GB2175007A - Culturing epithelial cells in a medium containing a barbituric acid derivative - Google Patents

Abstract

Addition of a barbituric acid derivative enables normal epithelial cells, which might survive for only one week at the best in the absence of a barbituric acid derivative, to survive for as long as approximately one month, thus making prolonged culture possible. The method of the present invention contributes to effective studies on normal epithelial cells and efficient preparation of large amounts of various physiologically active substances produced by normal epithelial cells.

Description

SPECIFICATION Method lox culture of normal epithelial cells Field of the Invention This invention relates to a method for prolonged culture of normal epithelial cells originating from an animal.

Background of the Invention As a result of recent progress in culture techniques and media, culture of animal tissues has gained importance not only in the field of basic researches but in industrial production.

Particular treatments make it possible to culture any cancerous cells which possess biological functions in vivo. On the other hand, prolonged culture, in particular, primary culture of normal epithelial cells possessing normal functions is an important problem to be solved.

Prolonged culture of animal cells might frequently bring about properties different from those of the mother cells, e.g. tumorigenesis caused by transformation. Establishment of a culture which can withstand prolonged culture while maintaining normal functions would significantly contribute to cellular analysis of the biological processes in vivo, studies on carcinogenesis and production of physiologically active substances. The determination of whether a physiologically active substance produced by a cancerous cell is identical or different from that produced by its mother cell will contribute to the utilization of such a substance and serve as a diagnostic means for the discrimination between cancerous and normal cells. Accordingly culture of normal epithelial cells possessing normal functions has been worthy of remark.

Primary culture of normal epithelial cells in vitro would generally result in a very short survival period of the cells, which is a serious obstacle to the examination of their physiological functions or to the production of physiologically active substances. The survival period of matured hepatocytes, which possess the largest number of functions among epithelial cells, in primary culture is approximately one week.

With regard to nonepithelial cells, Hayflick and Moorhead have reported culture of lung fibroblasts for approximately 10 months in 1961. Various improved methods for monolayer culture of epithelial cells have been attempted hitherto. For example, G. Michalopoulos and H.C. Pitot have reported a method for monolayer culture of matured hepatocytes possessing liver-specific functions wherein said cells are cultured on a floating collagen membrane (cf. Exp. Cell Res., 94, 70 (1975)). Since this method exhibits a disadvantage that contraction of the collagen membrane would wither the cells, there have been developed improved methods which include employing a nitrocellulose filter instead of the collagen membrane (cf. Savage, C.R., Jr. and Bonney, R.J., Exp. Cell Res., 114, 307 (1978)) and culturing the cells on a collagen gel nylon mesh (cf. Sirica, A.E., et al., Proc. Natl. Acad.Sci., USA, 76, 283 (1979); Cancer Res., 40, 3259 (1980)). In addition, other methods comprising culture on a biomatrix (cf. Rojkind, M., et al., J. Cell. Biol., 87, 255 (1980)) and use of a fibroblast as the feeder layer (cf. Michalopoulos, G., et al. In Vitro, 15, 796 (1979) and Langenbach, R., et al. Cancer Res., 39, 3509 (1979)) have been reported.

However these improvements would result in troublesome operations or difficult morphological observation. Moreover, the complicated factors thus added are not readily analyzed. In addition, there has been no report presenting a remarkable effect obtained by addition of chemical substances in primary monolayer culture in spite of the various improvements in culture technique as described above.

Summary of the Invention A convenient and practical method for culture may be established by adding a chemical substance which is effective in maintaining cells. As a result of our studies based on the foregoing viewpoint, we have found that barbituric acid derivatives which are known as hypnotic and antiepileptic agents would be effective in maintaining normal epithelial cells for a long time.

More particularly, we have found that addition of phenobarbital in a high concentration to the medium during a carcinogenesis accelerating test with the use of primary cultured matured rat hepatocytes would bring about the survival of the cells, which might die within one week in the absence of said barbituric acid derivatives, 49 days after the initiation of the primary culture in vitro.

The present invention has been completed based on the abovementioned finding and relates to a method for prolonged culture of normal epithelial cells which comprises culturing the normal epithelial cells in a medium containing a barbituric acid derivative.

Brief Description of the Drawings Figure 1 is a graph which shows changes in the survival ratio of hepatocytes with the passage of time.

Figures 2, 3, 4 and 5 are phase-contrast-microscopic photographs of hepatocytes in the presence or absence of PB.

Figure 6 is a graph which shows a cytotoxic effect of 3'-Me-DAB on culture of hepatocytes.

Figure 7 is a phase-contrast-microscopic photograph of hepatocytes treated with MC.

Detailed Description of Preferred Embodiments The present invention makes possible prolonged culture and survival of normal epithelial cells originating from animals including man by adding a barbituric acid derivative to the medium.

Normal epithelial cells to be cultured may be matured rat hepatocytes.

Any medium which is generally available in cell culture and suitable for the growth of normal epithelial cells may be employed.

The barbituric acid derivatives to be added to the medium are those of the following general formula (1):

wherein R1 and R2 represent each a hydrogen atom, an alkyl group, an optionally substituted linear or branched alkyl group having one to six carbon atoms, a saturated or unsaturated cyclic hydrocarbon group or a substituted amino group; and R3 and R4 represent each a hydrogen atom, an alkyl group or a substituted alkyl group having one or two carbon atoms.

Examples of these compound are as follows: phenobarbital mephobarbital

pentobarbital , -3-dimethyl-barbituric acid

cyclobarbital

uramil-N,N'-diacetic acid

hexobarbital

N-methylphenobarbital

5-(1 -hydroxyethyl)-trioxohexahydropyrimidine

5-(1 -aminoethyl)-trioxohexahydropyrimidine

5-( 1 -hydroxyethyl)-3-carboxymethyl-trioxohexahydropyrimidine

5-( 1 -hydroxyethyl)-3-hydroxyethyl-trioxohexahydropyrimidine

5-(1 -aminoethyl)-3-aminoethyl-trioxohexahydropyrimidine

Various methods of culture such as plate culture, roller bottle culture or spinner culture may be employed.

The culture may be carried out in a humidified atmosphere of 5% of carbon dioxide gas at 37"C.

In the method of the present invention, the addition of a barbituric acid derivative enables normal epithelial cells, which would otherwise die within one week, to survive for at least one month.

As a result of an experiment wherein indomethacin, which is a stabilizer of erythrocyte membrane, was added instead of the barbituric acid derivative, it has been found that the former compound would be effective not in maintaining hepatocytes for a long period but in preventing morphological degeneration thereof, thus stabilizing the functions of cell membranes.

To further illustrate the present invention, the following example will be given.

Example I. Experimental culture of primary cultured matured rat hepatocytes: 1-1 Preparation of isolated hepatocytes The liver of a male Donryu rat aged three months was perfused with collagenase to isolate hepatocytes to be tested.

1-2 Medium and culture The basal culture medium used in this study was Eagle's MEM supplemented with heatinactivated bovine serum at 20%, penicillin at 100 U/ml, streptomycin at 100 Ag/ml, kanamycin at 60 CLg/ml and fungizone at 1 ,ug/ml. Dexamethasone and insulin were added to the basal medium described above at final concentrations of 10 juM and 10 ,ag/ml, respectively. The hormone-supplemented medium was named as Dl medium. PB was dissolved in the basal and Dl medium at a final concentration of 3 mM, unless otherwise indicated. These PB-containing basal and Dl medium were named as PB medium and PBDI medium, respectively.

The isolated hepatocytes were finally suspended at a concentration of 3.5X105 cells/ml in the Dl medium. Four and 10 ml aliquots of the cell suspensions were inoculated into 60 and 100 mm Falcon plastic dishes, respectively, and cultured in a humidified atmosphere of 5% CO2 and 95% air at 37"C. Following 1-day attachment period, the culture media were replaced by the basal, PB or PBDI medium and thereafter the culture media were renewed every 2-3 days. Some cultures were continued to be maintained in the Dl medium in the same way.

1-3 Phenobarbital addition test 1-3.1 Methods of test After attachment period- under the condition as defined in 1-2 for 24 hours, the medium was switched over to the following four media: (1) basal medium (Eagle's MEM), (2) PB medium prepared by adding 3 mM of phenobarbital (a product of Maruishi Pharmaceuti cal Co., Ltd., referred to as PB hereinafter) to the basal medium, (3) PBDI medium prepared by adding 3 mM of PB to the Dl medium, and (4) Dl medium.

These media were replaced every two or three days.

The following assays were periodically carried out until the 35th days of the culture: (a) determination of surviving cell number, (b) microscopic observation, (c) assay of the amount of albumin in a medium by radio-immunoassay, (d) assay of the activity of glucose-6-phosphatase (G6 Pase) in cells, (e) assay of the activity of tyrosine aminotransferase (TAT) in cells, (f) induction effects of dexamethasone on the activities as described in (d) and (e), (g) assay of cytochrome P-450 (P-450) contents, (h) assay of P-450 contents induced by PB (P-450PB), and (i) assay of P-450 contents induced by methylcholanthrene (P-450MC).

1-3.2 Results (1) Maintenance effect on survival cells Fig. 1 shows changes in viable cell numbers in the four media with the passage of time. The numbers (1), (2), (3) and (4) shown in Fig. 1 refer to the media (1), (2), (3), and (4) as defined above, respectively.

Figs. 2, 3, 4 and 5 are phase-contrast-microscopic photographs which show morphological changes of the cells with the passage of time. The numbers (1), (2), (3) and (4) shown in these photographs refer to the media (1), (2), (3) and (4) as defined above, respectively.

Fig. 2 shows the condition of hepatocytes which were cultured in the four media for 7 days (X200).

Fig. 3 shows the condition of hepatocytes which were cultured in the four media for 14 days (X100).

Fig. 4 shows the condition of hepatocytes which were cultured in the PB medium (2) for 35 days (X200).

Fig. 5 shows the effect of the absence of PB on the survival of matured hepatocytes in primary culture, wherein A shows the condition of the cells which were continuously treated in the PB medium (2) for 7 days, B shows the condition of the cells which were cultured in the PB medium (2) for 6 days followed by culturing in the absence of PB for one day, C shows the condition of the cells which were continuously treated in the PB medium (2) for 14 days and D shows the condition of the cells which were cultured in the PB medium (2) for 6 days followed by culturing in the absence of PB for 7 days (X100).

Figs. 1 and 2 clearly indicate that the matured hepatocytes can rapidly degenerate and die one week after the initiation of the culture in the system containing no PB (i.e. the basal and Dl media). From the 14th day of the culture, growth of epithelial-like clear cells was observed (cf.

Fig. 3). On the other hand, the matured hepatocytes would gradually decrease in number with the passage of time but could be observed at least until the 49th day of the culture with little morphological degeneration except that nucleoluses were more pronounced than at the initial stage in the system containing 3 mM of PB (i.e. the PB and PBDI media) (cf. Figs. 1, 2, 3 and 4). The PBDI medium turned out to be the most suitable in maintaining the matured hepatocytes (cf. Fig. 1). In the presence of PB, the growth of the abovementioned epithelial-like clear cells was hardly observed (cf. Figs. 2, 3 and 4).

Table 1 shows numbers of surviving hepatocytes treated with PB in various concentrations for six days.

Table 1. Effect of PB concentration on survival of hepatocytes in primary culture PB PB Culture Number of conc-. treatment age hepatocytes* (mM) period (day) (x 104/dish) (day) 0.75 6 7 43.3 + 1.3 1.5 6 7 48.1 + 0.6 3.0 6 7 92.4 + 3.5 *Inoculum size: 140X104 cells/60 mm dish/4 ml DI-medium.

Mean+SD from 3 different dishes.

In order to investigate the effect of PB concentration on survival of hepatocytes in primary culture, the cultures were treated with 0.75, 1.5 and 3 mM PB in the basal medium for 6 days after 1-day attachment period in the Dl medium. The PB-containing media were replaced every 2 days. Numbers of surviving hepatocytes in the presence of 0.75 and 1.5 mM PB were about a half of that in the presence of 3 mM PB (table 1). Furthermore, surviving hepatocytes in the media containing 0.75 and 1.5 mM PB showed morphological degeneration which was very similar to that observed in the absence of PB.

In addition, no maintaining effect could be obtained unless PB was present persistently (cf. Fig.

5).

(2) Effect of maintaining cell functions Albumin Secretion Surviving hepatocytes in the presence of 3 mM PB showed remarkable secretion of albumin into the culture media for at least 35 days in primary culture. Although albumin secretions by hepatocytes in all of the basal, Dl, PB and PBDI medium declined with time in culture, those in the PB and PBDI medium hardly changed for at least 28 days after day 7 of primary culture, showing a relatively high rate of several,ug/ml/2 days (table 2). In the absence of PB combined supplementation of dexamethasone and insulin markedly increased the secretion of albumin, while in the presence of 3 mM PB those hormones hardly affected the secretion of albumin by hepatocytes.

Table 2. Time courses of albumin production of primary cultured hepatocytes in presence or absence of PB Culture Secreted albumin ( g/ml) days Basal DI PB PBDI 0 - 1 15.6 j 2.3 1 - 3 17.9 + 1.3 23.9 + 0.7 35.0 + 1.1 32.3 + 1.6 3 - 5 5.6 + 0.5 10.2 # 0.3 10.4 # 0.7 11.5 j 0.6 5 - 7 0.8 # 0.2 2.0 t 0.7 4.7 + 0.3 6.6 + 0.7 12 - 14 0.2 e 0.03 0.4 + 0.2 3.0 + 0.5 3.9 + 0.2 19 - 21 3.1 j 0.3 5.1 j 0.6 26 - 28 5.1 j 0.6 4.8 # 0.1 33 - 35 6.0 + 0.1 4.2 + 0.5 Mean+SD from 3 different determinations.

The freshly isolated hepatocytes were inoculated at 1.4X 106 cells/60 mm dish containing 4 ml of the Dl medium, incubated for 24 h, and then maintained in the basal, Di, PB or PBDI medium.

Albumin secretion is expressed in zg/ml of medium/2 days of culture, and values are expressed as mean+SD from three different dishes.

G6Pase and TAT Activities G6Pase activities in the cultures maintained in the basal, Dl, PB and PBDI medium were hardly different from each other (table 3). The activities of G6Pase in the all cultures decreased rapidly with time in culture. All of the cultures on the 7th day of primary culture showed merely about 15% of G6Pase activity in rat liver homogenate (53+5 mU/mg protein).

Table 3 shows the result.

Table 3. Time courses of G6Pase activities in primary cultured hepatocytes in presence or absence of PB Culture G6Pase (mU/mg protein) days Basal DI PB PBDI 1 53.2 + 7.2 2 38.1 ±5.8 31.3 + 9.2 40.7 + 5.6 44.1 + 3.4 3 28.8 + 1.6 19.4 + 4.3 30.6 # 3.1 31.3 + 3.6 5 14.2 + 2.7 12.6 + 0.7 14.3 + 2.4 14.9 t 3.0 7 8.1 + 0.9 7.0 + 1.7 8.3 + 0.6 9.8 t 1.4 10 4.4 + 2.1 3.7 + 2.6 5.0 + 0.7 5.3 + 1.0 14 2.5 # 1.4 2.6 # 1.9 5.1 # 1.2 4.9 # 1.0 21 4.1 + 1.6 3.3 + 0.4 28 4.7 + 1.5 3.1 + 0.6 35 5.5 + 2.2 2.0 + 0.1 Mean+SD from 3 different experiments.

Liver homogenate: 53+5 mU/mg protein.

The freshly isolated hepatocytes were inoculated at 3.5X106 cells/100 mm dish containing 10 ml of the Dl medium, incubated for 24 h, and then maintained in the basal, Dl, PB or PBDI medium as indicated. The cultured cells were pooled from four dishes and homogenized. The results are expressed as mean+SD from three different experiments.

TAT activities in the cultures maintained in the basal and Dl medium decreased only with time in culturn, and these cultures on the 7th day of primary culture showed 10 and 15%, respectively, of TAT activity in rat liver supernatant (44+7 mU/mg protein) (table 4). On the other hand, TAT activities in the cultures maintained in the PB and PBDI medium also decreased gradually with time in culture, but hardly changed after the 7th day of primary culture. The cultures maintained in the PB and PBDI medium showed 27 and 57%, respectively, of the TAT activity in rat liver supernatant even on the 35th day of primary culture (table 4).

Table 4 shows the result.

Table 4. Time courses of TAT activities in primary cultured hepatocytes in presence or absence of PB Culture TAT (mU/mg protein) days Basal DI PB PBDI 1 165.3 + 35.0 2 48.4 + 4.5 90.2 + 32.1 96.0 + 16.5 92.9 + 20.1 3 16.9 ::: + 4.0 21.6 + 3.4 34.5 + 8.3 51.3 + 6.4 5 6.7 + 1.6 15.3 + 3.6 22.7 + 2.8 32.3 + 5.9 7 4.3 + 0.2 6.9 + 1.4 13.3 + 3.0 25.7 + 1.2 10 1.9 + 0.7 3.5 + 0.3 9.5 + 0.6 20.2 + 0.8 14 1.1 + 0.2 3.1 + 1.0 10.1 + 0.7 19.0 + 2.3 21 10.5 + 0.4 18.8 + 2.1 28 9.4 + 0.2 20.1 + 8.4 35 11.9 + 1.9 25.1 t12.9 Mean+SD from 3 different experiments.

Liver supernatant: 4+7 mU/mg protein.

The freshly isolated hepatocytes were inoculated at 3.5X106 cells/100 mm dish containing 10 ml of the Dl medium, incubated for 24 h, and then maintained in the basal, Dl, PB or PBDI medium as indicated. The cultured cells were pooled from four dishes and homogenized. The results are expressed as mean+SD from three different experiments.

A well accepted expression of differentiated liver cell function is the ability of hepatocytes to induce liver-specific enzymes in response to hormones. Therefore, this parameter was also used as a marker for the maintenance of differentiated function in the hepatocytes surviving in the presence of 3 mM PB. The cultures maintained in the PB medium were incubated with dexamethasone (10 zM) for 18 h before cell-harvest. The addition of dexamethasone caused a 3-5 fold induction in TAT activity, but caused no induction in G6Pase activity (table 5). The hepatocytes surviving in the PB medium were capable of responding to dexamethasone in this manner at least up to day 28 of primary culture.

Table 5 shows the result.

Table 5. Induction of TAT and G6Pase activities in primary cultured hepatocytes in PB medium by dexamethasone Culture G6Pase (mU/mg protein) TAT (mU/mg protein) days PB PB + Dex* PB PB + Dex* 3 30.6 + 3.1 25.5 + 6.5 34.5 + 8.3 61.6 + 8.2 7 8.3 + 0.6 7.6 + 2.3 13.3 + 3.0 39.5 + 7.8 10 5.0 + 0.7 2.9 + 0.9 9.5 + 0.6 35.2 + 1.5 14 5.1 + 1.2 5.1 + 0.7 10.1 + 0.7 44.0 + 9.2 21 4.1 + 1.6 3.7 + 0.3 10.5 + 0.4 55.2 + 9.5 28 4.7 + 1.5 6.6 + 2.3 9.4 + 0.2 51.6 + 1.0 Mean+SD from 3 different experiments.

Dexamethasone (10 pM) was added 18 hr before cell-harvest.

C yto toxic Effect of 3'-Methyl-4-dimethylaminoazobenzene (3 '-Me-DAB) on Hepatocytes Cultured in the Presence or Absence of PB PB has been well known to induce hepatic microsomal drug-metabolizing-enzymes in animals (Synder, R. et al, "Hepatic Cytochrome P-45 Monooxygenase System", p.227, Pergamon Press, Oxford (1982)). So we investigated the effect of PB-treatment on cytotoxicity of 3'-Me-DAB on the primary cultured hepatocytes. Freshly isolated hepatocytes were attached to dishes in the Dl medium for the initial 24 h. Thereafter, the cultures were continued to be maintained in the same medium or maintained in the PBDI medium for further 2-10 days. The cultures were exposed to 3'-Me-DAB at 0.24 mM from day 1, 2 or 3 of primary culture.Survival number of hepatocytes were much higher in the PBDI medium than in the Dl medium (fig. 6). In other words, 3'-Me-DAB-sensitivity of hepatocytes was reduced remarkably by PB treatment. Furthermore, the longer the PB-treatment period prior to the exposure to 3'-Me-DAB became, the more insensitive to 3'-Me-DAB the hepatocytes became.

Fig. 6 shows the cytotoxic effect of 3'-Me-DAB on the primary cultured hapatocytes in the presence or absence of PB.

Cytochrome P-450 Contents Biochemical determinations of total cytochrome P-450 contents and immunochemical determinations of P-45OPB and P-450MC contents were carried out using homogenates of primary cultured hepatocytes treated with or without 3 mM PB or 10 pM MC.

The total cytochrome P-450 content in the hepatocytes cultured in the Dl medium for 24 h after inoculation was 67.8% of that in the freshly-isolated hepatocytes (tables 6, 7). At this time of culture, PB and MC were added to the primary hepatocyte cultures. The PB-containing medium was renewed every 2 days. Although PB treatment decreased effectively the falling rate of total cytochrome P-450 content in the primary cultured hepatocytes, it failed to induce P 450p, in the cells (table 6). On the other hand, by MC treatment the content of total cytochrome P-450 tended to increase in the primary cultured hepatocytes (table 7). Furthermore, P-450MC was evidently induced in the cells by MC treatment (table 7).

Table 6. Effect of PB on cytochrome P-450 contents in primary cultured hepatocytes Culture Total P-450 content (pmol/mg homogenate protein) days None Basal DI PB PBDI 0 146 (7.2) 1 99 (3.0) 3 40 (0.07) 50 (0) 70 (3.0) 69 (1.9) 5 nd nd 49 56 7 nd nd 46 36 Numbers in parentheses show specific P-450,, contents in pmol/mg homogenate protein. nd: not detectable.

The freshly isolated hepatocytes were inoculated at 3.5X106 cells/100 mm dish containing 10 ml of the Dl medium, indubated for 24 h, and then maintained in the basal, Dl, PB or PBDI medium as indicated. Numbers in parenthese show P-450PB contents in pmol/mg homogenate protein.

'Table 7. Effect of MC on cytochrome P-450 contents in primary cultured hepatocytes Culture Total P-450 content (pmol/mg homogenate protein) days None Basal+MC* DI + MC* PB + MC* PBDI + MC* 0 146 (8.0) 1 99 (7.0)** 3 93 (40.1) 106 (38.8) 115 (30.5) 146 (12.9) Numbers in parentheses show specific P-450MC contents in pmol/mg homogenate protein. *MC (10,uM) was added to the cultures 24 hr after inoculation. **-MC.

MC was dissolved in DMSO and added to the culture media to give a final concentration of 10 XtM, thereby giving a final concentration of 0.5% DMSO. The freshly isolated hepatocytes were inoculated at 3.5X106 cells/100 mm dish containing 10 ml of the Dl medium, incubated for 24 h, and then maintained in the basal, Dl, PB or PBDI medium supplemented with 10,uM MC as indicated. Numbers in parentheses show P-450MC contents in pmol/mg homogenate protein.

Effect of MC on Survival of Hepatocytes MC was added at 1, 10 or 100 M to the primary hepatocyte cultures following 1-day attachment period to examine whether or not MC might be effective for maintenance of hepatocytes in primary culture. Numbers of surviving hepatocytes, which were cultured for 6 days in the basal medium containing 1 and 10 stM MC, were 53.7 and 50.8%, respectively, of that cultured in the PB medium for the same period (table 8). In the basal medium, MC at 100 ,uM was very toxic to hepatocytes. Similarly, in the Dl medium containing MC at 1, 10 and 100 pom, numbers of surviving hepatocytes on the 7th day of primary culture were about a half of that in the PBDI medium (table 8). During the next 1 week in culture, hepatocytes in both of the basal and Dl medium containing MC decreased further in number. Furthermore, hepatocytes surviving in both of the basal and Dl medium containing MC resembled morphologically those in the MCfree basal and Dl medium, showing degenerating features such as degranulation in cytoplasm and multinucleation. Epithelial-like clear cells and fibroblast-like cells proliferated in the MCcontaining media, but hardly did in the PB and PBDI medium.

Stable 8. Effect of MC on survival of hepatocytes in primary culture PB MC Number of hepatocytes -(-x 104/dish) conc.-conc. Basal - DI (mM) (uM) Day 7 Day 14 Day 7 Day 14 0 0 38.2 + 2.3* 6.6 + 4.3* 46.0 + 1.9* 7.1 + 0.3* 0- 1 48.2 + 6.7 10.8 + 0.8 42.8 + 1.0 5.3 + 0.2 0 10 45.6 i 1.5 6.1 + 0.9 54.6 + 2.8 8.8 + 0.6 0 100 5.4 + 2.5 0.6 + 0.3 44.2 t 1.9 10.0 + 0.9 3 0 89.8 + 2.0 65.8 + 4.7 92.3 + 5.0 68.2 + 2.3 Inoculum size: 140X104 cells/60 mm dish/4 ml DI-medium.

Mean+SD from 3 different dishes. *0.5% DMSO.

The freshly isolated hepatocytes were inoculated at 1 .4X 106 cells/60 mm dish containing 4 ml of the Dl medium1 incubated for 24 h, and then maintained in the basal or Dl medium supplemented with MC at 1, 10 or 100 pM. Control cultures were maintained in the basal medium containing 0.5% DMSO or the PB medium following 24-hour attachment period. On the 7th and 14th day of primary culture, the number of surviving hepatocytes were determined by trypan blue dye exclusion in a haemocytometer. The results are expressed as mean+SD from three different dishes.

Fig. 7 shows the condition of primary cultured hepatocytes on the 7th day of the culture (X 100), wherein E refers to those treated with 10 pM of MC one day after the inoculation while F refers to those treated with 3 mM of PB for 6 days.

These data suggest that the maintenance effect of PB on the matured hepatocytes might not be caused by an increase in the detoxication.

1-4 Maintenance effects on cells of hepatocarcinogenic promoters other than PB: PB has been known as a hepatocarcinogenic promoter. Accordingly BHT and DDT, which have been also known as hepatocarcinogenic promoters, and DDE, which is an analog of DDT, were examined whether they would exhibit similar effects to that of PB.

Consequently it has been revealed that each compound would exhibit cytotoxicity but no maintenance effect on matured hepatocytes as shown in the following Table 9.

Table 9. Effect of various chemicals on survival of hepatocytes in primary culture Con- Number of hepato- Secreted albumin Reagent centra- cytes (x 104/dish) ( g/ml) tion 7 days 14 days 5-7 days 12-14 days (nM) BHT 3 0 +0 0 +0 1.5' 0 +0 0 +0 0.75 0 +0 0 FO 0.1 25.7+1.9 2.7+0.7 0.01 27.2+1.3 2.6+0.2 0.001 26.7+0.5 3.2+1.3 DDT 1 0 +0 0 +0 0.5 12.2 # 2.1 0 +0 0.1 26.2 # 2.0 1.8 # 0.8 0.01 24.2 # 2.7 1.9 # 0.4 0.001 23.8 # 2.9 1.7 # 0.5 Con- Number of hepato- Secreted albumin Reagent centra- cytes (x 104/dish) (ug/ml) tion (nM) 7 days 14 days 5-7 days 12-14 days DDE 1 0 +0 0 #0 0.1 38.3 # 5.9 3.3 # 0.5 0.01 39.3 # 2.9 3.8 # 0.7 0.001 41.9 # 3.8 3.9 # 0.4 Pento 3 0 #0 0 +0 1.5 2.8+0.8 0 +0 1 68.4 # 7.3 39.3 # 1.6 2.74 # 0.22 1.82 # 0.06 0.75 82.1 # 1.6 63.4 # 4.3 3.29 # 0.19 3.18 # 0.38 0.5 49.7 # 5.9 26.7 # 4.1 1.51 # 0.12 1.59 # 0.07 0.1 30.8 # 5.7 9.2 # 3.3 Indo 2 0 +0 0 tO 1 76.4 # 6.7 22.5 # 1.1 4.06 # 0.08 2.40 # 0.05 Tal 0.75 41.1 # 2.9 14.7 # 0.7 3.70 # 0.61 0.80 # 0.08 0.5 43.3 # 2.4 8.4 # 1.2 2.61 # 0.28 0.41 # 0.03 0.05 45.7 # 1.2 6.9 # 1.1 Chlor 1 0 +0 0 +0 0.5 1.0+0.5 0 +0 0.1 3.4+1.0 0 +0 0.05 2.2#0.3 0 +0 0.01 32.5 # 4.2 6.2 # 0.7 PB 3 101.7 # 21.0 71.5 # 5.4 3.95 # 0.18 3.53 # 0.28 DMSO 70.4 57.1 # 2.7 3.7 # 0.5 1.25 # 0.04 0.52 # 0.03 None - 47.4 # 4.1 4.9 # 0.4 0.85 # 0.23 0.21 # 0.03 Inoculum size: 140X 104 cells/60 mm dish/4 ml Dl medium.

Each reagent was added 24 hours after the initiation of the culture.

Mean+SD from three different dishes.

As previously described, amobarbital, which is a derivative of PB, has no effect of promoting carcinogesis (Peraino C. et al., Cancer Res., 35, 2884 (1975)). As a result of an examination on the cell maintenance effect of the amobarbital, it has been found that it would exhibit a similar effect of maintaining matured hepatocytes to that of PB at a concentration of 0.75 mM. Table 10 shows the result.

Table 10. Cell maintenance effect of amobarbital Concen- Viable cell ratio % (n=3) Reagent tration 7th day of 14th day of the culture the culture amobarbital 0.75mM 80.2 + 4.0 36.3 + 5.7 pB 3 mum 77.5,+ 4.7 50.3 + 8.7 (blank) - 38.2 + 3.7 4.3 + 0.4 Inoculum size: 140X104 cells/60 mm dish/4 ml These data suggest that the maintenance effect on matured hepatocytes is not common to all hepatocarcinogenic promoters.

On the other hand, pentobarbital, which is a derivative of PB and referred to as Pento in Table 9, exhibited a maintenance effect on matured hepatocytes at a concentration of 0.75 mM (cf.

Table 9).

1-5 Cell maintenance effect of erythromembrane stabilizers: Indomethacin and-chlorpromazine, which are known as erythromembrane stabilizers, were examined in a similar manner.

Chlorpromazine exhibited strong cytotoxicity and no maintenance effect on matured hepatocytes, while indomethacin effectively inhibited morphological degeneration of matured hepatocytes at a concentration of 1 mM while exhibiting cytotoxicity. Accordingly the chlorpromazine could not maintain the cells for a long period as different from PB (cf Table 9).

These data suggest that the stabilized functions of cell membranes might result in the prolonged maintenance of matured hepatocytes by PB.

II. Cell maintenance effect of barbituric acid derivatives: Since amobarbital and pentobarbital would exhibit a cell maintenance effect similar to that of PB, said effect of various barbituric acid derivatives were examined. Each reagent was employed at concentrations of 3 mM, 1.5 mM and 0.75 mM.

Each medium ways introduced into a Petri dish (0 60 mm) and inoculated with 140X104 of matured rat hepatocytes. After culturing for 24 hours to give a cell concentration of 106.9X13.6X104 cells/dish, each reagent of the foregoing concentration was added to the medium. Hexobarbital and mephobarbital were added in the form of 0.5% DMSO solutions while other reagents were directly added to each medium.

Viable cell ratio was assayed 7 and 14 days after the inoculation. Table 11 shows the result.

Table 11 Table 11 Concen- Viable cell ratio * (n=3) Reagent tration 7 days after 14 days after (mM) inoculation inoculation amobarbital 3 0.3 + 0.3 0 + 0 1.5 3.4 + 1.4 2.6 + 0.6 0.75 80.2 + 4.0 36.3 + 5.7 Table 11 hexobarbital 3 88.1 + 2.4 43.8 + 1.3 1.5 53.8 + 4.3 30.2 + 5.0 0.75 40.7 + 1.2 12.6 + 1.7 mephobarbital 3 45.5 + 4.5 4.7 + 1.0 1.5 70.2 + 1.8 23.1 + 2.3 0.75 51.9 + 6.8 19.1 + 4.6 pentobarbital 3 0.1 + 0.1 0 t 0 1.5 2.4 + 0.6 1.6 + 0.8 0.75 72.3 + 4.2 40.8 + 4.2 cyclobarbital 3 24.5 + 0.6 12.2 + 0.6 1.5 62.2 + 5.3 31.3 + 4.8 0.75 32.3 + 1.5 6.5 + 1.5 1-3-dimethyl- 3 65.5 + 4.0 16.7 + 1.4 barbituric acid 1.5 56.5 + 3.6 3.8 + 0.8 0.75 43.1 + 5.6 3.5 + 0.8 uramil-N,N'- 3 0 + 0 0 + 0 diacetic acid 1.5 69.8 + 7.8 36.6 + 4.4 0.75 52.5 i 3.3 25.2 + 1.1 barbituric acid 3 38.0 + 1.8 5.5 + 1.4 1.5 37.8 t 2.6 4.4 t 1.1 0.75 35.8 + 2.2 3.5 + 0.7 phenobarbital 3 77.5 + 4.7 50.3 + 8.7 Medium contain- 70.4 38.8 e11.5 3.2 + 0.5 ing DMSO Medium contain- ~ 38.2 + 3.7 4.3 + 0.4 ing no DMSO As shown in Table 11, barbituric acid exhibited no effect while amobarbital, hexobarbital, mephobarbital. cyclobarbital, 1 ,3-dimethylbarbituric acid, pentobarbital, uramil-N,N'-diacetic acid and phenobarbital exhibited a cell maintenance effect although the optimum concentration would depend on reagents. Consequently it has been confirmed that barbituric acid derivatives are effective in maintaining cells.

Claims (3)

1. A method for prolonged culture of normal epithelial cells which comprises culturing the normal epithelial cells in a medium containing a barbituric acid derivative.

2. A method as set forth in Claim 1, wherein said normal epithelial cells are primary cells originating from liver.

3. A method as set forth in Claim 1, wherein said barbituric acid derivative is a compound of the general formula (I)

wherein R1 and R2 represent each a hydrogen atom, an alkyl group, an optionally substituted linear or branched alkyl group having one to six carbon atoms, a saturated or unsaturated cyclic hydrocarbon group or a substituted- amino group and R3 and R4 represent each a hydrogen atom, an alkyl group or a substituted alkyl group having one or two carbon atoms.