Bovine liver cells in long-term cultivation and their specific properties; albumin production and glycogen storage

The liver cells obtained from a calf have been cultured continuously for 257 days in total at present (May 31, 1967). The primary culture was maintained in rotatory culture for about 2 months with gradual and continuous cell proliferation. The two original strains, LD-BS20 and LD-CS20, have been maintained in static culture since 4th subcultivation. Three substrains, LD-BS10, YLE-BS20 and LD-CS10, were derived from the original strains. Two kinds of appropriate media, in which the cells could be subcultured with trypsin without severe damages and maintained with some characteristic functions of liver cells, were reported. The one consisted of 20 per cent bovine serum, 0.4 per cent lactalbumin hydrolysate and saline D, and the other was added with 0.08 per cent yeast extract to the above mentioned medium. Calf serum examined was not so effective as bovine serum for cell proliferation. Morphologically, the cultured cells resembled parenchymal liver cells quite well. The cells spread wide with abundant pale staining cytoplasm and their large nuclei, oval or round, generally contained one to several nucleoli. The cells as well as the nuclei varied considerably in size, some being two to four times larger than others. Binuclear, trinuclear or polynuclear cells were also observed. No silver impregnated fiber was detected among the epithelial cells. Two attempts to characterize cell types in culture were made. First, the presence of glycogen was tested with PAS reaction and saliva digestion procedure. Secondly, the albumin formation in cultured liver cells was examined with the fluorescent antibody technique. The fact that both albumin and glycogen were observed in the cells suggests strongly that there is a possibility of the continuous cultivation of liver cells by the present method, and by these procedures it seems possible to identify functionally the cultured cells with the parenchymal liver cells.</p

Function of the liver cells in the short-term and the long-term cultures. I. Albumim production of the liver cells in vitro

In the experiments with cultured liver cells it is very important to know whether or not the cells in vitro have the same properties and functions as in vivo. The purposes of this work were to investigate the functions of the cultured liver cells and to identify functionally the liver cells cultured by our present method with the parenchymal liver cells. At first, the albumin production of the cultured liver cells, one of the well known functions of the liver cells, was examined by the immunological methods, especially, the fluorescent antibody technique and the complement fixation test. Culture methods which could display the functions of the liver cells as much as possible were explored simultaneously. The results were as follows: 1. Albumin production was detected in the strain RLN·10 liver cells established from the liver tissues of a Donryu rat with immunofluorescent method and complement fixation test. This confirms that the cultured liver cells maintain the function to produce albumin and these cells have originated from the parenchymal liver cells. 2. Hepatoma strains (AH 66-TC-l, AH 7974-TC-l) also showed the albumin production but the extent of its production was less than that of the strain RLN-10. 3. In the short-term cultured liver cells, the albumin production was testified only slight in one month and was exhibited in a small amount in three months. 4. Every culture method examined exhibited no appreciable difference in the albumin production in the cultured liver cells.</p

Contribution of cell cultures to the Nobel Prize

約1世紀前に, 生きた組織や細胞を動物の体外に取り出した研究―組織培養あるいは細胞培養―が始まった．その後の研究の流れの中で, 1) 組織・細胞培養の特性を生かして行われた研究でノーベル賞に輝いた研究, 2) 組織・細胞培養の経験をもつノーベル賞受賞者, 3) ノーベル賞にはならなかったけれども特記できると私が考える細胞培養の研究，などについて取り上げてみたい

Renato Dulbecco: Dulbecco’s modified Eagle’s medium

現在，医学分野の多くの研究は培養細胞や培養組織を用いてなされている．それらの研究成果が報告されている論文の“材料と方法”の項で，ダルベッコ培地を使用して細胞を培養したといった記載を読んだ研究者は少なくないであろう．また，実際にダルベッコ培地を使用した研究者もいると思う．今回は，この培地を報告したDulbecco（1975年，がんウイルスの研究によってノーベル賞）の波乱万丈の経歴，彼の人脈（多くの人物がノーベル賞を受賞），彼の研究概略などについて紹介したい．そして，筆者のダルベッコ培地に対する見解とその培地を使用した経験について述べる．筆者の結論は，初代細胞培養にはダルベッコ培地が勧められること，また，培養細胞を用いての毒性検定に際しては，使用する培地によって結果が異なることがあるので注意する必要があることである

Properties and identification of rat liver cells in long-term cultivation

When cultured cells are used in experiments, It is very important to know from what kinds of cells the cultured cells are originated, and what characteristics the cultured cells maintain continuously in vitro Some properties of rat liver cells in long-term cultivation were examined for the purpose of identifying the cultured cells with parenchymal liver cells by investigating their functions. The production of rat serum albumin and &#945;-globulin which is regarded as specific functions of liver parenchymal cells was detected in these cultured rat liver cells with the method of immunoelectrophoresis. Histochemically, acid phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, and adenosine triphosphatase were demonstrated in the cultured rat liver cells which were morphologically epithelial. Alkaline phosphatase showed little activity in these cells. Glycogen was recognized by the periodic acid-Schiff technique, when bovine serum concentration in the culture fluid was reduced to 5 per cent. These histochemical findings of cultured rat liver cells were identical with those of parenchymal liver cells in vivo. These facts suggest that there is a possibility of the continuous cultivation of liver cells by the present methods and of the identification of the cultured cells with the parenchymal liver cells from their functions.</p

Effects of antioxidants on survival of adult rat hepatocytes under various oxygen tensions in serum-free primary culture.

Effects of antioxidants, such as superoxide dismutase, vitamin C, vitamin E, 4-(0-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane), and selenite on survival of adult rat hepatocytes were examined under normoxic and hyperoxic conditions in serum-free primary culture. The tested antioxidants, except for vitamin C, significantly increased the survival rate of hepatocytes under the normoxic condition (under air). Thus, even the normoxic culture condition is hyperoxic for hepatocytes. Elevation of oxygen tension (40% O2) caused severe morphologic degeneration of hepatocytes and remarkable decrease in the survival rate of the cells. Addition of the antioxidants effectively protected hepatocytes from the morphologic degeneration, and significantly improved the survival of the cells under the hyperoxic condition. These findings indicate that the antioxidants can maintain the long-term survival of hepatocytes in serum-free primary culture.</p

Presence of Microplastics in Four Types of Shellfish Purchased at Fish Markets in Okayama City, Japan

The worldwide microplastic pollution in our environment is a matter of great concern. Harmful effects of plastics have been reported in various types of organisms including murine animals. We examined the presence of microplastics in four types of shellfish purchased from fish markets in Okayama, Japan and served to the public: short-neck clam (Ruditapes philippinarum, asari in Japanese), hard-shell clam (Meretrix lusoria, hamaguri), brackishwater clam (Cyrenidae, shijimi), and oyster (Crassostrea gigas, kaki). Our analyses demonstrated that approx. 3 pieces of microplastics were present per single shellfish, based on the division of the total number of pieces of microplastic obtained from all 4 types of shellfish by the total number of shellfish examined. Since health problems in humans due to microplastics have not yet been confirmed, further examinations of the effects of ingested microplastics are needed