JPH01296982A - Culture method for hepatic cell - Google Patents

Abstract

PURPOSE:To provide the title culture method in such a manner that hepatocytes are put to culture under specified conditions, and using the resultant culture solution separated, hepatic cells are subjected to culture for a long period in such a state as to be suspended in a spherically aggregated form, while retaining stabilized functions and form. CONSTITUTION:Hepatocytes are put to culture in a negative electric charge-free plastic container, using a serumless medium absent from any substrate for cell adhesion and interstitial matter out of hepatic cell, and the resultant cells are separated by, e.g., ultrafiltration to prepare a culture solution. Thence, a spherical cluster of hepatic cell is added to said culture solution to make a culture of the hepatic cells.

Description

[Detailed Description of the Invention] (Industrial Application Field) Hepatocytes are cells that perform biosynthesis of various proteins such as albumin and fibrinogen, and have extremely complex metabolic functions such as detoxification.

Establishing this hepatocyte culture system makes it possible to reproduce in vitro various functions important to these living organisms. In other words, it provides a model for elucidating the still unknown functions of liver cells, and is widely used as a major research method in cell biology. This makes it possible to perform this in an in vitro system without using.

Furthermore, there are great expectations for the production of an artificial liver support device that can perform the various functions described above as a biological artificial liver.

The present invention relates to a method for culturing hepatocytes that have stable functions over a long period of time.

(Prior art) There are many possible causes that influence the biological activity of cultured hepatocytes, but serum factors and medium single-layer substrate are considered to be particularly important. Serum factors include insulin, epithelial growth, It is known that various hormones such as factors play an important role in the engraftment and proliferation of hepatocytes.
There is no consensus on the effects of many other factors in serum on the long-term survival of hepatocytes, and further investigation is required. In addition to collagen and junction proteins, extracellular interstitial substances in which these materials form complexes are known as culture substrates.

Conventionally, hepatocytes are separated from the liver of an adult animal by a collagenase perfusion method, and monolayer culture is often used on a collagen or junctional protein substrate. In addition, a primary culture method using a primary culture container is also used.

Furthermore, subcultured cells with self-propagation ability are also used.

(Problem to be solved by the invention) Regarding the conventionally frequently used monolayer culture method of primary hepatocytes-C
There are many problems with long-term functional maintenance. In other words, the biosynthesis and secretion amount of secreted substances such as albumin are extremely low compared to those in vivo.

Furthermore, when culture is performed in a serum-supplemented medium, parenchymal cells die within one week and non-parenchymal cells appear in their place, making long-term culture impossible.

For long-term culture, it is possible to subculture cells that have undergone transformation and become cancerous, which serves the purpose, but in many cases, they have lost the differentiation function that normal hepatocytes have, and it is difficult to maintain the function. remains a big question mark.

By culturing in a serum-free medium using hepatic extracellular interstitial material as a culture substrate, the cells aggregate into a spherical shape and take on a floating form, which can be cultured for two weeks.Albumin, transferrin, and The secretion of proteins such as fibrinogen was confirmed, and the secretion port remained in vivo for two weeks.
can maintain the level of However, to obtain this hepatic extracellular interstitial material, tSl! >V operation is required.

[Means for Solving the Problems] In order to solve the problems mentioned above, the present inventors have made extensive studies and found that it is possible to culture for a relatively long period of time, and cells aggregate in a spherical shape and float. However, we have established a culture method that maintains the same morphology and function as in vivo, such as the ability to secrete proteins such as albumin, and is easy to operate.

That is, in the present invention, hepatic parenchymal cells separated from the liver are cultured in a non-negatively charged plastic culture vessel without adding a substrate for cell adhesion or extrahepatic interstitial material.
A method for culturing hepatocytes characterized by adding hepatocyte spherules to a culture medium cultured using a serum-free medium, and a method for culturing hepatocytes separated from the liver using a non-negatively charged plastic material. The method is characterized in that hepatocyte spherules are added and cultured in a culture container that has been cultured using a serum-free medium without adding a substrate for cell adhesion or an extrahepatic interstitial substance. The present invention provides a method for culturing hepatocytes. The present invention will be explained in detail below.

The culture solution or culture container for culturing hepatocyte spherules is
The cells may be cultured in accordance with the "Method for spherical culture of hepatocytes" for which the present inventors filed a patent application dated April 30, 1986, and a scented culture solution or culture vessel may be used.

That is, hepatocytes may be obtained from intact hepatic parenchymal cells from the fresh liver of an adult animal, and the method is not particularly limited. For example, the animal is laparotomyd under anesthesia, a cannula is inserted into the portal vein, precancerous cells are flushed out, hepatocytes are dispersed by perfusion with 0.05% collagenase solution, and parenchymal cells are separated by low-speed centrifugation. I can do it.

The serum-free medium used may be one that is normally used for primary culture of animal cells. Specifically, insulin was added to William Medium E in μg/Φ1. Glucagon lOμg/ml. Epidermal growth factor 501g/ll,
Prolactin 20iLl/+Ill, growth hormone 10
uU/1, copper 0.1μ, selenium 3μM, zinc 5
For example, a serum-free medium supplemented with 0 pM may be used.

The culture container may be one that is made of plastic that does not have a negative charge as a base material and is not coated with a substrate for cell contact 7'j such as collagen. Specifically, Falcon Tissue Culture Dish, Falcon Petri Dish, and Brimaria Dish (manufactured by Becton & Dickington).

Coaster dates (manufactured by Data Packaging)
, Sumitomo Bakelite cell culture petri dish (manufactured by Sumitomo Bakelite Co., Ltd.), and the like.

Culture is performed using the materials described above to obtain a culture solution or a culture container. This culture may be performed in a carbon dioxide incubator using a known method. The initial concentration of cells to be cultured is LO5
A range of 11 to 106 cells/ml is desirable. The culture time may be determined in advance by culturing hepatocytes with the culture solution to obtain good results, and is not particularly limited. According to studies by the present inventors, when obtaining a culture solution, it is sufficient to culture for several hours, preferably 3.4 hours. Moreover, when obtaining a culture container, the culture may be carried out for several days, preferably for 4 days or more.

After such culturing, ■1 by ultrafiltration, centrifugation, etc.
A hepatocyte spherical mass is added and cultured using a culture medium or a culture medium from which one cell has been removed and a culture vessel from which cells have been removed. When culturing is carried out using a culture solution, various types of containers can be used, and there are no particular limitations.

In addition, when culturing is performed using a culture container, the culture solution may be a serum-free medium that is usually used for primary culture of animal cells.
There is no need to add special substrates.

The hepatocyte spherical mass is cultured using either the culture solution or culture container thus obtained. What is hepatocyte spheroid?
It refers to a cell mass in which hepatocytes aggregate in a spherical shape and float. There are no particular limitations on the method for obtaining this cell mass. - For example, hepatocytes may be cultured in accordance with the patent application filed by the present inventors on April 30, 1986 entitled "Method for culturing spherical hepatocytes" to obtain spherical hepatocyte masses. Specifically, if a plastic culture container with no negative charge is used and cultured for 4 days under the same conditions as described above, this cell mass will be formed, which will be collected together with the culture medium and subjected to low-speed centrifugation. It can be obtained by separating it from the previous culture solution.

The final goal of culturing hepatocyte spherules requires a culture environment,
Regarding the number of cells, any known method may be used, and there is no particular limitation, but for example, the cells may be cultured in a 5% CO2 incubator at 37°C.

(Effect) The reason why hepatocyte spherules can be cultured in a suspended state for a long time is because proteoglycans were secreted from the previously cultured hepatocytes into the culture medium or culture container used for culturing the hepatocyte spherules. it is conceivable that.

(Effect of the invention) According to the present invention, hepatocytes that are waiting for stable function and morphology,
Long-term culture is possible in a spherical, floating state.

In addition, when culturing hepatocyte spherules using a hepatocyte culture medium, the same culture medium can be used repeatedly, and there are no particular limitations on the container used, and the culture can be easily carried out using normal methods. can. When culturing hepatocyte spherules using a container after culturing, a commonly used culture solution can be used, and there is no need to add a special substrate, and the culture can be carried out easily.

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Adult rats weighing 200 to 250 g were subjected to laparotomy under Nembutal anesthesia, and precancerous flow was performed using Hank's solution containing no calcium ions through a cannula inserted into the hepatic portal vein. 0
．． Perfusion was performed for 6 to 8 minutes with 0.05% collagenase solution. Thereafter, the hepatocytes were dispersed and separated, and non-parenchymal cells were removed by low-speed centrifugation at 50×g for 1 minute three times. William Medium E with insulin IO μg/a I,
Glucagon IO μg/m I, epidermal growth factor 50 ng
/ml, prolactin 2011111011, growth hormone lOμU/ml. Copper 0゜■μM, selenium 3μ
M, 5XlO' of the above hepatocytes were suspended in a serum-free medium (hereinafter abbreviated as HDM) 61 supplemented with 509M zinc, and incubated with 5% carbon dioxide gas for 4 hours in Primary Apotol (manufactured by Becton & Dickinson). The cells were cultured in a carbon dioxide gas incubator at 37°C. After culturing, the culture supernatant was collected and centrifuged at low speed, and all cells were removed.

The hepatocytes obtained in the same manner were cultured under the same conditions for 40 hours, and the cells aggregated into a spherical shape to form a floating mass, which was recovered together with the culture medium, and the cell mass was separated by low-speed centrifugation. It was separated from the culture solution. This cell mass was mixed with the above-mentioned culture supernatant and 1.5 ml each of Primaria, Falcon 100B, and Falcon 3001 (
The cells were transplanted to 3 types of 351 culture dishes manufactured by Becton & Dickinson. 1 per datesh
00 cell clusters were transplanted. The cells were cultured in a 5% carbon dioxide incubator at 37°C, and after 24 hours, the morphology of the cells was classified into the following five types, and 50 to 80 morphologies were observed using a phase contrast microscope to determine their percentages. . As a control experiment, cell clumps were cultured in each container using fresh HDM instead of Culture-L supernatant.

A: Cells aggregated in a spherical shape and suspended.

B: Cells aggregated in a spherical shape but attached to the culture container.

C: A spherical mass adheres to the surface of the culture container and partially extends.

D: The central part of cells that have adhered and extended form a layer.

E: Cells that have adhered and spread form a monolayer.

As shown in Table 1, there is a clear difference in cell morphology when compared to when unused HDM is used, indicating that hepatocytes maintain the A and B morphology suitable for long-term culture. I understand. In other words, the culture supernatant used this time has such an effect. Moreover, even after culturing for more than one week, the morphology was maintained. Furthermore, when observed with an electron microscope, the intracellular organelles retain the same morphology as that of in vivo hepatocytes, and intercellular adhesion spots and small bile ducts are also formed, which also shows that the organelles are morphologically stable. considered to be maintained.

Table 1 Example 2 A cell mass was cultured using a Falcon 1008 Dish under the same conditions as in Example 1, and the amount of albumin secreted per day was measured for two weeks after the culture, and the amount of deoxyribonucleic acid at that time was measured. I found the value. A control experiment was performed using a monolayer layer on a collagen matrix.

The amount of albumin secretion is 1.0 μg/μg at 20 eyes DN
A7 days, this is 611 [1.5 μg by 1
/μg DNA/day and remained at this level for up to 2 weeks [1]. On the other hand, in monolayer culture, 1.0μ
g/μg DNA/day gradually decreased over two weeks to 0.15μg/μgl)N/[l
It became.

From the above, it can be seen that according to the present invention, a culture system that is stable in terms of differentiation function such as protein secretion ability can be established.

Example 3 Culture was carried out under the same conditions as in Example 1 when the supernatant was obtained, and the culture was carried out for one month. The culture solution and cells were removed by suction from the culture container, and used to culture hepatocytes. still,
Unused HDM was used as the culture medium, and the pretreatment of the transplanted cells, the number of cells, and the culture environment conditions were the same as in Example 1.

The cells were cultured for one week without replacing the culture solution, and their morphology was observed using a phase contrast microscope. As a result, the spherical mass of cells maintained its shape for one week after transplantation, and attachment to the bottom surface was extremely slight.

Claims (2)

[Claims] (1) Hepatic parenchymal cells isolated from the liver were cultured in a non-negatively charged plastic culture container using a serum-free medium without the addition of a substrate for cell adhesion or hepatic extracellular interstitial material. A method for culturing hepatocytes, which comprises adding hepatocyte spherules to a cultured medium and culturing them. (2) Hepatic parenchymal cells isolated from the liver were cultured in a non-negatively charged plastic culture container using a serum-free medium without the addition of a substrate for cell adhesion or hepatic extracellular interstitial material. A method for culturing hepatocytes, which comprises adding hepatocyte spherules to a cultured culture vessel and culturing them.