JPH10179148A - Obtaining of human small liver cell and primary culture and successive culture of the cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently obtaining a human small liver cell having an ability for forming a colony estimated to contain liver precursor cells. SOLUTION: This method for obtaining a human small liver cell comprises treating a normal liver cell collected from a human liver with a mixture solution containing collagenase and dispase, separating the dispersed liver cells into a heavy fraction and a light fraction by a low speed centrifugal method and subsequently collecting the small liver cells in the light fraction. The method for primarily culturing the cell comprises separating the dispersed liver cells into the heavy fraction and the light fraction by a low speed centrifugal method, and subsequently primarily culturing the small liver cells in the light fraction in the mixture of a culture medium containing human serum and an ascorbic acid compound as essential components with a conditioning medium for 3T3 cells to form a colony for obtaining the human small liver cells having a clonic proliferation ability. The method for successively culturing the cell comprises peeling the colony from a culture medium with an EDTA/trypsin solution and again culturing the small liver cells of the peeled colony in the mixture culture medium to form a colony.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for obtaining small human hepatocytes and a method for primary and subculture of these cells. More specifically, the present invention relates to a material for cell biology and molecular biology research relating to the development / differentiation and division / proliferation processes of human hepatocytes, its canceration mechanism, and the like, or a medical material such as a hybrid artificial liver. The present invention relates to a method for obtaining human small hepatocytes useful as a method, and a culture method for obtaining a large amount of human small hepatocytes.

[0002]

2. Description of the Related Art An animal individual is a multicellular organism in which one fertilized egg repeats division and differentiates into various tissues (cell aggregates) sharing different functions. In the case of each tissue constituting the body, each cell constantly divides and proliferates, and the individual is maintained by actively producing cells capable of expressing a differentiation function one after another. Therefore, in order to understand the biological entity of human and other animal individuals, or to elucidate the mechanisms of carcinogenesis and develop therapeutic methods, the cells that make up each tissue must be analyzed by cell biology and molecular biology. It is important to analyze in detail biologically and to clarify the development / differentiation process and the mechanism of division and proliferation.

Conventionally, as a means for analyzing cells of a living tissue in detail, a method has been established in which cells taken out of a living body are cultured, and further, the cultured cells are divided and proliferated to survive in a subculture. I have. However, for rat and human hepatocytes, it has been considered that it is impossible to subculture primary cells isolated from mature individuals. That is, the adhesion-dependent mature hepatocytes are greatly damaged when detached from the culture substrate due to the subculture operation, and it is difficult to reattach to the culture substrate. It was impossible to study the developmental process of hepatocytes and the state of mitotic proliferation.

The inventors of the present invention have overcome the above difficulties by devising the components of the culture medium and succeeded in subculturing primary cells collected from adult rat liver. A method has already been applied for a patent (Japanese Patent Application No. 6-89056). Furthermore, the inventors of the present invention have proposed hepatic progenitor cells (progen
Hepatocytes having clonal proliferative potential, which are thought to contain itor cells), a method for obtaining the same, and a method for continuously culturing those cells have been invented, and a patent application has already been filed (Japanese Patent Application No. Hei. 7-21686). In particular, the subculturing method in the prior application invention involves separating hepatocytes from the liver of an adult mammal, separating the hepatocytes into a heavy fraction and a light fraction by low-speed centrifugation, and The cells are primarily cultured in a medium containing bovine fetal serum and ascorbic acids as essential components to form colonies in hepatocytes belonging to small cells, and the colony cells are detached from the medium with an EDTA solution or an EDTA / trypsin solution. Or reculturing the cells in a medium having the same composition as the primary culture medium or in a medium (conditioned medium) used at the beginning of the primary culture.

Further, the inventors of the present invention have proposed a method of culturing the above-mentioned hepatic parenchymal cells having clonal proliferation ability with a small number of seeded cells, using a medium using small hepatocytes for the above-mentioned primary culture. A method characterized by culturing 3T3 cells in a mixed medium with a conditioned medium or co-culturing small hepatocytes with 3T3 cells has been developed, and a patent application has already been filed (Japanese Patent Application No. 8-133985).
issue).

[0006]

In recent years, the development of an artificial liver incorporating a cultured hepatocyte (hybrid artificial liver) has been actively studied. Hepatocytes isolated from such heterologous animals and cell lines derived from cancer cells have been used. However, when these hepatocytes are implanted in a human body, there have been problems such as an immune response to the invasion of foreign animal cells and carcinogenesis of other organs due to activation of cancer cells.

[0007] For this reason, human normal hepatocytes are expected to be used as cultured hepatocytes using the hybrid artificial liver, but it is difficult to obtain a large amount of normal human hepatocytes. The lack of a probable culturing method for incorporation into a device has been an obstacle to the development of a hybrid artificial liver using human hepatocytes.

[0008] The present invention has been made in view of the above-mentioned circumstances, and develops the knowledge of rat hepatocytes already developed by the present inventors to improve the ability to form colonies from a small amount of normal human liver tissue. It is an object of the present invention to provide a method for obtaining a small hepatocyte having the following, and a method for efficiently subculturing the cell.

[0009]

Means for Solving the Problems The present invention solves the above-mentioned problems by treating normal hepatocytes collected from human liver with a dispase-containing collagenase solution, and then dispersing the dispersed hepatocytes at a low speed. A method for obtaining human small hepatocytes, comprising separating the light fraction into a heavy fraction and a light fraction, and collecting the small hepatocytes in the light fraction (Claim 1).
And human small hepatocytes (claim 2) obtained by this method.

Further, the present invention is characterized in that the human small hepatocytes are cultured in a mixed medium of a medium containing human serum and ascorbic acids as essential components and a conditioned medium of 3T3 cells to form colonies. The present invention also provides a primary culture method for human small hepatocytes (claim 3), and a human small hepatocyte having colony forming ability obtained by the primary culture method (claim 6).

Further, the present invention provides a method for removing human small hepatocytes having the above-mentioned ability to form colonies from a medium by using an EDTA / trypsin solution, and removing the small hepatocytes from a medium containing human serum and ascorbic acids as essential components. A method for subculturing human small hepatocytes (claim 7), which comprises forming colonies by culturing cells in a mixed medium with a conditioned medium.

In each of the above methods, the medium essentially containing fetal bovine serum and ascorbic acids is a DMEM medium containing fetal bovine serum, ascorbic acids, epidermal growth factor, nicotinamide and DMSO. Is a preferred embodiment. In a preferred embodiment, the conditioned medium of 3T3 cells is a culture supernatant obtained by culturing 3T3 cells in a DMEM medium containing fetal calf serum as an essential component.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The human small hepatocytes of the present invention are obtained, for example, by collecting a part of a normal non-tumor tissue from a liver cancer tissue removed by a surgical operation and treating it with a mixed solution of collagenase and dispase. The dispersed hepatocytes can be obtained by centrifuging the dispersed hepatocytes at low speed to separate them into a weight fraction and a weighed fraction, and collecting small hepatocytes in the weighed fraction. More specifically, after treating the collected liver tissue with a known perfusate, dispase (100 to 10,0
(00 PU / ml) and then perfused with a 0.01-1% collagenase solution, and then the liver tissue is digested with a 0.01-1% collagenase solution. Since the injection time of each solution varies depending on the state of the liver tissue, it may be appropriately set while observing the swelling of the tissue by the perfusate. Next, the digested tissue was pipetted with a washing solution to disperse the cells, undigested tissue was removed with sterilized gauze, mesh, or the like, and after filtration, a light-weight fraction was separated by low-speed centrifugation (50 G). Collect the cells contained in the fraction.

According to this method, human normal small hepatocytes can be efficiently obtained from a small amount of normal liver tissue contained in a surgically removed tissue or the like. Next, the human small hepatocytes thus obtained were subjected to the primary culture method described above, that is, a culture medium containing human serum and ascorbic acids (eg, L-ascorbic acid phosphate) as essential culture medium, and 3T3 cells. Conditioned Medium (3T3
And culture using a mixed medium with CM). By using this mixed medium, colonies can be efficiently formed even from a small number of small hepatocytes.

[0015] 3T3 cells are known cell lines obtained from Swiss mouse embryos or Balb / C mouse-derived cells.
Balb3T3 cells can be used, and the conditioned medium is a D medium containing bovine fetal serum as an essential component.
3T3 cells (5 × 10 ⁵ cells / 10 cm dis
h) can be obtained as a culture supernatant after culturing for about 3 days. Then, the conditioned medium is mixed with the medium containing the FBS and ascorbic acids as essential components in a ratio of 1: 9 to 9: 1, more preferably,
They can be mixed in approximately equal amounts for use in culturing small hepatocytes.

The medium containing human serum and ascorbic acids as essential components includes, specifically, human serum,
DMEM media containing ascorbic acids, epidermal growth factor, nicotinamides and DMSO can be used. That is, human serum and ascorbic acids cause colonization of small hepatocytes. Epidermal growth factor (EGF) and DMSO are not essential for colony formation, but have a colony formation promoting action, and nicotinamides are considered to suppress hepatocyte differentiation. It is preferred as a component to perform. Furthermore, the light-weight fraction obtained by low-speed centrifugation contains endothelial cells, Kupffer cells, stellate cells, bile duct epithelial cells, etc., in addition to small hepatocytes, and is considered to provide a special environment for small hepatocytes. However, the above-mentioned nicotinamides, ascorbic acids and DMSO suppress the growth of their non-parenchymal cells, and allow small hepatocytes to be selectively cultured and grown.

The amounts of these components added to the medium are, for example, 5-20% for human serum and 0.1-0.1% for ascorbic acids.
1.0 mM, EGF can be 1-100 ng / ml, nicotinamides can be 1-20 mM, and DMSO can be about 0.1-2%. Culture is performed under 5% CO ₂ conditions.
Perform at a temperature of around 37 ° C. By the culture as above,
Hepatocyte colonies containing human small hepatocytes are obtained. Furthermore, for cells forming these colonies, for example, the presence or absence of peroxisomes, reactivity to hepatocyte markers, reactivity to cancerous hepatocyte markers, reactivity to undifferentiated hepatocyte markers, and By performing screening using at least one of the reactivity of the oval cell with the antibody against the surface antigen as an index, the expression of a differentiation function as a hepatocyte can be confirmed. Of these, the presence or absence of peroxisomes can be confirmed by observation with a transmission electron microscope. Albumin and α ₁ − as hepatocyte markers
Antibodies such as antitrypsin and transferrin, GST-P as a marker of cancerous or undifferentiated hepatocytes,
Known antibodies (OC2, OC3) can be used as antibodies against α-fetoprotein antibody, γ-GTP staining, etc., and as antibodies against oval cell surface antigen. Furthermore, non-parenchymal cells in culture can be identified by using a marker for bile duct epithelial cells, a marker for stellate cells, and the like.

Next, the method for subculturing small hepatocytes of the present invention will be described. That is, this method is a method in which colonies of human small hepatocytes (primary cultured cells) obtained by primary culture are detached from a Petri dish, recultured in another Petri dish, and expanded. When detaching the colony, remove the medium from the Petri dish, and then add ED to the colony.
TA (0.002-0.2%) and trypsin (0.0
(0.5-0.5%) and treating for about 10 minutes, the colonies can be separated into small hepatocytes and non-parenchymal cells.

The small cells thus dispersed are recultured in a mixed medium of 3T3CM and a medium containing human serum and ascorbic acids as essential components. 3T3
Specific components of the CM and the medium mixed with the CM can be the same as those in the above-mentioned primary culture method. Each of the methods of the present invention as described above makes it possible to efficiently obtain a large amount of small hepatocytes capable of forming colonies from a small amount of normal human liver tissue. These hepatocytes are
It is useful not only as a material for cell biology and molecular biology research on the development / differentiation and division / proliferation processes of human hepatocytes, or its canceration mechanism, but also as a material for hybrid artificial livers. It is expected to bring new developments to the development of disease treatment technology.

Hereinafter, the subculturing method of the present invention will be described in more detail and specifically with reference to examples. of course,
The present invention is not limited to the following examples.

[0021]

【Example】

Example 1: Acquisition of human hepatocytes A part of non-tumor normal tissue was collected from human surgically removed liver tissue, and 0.5 mM ethylene glycol-bis (2-a
After injection of Hanks solution containing minoethylene ether) tetraacetic acid for about 10 minutes through a syringe inserted from the vascular stump of the hepatic transection surface to prevent air bubbles from entering, 1,000U
0.05% collagenase solution with P / ml dispase (3
(7 ° C.) was injected from the same syringe for 3 minutes, and a 0.05% collagenase solution (37 ° C.) alone was injected for 15 minutes to digest liver tissue.

Next, the digested tissue was incised in the liver capsule with a scalpel, and 0.4 U / ml in 10% bovine serum albumin (BSA).
After dispersing the hepatocytes by pipetting with a washing solution to which DNase was added, undigested tissue pieces were removed with sterilized gauze, and the mixture was filtered through a 70 μm nylon mesh. The obtained filtrate is centrifuged at low speed (50 G: 1 minute x 3 times)
Then, the sediment of a substantial fraction containing a large amount of mature hepatocytes was removed to obtain a centrifuged supernatant containing a weighed fraction. The non-parenchymal cell fraction was further centrifuged at a low speed (150 G: 5 minutes × 3 times) to obtain human small hepatocytes containing non-parenchymal cells. Example 2 Primary Culture of Small Hepatocytes 3T3 cells (5 × 10 ⁵ cells / 10 cm dish) were cultured in D containing 10% fetal bovine serum, penicillin and streptomycin.
The cells were cultured in a MEM medium at 37 ° C. under 5% Co ₂ conditions. The culture supernatant of the culture medium used for this culture was defined as a conditioned medium (3T3CM).

The human small hepatocytes obtained in Example 1 were subjected to 3.5
seeded in culture dishes cm diameter by 9 × 10 ⁵ cells, DMEM medium (10% human _{serum, 44mM NaHCO 3, 20mM HEPES,} 0.5mg / l insulin, 10 ^-7 M dexamethasone, 30 mg / l L-proline, penicillin and streptomycin Content) and 3T
In a medium (50% 3T3CM) mixed with an equal volume of 3CM,
The cells were cultured at 37 ° C. under 5% CO ₂ for 2 to 3 hours. Next, the culture medium was replaced with a DMEM medium in which 10 mM nicotinamide, 10 ng / ml EGF and 0.2 mM L-ascorbic acid phosphate were added to the above-mentioned mixed medium, and from day 4, 1% DMSO was added to the medium. , Culture for 35 days,
The number of formed colonies was counted. Also, as a control,
The number of colonies when cultured in a DMEM medium not containing 3T3CM was also counted.

The results are as shown in FIGS. 1 (A) and 1 (B). When the cells were cultured in a DMEM medium not containing 3T3CM (FIG. 1: A), the cells were cultured in a 50% 3T3CM medium (FIG. 1A). : B), it was observed that more colonies were formed. Example 3 Subculture of Small Hepatocytes The medium was removed from the petri dish in which the hepatocyte colonies containing the small hepatocytes of Example 2 were formed, and the colonies were reduced to 0.02%.
The cells were treated with EDTA and 0.05% trypsin to disperse colony small hepatocytes and non-parenchymal cells in the solution.
This cell dispersion was pipetted to obtain each dispersion of non-parenchymal cells around the colony and small hepatocytes. Then
A mixed medium of the above 3T3CM and DMAEM medium (50
% 3T3CM).

As a result, it was confirmed that a colony composed of many cells was formed.

[0026]

As described above in detail, according to the present invention, it is possible to efficiently obtain a large amount of small hepatocytes having colony viability from a small amount of normal human liver tissue. These hepatocytes are used not only as materials for cell biology and molecular biology research on the development / differentiation and division / proliferation processes of human hepatocytes, or their canceration mechanisms, but also as materials for hybrid artificial livers. Is also useful, and is expected to bring new developments in the development of treatment techniques for liver diseases.

[Brief description of the drawings]

FIG. 1A is a histogram showing the number of colonies of human small hepatocytes cultured in DMEM medium not containing 3T3CM, and B is a histogram showing the number of colonies of human small hepatocytes cultured in 50% 3T3CM medium. It is.

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[Procedure amendment]

[Submission date] April 16, 1997

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

Claims (9)

[Claims] 1. A method for treating normal hepatocytes collected from a human liver with a dispase-containing collagenase solution, followed by low-speed centrifugation of the dispersed hepatocytes into a heavy fraction and a light fraction. A method for obtaining human small hepatocytes, comprising collecting small hepatocytes. 2. A small human hepatocyte obtained by the method of claim 1. 3. A medium comprising the human small hepatocytes according to claim 2 and human serum and ascorbic acids as essential components.
A primary culture method for human small hepatocytes, which comprises culturing cells in a mixed medium with a conditioned medium to form colonies. 4. The primary human small hepatocyte of claim 3, wherein the medium containing human serum and ascorbic acids as essential components is a DMEM medium containing human serum, ascorbic acids, epidermal growth factor, nicotinamide and DMSO. Culture method. 5. The conditioned medium of 3T3 cells is 3T in DMEM medium containing bovine fetal serum as essential.
4. The primary culture method for small hepatocytes according to claim 3, which is a culture supernatant after culturing three cells. 6. A small human hepatocyte which forms a colony by the primary culture method according to any one of claims 3 to 5. 7. The human small hepatocytes according to claim 6, which have formed colonies, are detached from the medium with an EDTA / trypsin solution, and these small hepatocytes are conditioned with a medium containing human serum and ascorbic acids as essential components and 3T3 cells. A subculture method for human small hepatocytes, which comprises forming colonies by culturing in a mixed medium with Domedium. 8. The subculture of small hepatocytes according to claim 7, wherein the medium containing human serum and ascorbic acids as essential components is a DMEM medium containing human serum, ascorbic acids, epidermal growth factor, nicotinamides and DMSO. Culture method. 9. The conditioned medium of 3T3 cells is a 3T3 medium in DMEM medium containing fetal calf serum as an essential component.
The method for subculturing small hepatocytes according to claim 7, which is a culture supernatant after culturing three cells.