe HNANB inflammation After incubating for 2 hours, inoculum was completely removed, complete medium 2m
l and the culture was maintained. Change the medium every 3-4 days,
The cells were collected every week by trypsin treatment, replated on an 8-well chamber slide (Miles), and the HNANB virus antigen was detected by the following fluorescent antibody method.

The medium was removed from the chamber slide with the cells attached, washed three times with PBS and dried. The sample was fixed by immersion in acetone at 4 ° C. for 10 minutes, and dried again. HN
Chimpanzees covered with HNANB convalescent hepatitis serum (1:50 dilution), which may contain ANB virus-specific antibodies. After overnight reaction at 4 ° C. in a wet box, the plate was washed three times with PBS, and an anti-human IgG-FITC-conjugated antibody (MBL) diluted 1: 100 was added as a secondary antibody. After reaction at 37 ° C. for 2 hours, the plate was washed with PBS, embedded in 80% glycerin, and observed with a fluorescence microscope. As a result, cytoplasm-specific granular fluorescence was observed 4 to 6 weeks after inoculation, but was not observed in the negative control (Table 5).

After 8 weeks, the fluorescence disappeared, which is probably due to the fact that the cells began to detach from the culture dish after 6 to 8 weeks. Such detachment of cells was different from the characteristics of CPE due to virus propagation, and was considered to be due to the limitation of culture maintenance. Therefore, the passage of the infectious agent (virus) was proved by the following method.

Five weeks after the inoculation, the cells were collected together with the medium using a cell scraper, the cell membrane was broken by freeze-thawing three times, only the supernatant was obtained by low-speed centrifugation, and 0.5 ml of the supernatant was newly inoculated into H6Ad-1. This operation was repeated (passage infection), and each passage was analyzed by the fluorescent antibody method. Table 6 shows the results. The specific fluorescence shown in Table 5 was proved to be due to a passable infectious agent, and this factor was not identified.
The HNANB virus was strongly supported.

From the above results, it has been clarified that immortalized hepatocytes of humans and non-human primates can confer proliferative properties with any of hepatocyte-specific viruses (HAV, HBV, HNANBV).

[Brief description of the drawings]

FIG. 1 shows HB in immortalized hepatocytes obtained by the present invention.
4 shows the production of virus-specific proteins.

──────────────────────────────────────────────────続 き Continued on the front page (56) References Proc. Am. Assoc. Cancer Res. Annu. Meet. , Vol. 28, No. 0 (1997) p. 123 Proc. Natl. Acad. Sc i. USA, Vol. 86, no. 6 (1989) p. 1875-1879 Mol. Cell. Biol. , Vol. 4, No. 8 (1984) p. 1653-1656 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5/10 C12N 15/86 C12N 7/00 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (10)

(57) [Claims] 1. An immortalized hepatocyte derived from a non-human primate having susceptibility to hepatitis virus, which cell can be obtained by a method comprising the following steps: Isolating liver parenchymal cells by allowing collagenase and dispase to act on liver tissue that has been surgically excised; infecting the hepatocytes with a recombinant adenovirus in which the SV40T gene has been inserted in a floating state; Culturing three-dimensionally in an intercellular matrix. 2. The cell according to claim 1, wherein the hepatitis virus is selected from the group consisting of hepatitis A virus, hepatitis B virus and non-A non-B hepatitis virus. 3. The cell according to claim 1, wherein the non-human primate is selected from the group consisting of chimpanzees, cynomolgus monkeys, green monkeys, rhesus monkeys, Japanese monkeys, and marmosets. 4. The cell according to any one of claims 1 to 3, wherein the intercellular matrix is composed of a substance selected from the group consisting of collagen, fibronectin, laminin, and proteoglycan. . 5. A method for preparing immortalized hepatocytes derived from a non-human primate having susceptibility to hepatitis virus, which comprises the following steps: Isolating liver parenchymal cells by allowing collagenase and dispase to act on liver tissue that has been surgically excised; infecting the hepatocytes with a recombinant adenovirus in which the SV40T gene has been inserted in a floating state; Culturing three-dimensionally in an intercellular matrix. 6. The method according to claim 5, wherein the hepatitis virus is selected from the group consisting of hepatitis A virus, hepatitis B virus and non-A non-B hepatitis virus. 7. The cell according to claim 5, wherein the non-human primate is selected from the group consisting of chimpanzees, cynomolgus monkeys, green monkeys, rhesus monkeys, Japanese monkeys and marmosets. 8. The cell according to any one of claims (5) to (7), wherein the intercellular matrix is composed of a substance selected from the group consisting of collagen, fibronectin, laminin, and proteoglycan. . 9. A method for propagating a hepatitis virus, comprising using the cell according to any one of claims (1) to (4). 10. The hepatitis virus is a hepatitis A virus, B
The method according to claim 9, wherein said method is selected from the group consisting of hepatitis virus and non-A non-B hepatitis virus.