JP3014322B2 - Culture method of small hepatocytes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for culturing small hepatocytes. More specifically, the present invention is used as a material for cell biology and molecular biology research on the development / differentiation and division / proliferation processes of hepatocytes, or its canceration mechanism, or for the development of therapeutic techniques for various liver diseases. For efficient primary and subculture of small hepatocytes (small hepatic parenchymal cells) useful as medical materials for medical treatment.

[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. The cells were re-cultured in a medium having the same composition as the primary culture medium, or were re-cultured in a medium (conditioned medium) used in the early stage of the primary culture.

[0005]

In the above-described method for subculturing hepatic parenchymal cells having clonal proliferation ability, which has been proposed by the inventors of the present invention, it is necessary to form colonies on cells under subculture conditions. Cell seeding density above a certain level
(For example, 5 × 10 ⁴ cells / 35 mm dish).

[0006] Therefore, an object of the present invention is to provide an improved method capable of forming colonies reliably even with a smaller number of cells and efficiently subculturing small hepatocytes.

[0007]

According to the present invention, as a first invention for solving the above-mentioned problems, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are centrifuged at a low speed to obtain a weight fraction. And culturing the small hepatocytes in the lightweight fraction in a mixed medium of a medium containing essential fetal bovine serum and ascorbic acids and a conditioned medium of 3T3 cells to form colonies. A method for culturing small hepatocytes (claim 1) is provided.

According to a second aspect of the present invention, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated by centrifugation at a low speed into a heavy fraction and a light fraction. A method for culturing small hepatocytes, comprising co-culturing cells and 3T3 cells in a medium containing fetal calf serum and ascorbic acids as essential components to form colonies (claim 4).
I will provide a.

As a third invention, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated. After primary culture in a medium containing fetal calf serum and ascorbic acids as essential components to form colonies, EDTA
The cells of the colony were detached from the medium with a trypsin solution to individually disperse the small hepatocytes, and these dispersed small hepatocytes were separated from a medium containing bovine fetal serum and ascorbic acids as essential components and a conditioned medium of 3T3 cells. The present invention provides a method for subculturing small hepatocytes, characterized in that colonies are formed by culturing the cells in a mixed medium (claim 6).

As a fourth invention, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated by centrifugation at a low speed into a weight fraction and a light weight fraction. After a primary culture in a medium containing fetal serum and ascorbic acids as essential components to form colonies, EDTA /
Separating the colony cells from the medium with a trypsin solution to disperse the small hepatocytes individually, and co-culturing these dispersed small hepatocytes and 3T3 cells in a medium containing bovine fetal serum and ascorbic acids as essential components. The present invention provides a method for subculturing small hepatocytes (claim 9), wherein a colony is formed by the method.

As a fifth invention, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated. , A colony is formed by culturing in a mixed medium of a medium containing essential fetal bovine serum and ascorbic acids and a conditioned medium of 3T3 cells, and then the colony cells are detached from the medium with an EDTA / trypsin solution to remove small hepatocytes. Are individually dispersed, and a colony is formed by culturing these dispersed small hepatocytes in the mixed medium (claim 11).

Further, as a sixth invention, hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated by centrifugation at a low speed into a heavy fraction and a light fraction. The cells were cultured in a mixed medium of a medium containing bovine fetal serum and ascorbic acids as essential components and a conditioned medium of 3T3 cells to form colonies, followed by ED.
The colony cells are detached from the medium with a TA / trypsin solution to disperse the small hepatocytes individually, and the dispersed small hepatocytes and 3T3 cells are co-cultured in a medium containing essential fetal bovine serum and ascorbic acids. The present invention provides a method for subculturing small hepatocytes (claim 14), wherein a colony is formed by performing the method.

In these methods, the medium containing fetal bovine serum and ascorbic acids as essential components is a DMEM medium containing fetal bovine serum, ascorbic acids, epidermal growth factor, nicotinamide and DMSO. This is a preferred embodiment. In addition, the first and third
And in the fifth invention, the conditioned medium of 3T3 cells is a DM containing bovine fetal serum as essential.
A preferred embodiment is a culture supernatant obtained after culturing 3T3 cells in an EM medium.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Small hepatocytes serving as controls for primary culture in the first and second inventions can be obtained by the method of the above-mentioned prior invention (Japanese Patent Application No. 7-21686). That is, hepatocytes are collected from the liver of a mature mammal, a light fraction obtained by low-speed centrifugation (50 G) is separated, and the cells contained in this fraction are cultured. At this time, the first
According to the invention, a mixed medium of a medium containing essential fetal bovine serum (FBS) and ascorbic acids (for example, L-ascorbic acid phosphate) and a conditioned medium of 3T3 cells (3T3CM) is used as the culture medium. 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.

As a medium containing FBS and ascorbic acids as essential components, specifically, a DMEM medium containing FBS, ascorbic acids, epidermal growth factor, nicotinamides and DMSO can be used. That is, FBS 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 preferable as a component to be used. further,
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 thought to provide a special environment for small hepatocytes. 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, 5-30% for FBS and 0.1-1.% For ascorbic acids.
0 mM, EGF can be 1-100 ng / ml, nicotinamides can be 1-20 mM, and DMSO can be about 0.1-2%. Culture was performed under 5% CO ₂ conditions for 3 hours.
This is performed at a temperature of about 7 ° C. Hepatocyte colonies containing clonally growing small hepatocytes are obtained by the above culture. 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. Hepatocyte markers include antibodies such as albumin, α ₁ -antitrypsin, transferrin, etc., and GST-P, α-fetoprotein antibodies, γ-GTP
Antibodies against the surface antigen of oval cells were used for staining and the like. Antibodies (OC2, OC3) prepared by Hixson et al.
Can be used. 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.

On the other hand, in the primary culture in the second invention, small hepatocytes in the light-weight fraction obtained by low-speed centrifugation
This is a method for forming colonies efficiently by co-culturing with 3 cells. As the medium in this case, a medium containing essential fetal bovine serum and ascorbic acids as described above, specifically, a DMEM medium containing FBS, ascorbic acids, epidermal growth factor, nicotinamides, and DMSO should be used. Can be. Each component of the medium can be the same as described above. further,
About the same number of small hepatocytes and 3T3 cells may be sowed in a medium and co-cultured.

Next, the method for subculturing small hepatocytes of the present invention (third to sixth inventions) will be described. That is,
This method is a method in which hepatocyte colonies (primary cultured cells) obtained by primary culture are detached from a Petri dish, recultured in another Petri dish, and expanded. The primary culturing method may be the same method as the above-mentioned prior invention (Japanese Patent Application No. 7-21686) (third and fourth inventions), or
The primary culture method according to the first and second inventions may be used (fifth and sixth inventions). In the case of primary culture cells obtained by either method, when removing colonies, the medium was removed from the Petri dish, and EDTA (0.002) was added to the colonies.
~ 0.2%) and trypsin (0.005 ~ 0.5%).
The colony can be separated into small hepatocytes and non-parenchymal cells by adding the above solution and treating for about 10 minutes. The cells can be individually dispersed by filtering these cell separation solutions with a filter (pore size: about 20 μm) to remove small aggregations.

In the third and fifth aspects of the present invention, the small cells dispersed as described above are mixed with a medium containing essential fetal bovine serum and ascorbic acids in 3T3
Re-culture in a mixed medium with CM. As a result, even if the cell seeding density (for example, 5 × 10 ⁴ cells / 35 mm dish) is about 1/5 of the cell seeding density required for the colony formation in the subculture method of the prior application, it is possible to obtain a good colony. Can be formed. Specific components and the like of 3T3CM and a medium to be mixed with 3T3CM can be the same as those of the first invention.

On the other hand, the fourth and sixth inventions are methods for forming subcultured colonies even at a small cell seeding density by co-culturing dispersed small cells with 3T3 cells. The type of 3T3 cells, the medium for co-culture, the number of seeded cells, and the like can be the same as in the second invention. The method of the present invention as described above can be applied to all mammalian hepatocytes including humans,
Colonies can be formed in small hepatocytes (hepatic parenchymal cells) having clonal proliferation ability obtained from various animal species and primary culture is performed, and the primary culture cells can be further subcultured. And, for example, subcultured cells of hepatic parenchymal cells having clonal proliferative ability collected from human liver can be used for producing hybrid liver and the like, and bring new development to the development of treatment technology for liver diseases. Expected.

Hereinafter, the subculture 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.

[0023]

【Example】

Example 1 (1) Acquisition and Primary Culture of Hepatocytes Liver cells were collected from 10-week-old Fischer rats by collagenase perfusion and centrifuged at low speed (50 g, 1 minute × 3).
Times), the supernatant obtained was further centrifuged at low speed (150 g, 5 minutes × 3 times) to obtain a non-parenchymal cell fraction as a precipitate.
9 × 10 ⁵ cells were inoculated in a 3.5 cm diameter culture dish, and DMEM medium (10% FBS, 44 mM NaHCO ₃ , 20 mM HEPE
S, 0.5mg / l insulin, ^10-7M dexamethasone, 30mg
/ lL-proline, penicillin and streptomycin) at 37 ° C under 5% CO ₂ for 2-3 hours. Next, the culture medium was replaced with a DMEM medium obtained by adding 10 mM nicotinamide, 10 ng / ml EGF and 0.2 mM L-ascorbic acid phosphate to the above medium.
From the day, 1% DMSO was added to the medium, and the culture was continued. (2) Preparation of conditioned medium of 3T3 cells 3T3 cells (5 × 10 ⁵ cells / 10 cm dish) were prepared from 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. Then, the culture supernatant of the medium used for this culture was used as a conditioned medium. (3) Subculture of small hepatocytes The culture medium was removed from the petri dish in which hepatocyte colonies including small hepatocytes were formed, and the colonies were treated with 0.02% EDTA and 0.05% trypsin to obtain small hepatocytes of the colonies. And non-parenchymal cells were dispersed in the solution. Pipette this cell dispersion, and non-parenchymal cells around the colony,
A dispersion of each of the small hepatocytes was obtained. These dispersions were then filtered through a filter having a pore size of 20 μm to remove small cell aggregation and to substantially disperse the individual cells. The dispersed cells are slightly seeded on a petri dish at various cell seeding densities, and the conditioned medium of 3T3 cells and a DMAEM medium (10% FBS, 44 mM NaHCO ₃ , 20 mM)
M HEPES, 0.5 mg / l insulin, 10 ^-7 M dexamethasone, 30 mg / l L-proline, penicillin and streptomycin) were mixed in equal amounts in a medium (50% 3T).
3CM) and the number of colonies formed and the number of cells per colony were counted. As a control, culture was similarly performed using the fresh DMEM medium (Fresh Medium), and colony formation and cell number were similarly measured.

The results are as shown in FIG. 1 and FIG. That is, when cultured in a medium containing a 3T3 cell condition medium, the number of cells (1 × 10 ⁴ c) is 1/5 of the number of cells required for colony formation by the conventional method.
ells) (FIG. 1). More importantly, small hepatocyte colonies cultured in a medium containing conditioned medium of 3T3 cells were composed of about 10 times as many cells as colonies in the control medium (FIG. 2). Example 2 Small hepatocytes obtained by the same method as in Example 1 were
Similarly, after removing from the medium and dispersing in the same manner as in Example 1, the small hepatocytes (1 × 10 ⁴ cells) were mixed with the same number of 3T3 cells, and the same DMEM medium (10% FBS, 44 mM NaHCO ₃ , 20 mM HEPES, 0.5 mg / l insulin, 10 ⁻⁷ M dexamethasone, 30 mg / l L-proline, penicillin and streptomycin). The 3T3 cells used were those previously peeled from the medium with the same EDTA / trypsin solution as used for small hepatocytes.

The results are as shown in FIG. 3 and FIG. As is clear from these results, by co-culturing with 3T3 cells, colony formation was observed with 1/5 of the number of cells required for colony formation by the conventional method (1 × 10 ⁴ cells) (1). It was confirmed that the formed colony had a sufficient size (area) (FIG. 4).

[0026]

As described in detail above, according to the present invention, small hepatocytes having clonal proliferative potential, which are thought to contain hepatic progenitor cells, can be efficiently cloned even with a small number of cells, and furthermore, they can be subcultured. Can be cultured. As a result, the development and differentiation process of hepatocytes,
It will be possible to study the proliferation and function expression mechanism in detail, and to open up a new way to elucidate the mechanism of various human liver diseases including liver cancer and to develop therapeutic methods.

[Brief description of the drawings]

FIG. 1 is a graph showing the number of colonies on day 9 of culture formed in two types of media for each cell seeding density.

FIG. 2 is a graph showing the number of cells per colony on day 9 of culture formed in two types of media for each cell seeding density.

FIG. 3 is a graph showing the number of colonies on the fifth day of co-culture of small hepatocytes and 3T3 cells.

FIG. 4 is a graph showing the average of the area of each colony on day 5 of co-culture of small hepatocytes and 3T3 cells.

Continuation of the front page (56) References JP-A-8-112092 (JP, A) JP-A-7-274951 (JP, A) JP-A-7-274952 (JP, A) JP-A-62-83883 (JP) , A) JP-A-64-85918 (JP, A) JP-A-60-501190 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (15)

(57) [Claims] 1. A method for separating hepatocytes from the liver of an adult mammal, separating the hepatocytes by centrifugation at a low speed into a heavy fraction and a light fraction, and converting the small hepatocytes in the light fraction into fetal bovine serum. A method for culturing small hepatocytes, comprising culturing in a mixed medium of a medium containing essential ascorbic acids and a conditioned medium of 3T3 cells to form colonies. 2. A medium containing bovine fetal serum and ascorbic acids as essential components, comprising: fetal bovine serum, ascorbic acids,
The method for culturing small hepatocytes according to claim 1, which is a DMEM medium containing epidermal growth factor, nicotinamides and DMSO. 3. The conditioned medium of 3T3 cells is 3T in DMEM medium containing bovine fetal serum as essential.
The method for culturing small hepatocytes according to claim 1, which is a culture supernatant after culturing three cells. 4. A hepatocyte is separated from the liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes and the 3T3 cells in the light fraction are separated. A method for culturing small hepatocytes, comprising forming a colony by co-culturing in a medium containing bovine fetal serum and ascorbic acids as essential components. 5. A medium containing fetal bovine serum and ascorbic acids as essential components, wherein the medium comprises fetal bovine serum, ascorbic acids,
The method for culturing small hepatocytes according to claim 4, which is a DMEM medium containing epidermal growth factor, nicotinamides and DMSO. 6. A hepatocyte is isolated from the liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum and After primary culture in a medium containing ascorbic acid as an essential component to form colonies, the colony cells are detached from the medium with an EDTA / trypsin solution to disperse small hepatocytes individually, and these dispersed small hepatocytes are dispersed. Is cultured in a mixed medium of a medium containing essential fetal bovine serum and ascorbic acid and a conditioned medium of 3T3 cells to form colonies, whereby a method for subculturing small hepatocytes is characterized. 7. A medium comprising fetal bovine serum and ascorbic acids as essential components, wherein the medium comprises fetal bovine serum, ascorbic acids,
7. The method for subculturing small hepatocytes according to claim 6, which is a DMEM medium containing epidermal growth factor, nicotinamides and DMSO. 8. The conditioned medium of 3T3 cells is 3T in DMEM medium containing fetal calf serum as essential.
The subculture method of small hepatocytes according to claim 6, which is a culture supernatant after culturing three cells. 9. Hepatocytes are collected from the liver of an adult mammal, and the hepatocytes are separated by centrifugation at a low speed into a heavy fraction and a light fraction, and the small cells in the light fraction are separated from fetal bovine serum and ascorbin. After primary culture in a medium containing acids as essentials to form colonies, colony cells are detached from the medium with an EDTA / trypsin solution to disperse small hepatocytes individually. 3
A subculture method for small hepatocytes, which comprises forming a colony by co-culturing T3 cells with a medium containing fetal calf serum and ascorbic acids as essential components. 10. A medium containing fetal bovine serum and ascorbic acids as essential components, wherein the medium contains fetal bovine serum, ascorbic acids, epidermal growth factor, nicotinamide and DMS.
The method for subculturing small hepatocytes according to claim 9, which is a DMEM medium containing O. 11. A hepatocyte is separated from a liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum. After culturing in a mixed medium of a medium containing essential and ascorbic acids and a conditioned medium of 3T3 cells to form colonies, the colony cells are detached from the medium with an EDTA / trypsin solution to disperse small hepatocytes individually. And subculturing the dispersed small hepatocytes in the mixed medium to form colonies. 12. A medium comprising fetal bovine serum and ascorbic acids as essential components, wherein the medium comprises fetal bovine serum, ascorbic acids, epidermal growth factor, nicotinamide and DMS.
The method for subculturing small hepatocytes according to claim 11, which is a DMEM medium containing O. 13. The conditioned medium of 3T3 cells is expressed in DMEM medium containing bovine fetal serum as an essential component.
The method for subculturing small hepatocytes according to claim 11, which is a culture supernatant after culturing T3 cells. 14. A hepatocyte is separated from the liver of an adult mammal, and the hepatocyte is separated into a heavy fraction and a light fraction by low-speed centrifugation, and the small hepatocytes in the light fraction are separated from fetal bovine serum. After culturing in a mixed medium of a medium containing essential and ascorbic acids and a conditioned medium of 3T3 cells to form colonies, the colony cells are detached from the medium with an EDTA / trypsin solution to disperse small hepatocytes individually. These dispersed small hepatocytes and 3T
A method for subculturing small hepatocytes, comprising forming a colony by co-culturing three cells with a medium containing bovine fetal serum and ascorbic acids as essential components. 15. A medium containing fetal bovine serum and ascorbic acids as essential components, wherein the medium comprises fetal bovine serum, ascorbic acids, epidermal growth factor, nicotinamide and DMS.
The method for subculturing small hepatocytes according to claim 14, which is a DMEM medium containing O.