FR2582315A1 - PROCESS FOR CULTING NORMAL EPITHELIAL CELLS - Google Patents

Abstract

METHOD FOR PROLONGING THE LIFE OF NORMAL ANIMAL EPITHELIAL CELL CULTURES. IT CONSISTS OF ADDING TO THE CULTURE MEDIUM A DERIVATIVE OF BARBITURIC ACID OF FORMULA: (CF DRAWING IN BOPI) IN WHICH R AND R EACH REPRESENT A HYDROGEN ATOM; AN ALKYL GROUP; A LINEAR OR BRANCHED ALKYL GROUP, POSSIBLE SUBSTITUTE AND HAVING 1 TO 6 CARBON ATOMS; A CYCLIC HYDROCARBON GROUP, SATURATED OR UNSATURATED; OR A SUBSTITUTE AMINO GROUP; AND R AND R EACH REPRESENT A HYDROGEN ATOM; AN ALKYL GROUP; OR A SUBSTITUTE ALKYL GROUP WITH 1 OR 2 CARBON ATOMS. IT ALLOWS, ESPECIALLY IN THE CASE OF HEPATOCYTES, CELL SURVIVAL 49 DAYS AFTER THE START OF PRIMARY CULTURE, WHILE IN THE ABSENCE OF THESE DERIVATIVES IT DOES NOT EXCEED A WEEK.

Description

The present invention relates to a method

  prolonged culture of normal epithelial cells,

Animal Rig.

  Recent advances in culture techniques and media have resulted in the importance of animal tissue cultures not only in the field of basic research, but also in

  in that of production on an industrial scale.

  Special treatments make it possible to

  worm in vivo of cancer cells with functional

  biological conditions. On the other hand, the problem remains to be solved.

  prolonged cultures, particularly primary cultures of normal epithelial cells, with

normal functions.

  Prolonged cultures of animal cells can

  frequently cause the manifestation of properties different from those of the mother cells, for example a

  tumorigenesis caused by transformation. The establishments

  culture, which can withstand the long, while maintaining its normal functions, would bring a clear

  contribution to the cellular analysis of biological processes

  in vivo, studies on carcinogenesis, and

  production of physiologically active substances. The fact

  to be able to determine whether a physiologically

  produced by a cancer cell, is identical to or different from that produced by the parent cell, would be involved in the use of that substance, and

  serve as a means of diagnosis to discern

  cancerous cells of normal cells. As a result, normal epithelial cell culture

  normal functions, deserves a comment.

  Primary cultures of normal epithelial cells, in vitro, usually have only a very short period of cell survival,

  serious obstacle to the examination of their physiological functions.

  or the production of physiologically acceptable substances

  tives. The survival period of mature hepatocytes, which

  have the greatest number of functions among the cells

  epithelial cells, in primary culture, is of the order of 1

  Maine. With respect to non-epithelial cells, Hayflick and Moorhead mentioned in 1961 a duration of

  approximately 10 months of culture for pulmonary fibroblasts

  res. To date, various improved processes have been tried

  for the monolayer culture of epithelial cells.

  For example, G. Michalopoulos and H. C. Pitot describe a

  monolayer culture process of mature hepatocytes

  the specific functions of the liver, in which these

  lules are grown on a collagen membrane in addition to

  pension (see exp.Cell Res 94, 70 (1975).

  has the disadvantage that the contraction of the

  collagen brane wilts the cells, we have developed

  improved processes which include the use, on the

  of this collagen membrane, a nitrocellulose filter,

  lulose [cf. C.R. Savage Jr. and R.J.Bonney, Exp. Cell. Res., 114, 307 (1978)], and culturing the cells on a collagen gel nylon screen [cf. A.E. Sirica et al., Proc. Natl. Acad. Sci. USA, 76, 283 (1979); Cancer Res., 3259 (1980)]. In addition, other methods including

  culture on a biomatrix [cf. M. Rojkind et al., J.Cell.

  Biol., 87, 255 (1980) 7, and the use of a fibroblast as a feeder layer [cf. G. Michalopoulos et al., In Vitro, 15, 796 (1979), and R. Langenbach et al. Cancer

  Res., 39, 3509 (1979) have been reported.

  However, these improvements lead to

  difficult or difficult morphological observations.

  the. Moreover, the complicated factors thus added are not easily analyzed. Finally, there is no report

  having a remarkable effect, obtained by the addition of sub-

  chemical stains in the primary monolayer culture, despite the various improvements in

culture, reported above.

  The present invention is based on the observation

  addition of derivatives of barbituric acid, which

  are known as hypnotic and anti-epileptic agents

  is effective for cell conservation

  normal epithelial, for a long time; we have

  more particularly than the addition of phenobarbital in high concentration, in the middle, during a test

  acceleration of carcinogenesis, with use of

  ripe rat cells in primary culture, resulted in the survival of the cells, which could die within a

  week in the absence of these barbituric acid derivatives

  49 days after the beginning of the primary crop in vi

  tro. The new process for extending the cultivation of

  normal epithelial cells to cultivate these cells

  in a medium containing a barley acid derivative

  biturique. In the following drawings: FIG. 1 represents a graph which shows the changes in the survival rates of the hepatocytes (in ordinate the number of hepatic cells), as a function of

  the duration of the cultivation (range in number of days

cisse).

  Figures 2 to 5 are microscopic photographs

  Hepatocyte phase contrast mice, in the presence

  this or the absence of PB (phenobarbital); Figure 6 is a graph that shows the effect

  cytotoxic effect of 3'-Me-DAB (3-methyl-4-dimethylaminobenzene)

  zene) on hepatocyte culture; on the ordinate the rate% of living cells and in abscissa the number of days of culture; Figure 7 is a microscopic photograph

  in phase contrast of hepatocytes treated with MC (methyl-

  cholanthrne) The present invention allows the extended culture and survival of normal animal epithelial cells, including humans, by addition in the middle of a derivative

  barbituric acid. Normal epithelial cells

  the ones to be cultivated can be mature rat hepatocytes.

  Any medium generally used

  for cell cultures, suitable for growth

  this normal epithelial cells.

  The derivatives of barbituric acid, which can be added to the medium, are represented by the following general formula:

O R3

iR Y N

R

R N

O R4

  wherein R1 and R2 each represent a hydrogen atom

  gene, an alkyl group, a lindar or branched alkyl group

  , optionally substituted and having 1 to 6 carbon atoms

  bone; a cyclic hydrocarbon group, saturated or unsaturated

  or a substituted amino group; R3 and R4 each represent a hydrogen atom; an alkyl group; or one

  substituted alkyl group, having 2 carbon atoms.

  Examples of these compounds are listed below.

  after. mephobarbital phenobarbital

O O

C H NH NH

NH3 'N

0 o CH3

  pentobarbital acid dimethyl-1,3 barbituri-

than

O O0 CH3

C2 H5 ') "Nil H N / r- O

CH3CH2CH2CH

amobarbital CH3 0% C2H5

(CH3) 2CHHCH2CH2 N

NH o0 cyclobarbital

C2H5 <NH

  NHXu Go uramil acid N, N-diacetic

O

H NH

HOOCCH2 /

N NH H

HOOCCH2 /

2

o hexobartital

CH N / 3

<N 3

CHO

NO OH

N-methylphenobarbital

CH

O / c3

C H N

OH

NOT

  O- (5-hydroxyethyl) trioxohexahydropyrimidine

CH3 H

CH

HO \ -

HX

0

  (amino-ethyl) -5-trioxohexahydropyrimidine O

CH3. C H

NH2 C

NH \ -O

H H NH

  (5-hydroxyethyl) 3-carboxymethyl trioxohegahydro-

pyrimidine COO-I I

CH2

o I I -o HO N

H

  (hydroxy-ethyl) -5-hydroxyethyl-3-trioxohexahydropyridine

midine CH2OH: CH2 o!

CH3_CH N

I v

HO F

H NH

H /

  (amino-1'ethyl) -5-aminoethyl-3-trioxohexahydropyrimidine

CH2NH2

CH2

CH3- CH N ..

N2

H -N

  Various culture methods can be used, such as plate culture, rotational culture, rotating bottle culture or rotary tube, etc.

  It can be operated at 37 C, in humid atmosphere with

5% carbon dioxide.

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

  In conclusion, an experiment was

  tion, during which indometha-

  which is a stabilizer of the membrane of the erythrocyte,

  instead of the derivative of barbituric acid, that the first

  The first compound proved effective, not for the long-term preservation of hepatocytes, but to prevent their morphological degeneration, thus stabilizing the

  functions of cell membranes.

  The following example is given to better illustrate

the present invention

EXAMPLE

  I. Experimental culture of rat mice hepatocytes in culture

  primary ture 1) Preparation of isolated hepatocytes The liver of a male Donryu rat, 3 months old, is perfused with collagenase, in order to isolate the hepatocytes to be tested. 2) Environment and culture

  The basic culture medium, used during this

  The study is Eagle MEM medium supplemented with 20% heat-inactivated bovine serum, 100 U / ml penicillin, μg / ml streptomycin, 60 μg / ml kanamycin, and 1 μg / ml fungizone. At this basic medium, we add

  dexamethasone and insulin, at final concentrations of

  the respective ones of 10 gm and 10 gg / ml. This medium, supplemented with hormones, is hereinafter referred to as DI medium. Unless otherwise indicated, PB (phenobarbital) is dissolved in basal medium and DI medium to a final concentration of 3 mM. The base medium and this DI medium containing the PB are respectively designated thereafter as PB medium.

and PBDI medium.

  The isolated hepatocytes are finally suspended at a concentration of 3.5 × 10 5 cells / ml in DI medium. 4 and 10 ml portions of the cell suspensions are respectively seeded in 60 and 100 mm Falcon plastic boxes. , and are grown in a humid atmosphere at 37 C, 5% CO2 and 95% air. After a one-day fixation period, the culture media are replaced with basal medium, PB medium or

  medium, and then these culture media are renewed.

  Velvet every 2 to 3 days. We continue to keep some

  cultures in the DI environment in the same way.

  3) Phenobarbital addition test 1. Test methods After the fixation period under the conditions defined in I-2 for 24 hours, the medium is exchanged for the following four media: (1) base medium (Eagle MEM (2) PB medium prepared by addition of 3mM phenobarbital to basal medium (3) PBDI medium prepared by adding 3mM PB to DI medium, and

(4) DI medium.

  These media are replaced every two or three days.

  The following tests are carried out periodically until the day of the cultivation:

  (a) determining the number of surviving cells

  your; (b) microscopic observation; (c) determination of the amount of albumin in a medium, by radioimmunoassay;

  (d) assaying the activity of glucose-6 phos-

phatase (G6Pase) in the cells,

  (e) determination of the activity of the amino tyrosine

  transferase (TAT) in cells, (f) induction effect of dexamethasone on the activities described in (d) and (e), (g) determination of cytochrome P-450 levels

(P-450),

  (h) determination of P-450 levels induced by PB (P-450pB), and (i) assay of P-450 levels induced by

methylcholanthrene (P-450MC).

  Results (1) Preservation effect on cell survival Figure 1 shows the changes in the number of living cells in the four media as a function of elapsed time. The numbers (1), (2), (3) and (4), in this figure,

  relate to the respective environments, defined above.

  Figures 2 to 5 are microscopic photographs in

  contrast, which show the morphological changes

  cell logic as a function of time. Numbers

  (1), (2), (3) and (4) correspond to half the

  locations (1), (2), (3) and (4) above.

  Figure 2 shows the appearance of hepatocytes grown in

  the media in question for 7 days (x 200).

  Figure 3 illustrates the appearance of hepatocytes grown in

  these media for 14 days (x 100).

  Figure 4 refers to hepatocytes grown in the

  PB (2) medium for 35 days (x 200).

  Figure 5 shows the effect of the presence of PB on the survival of mature hepatocytes in primary culture; A shows the appearance of cells that have been continuously treated

  in the PB (2) medium for 7 days, while B corresponds to

  to the cells that were cultured in PB (2) medium for 6 days, then in the absence of PB for one day, C shows the condition of cells continuously treated in PB (2) medium for 14 days, and Cells cultured in the PB (2) medium for 6 days, followed by

  culture in the absence of PB for 7 days (x 100).

  Figures 1 and 2 clearly indicate that mature hepatocytes can rapidly degenerate and die within one week of the start of culture in the non-PB system (i.e., in the basal medium and the medium). DI). From the 14th day of culture, we observe the growth of non-pigmented cells, epithelial type (see Figure 3). On the other hand, mature hepatocytes gradually diminish in number as time passes, but can be observed at least until the 49th day of culture with little morphological degeneration, except that the nuclei are more pronounced than at the initial stage in the system containing 3 mM PB (ie PB and PBDI media) (see Figures 1 to 4). The environment

  PBDI appears to be the most appropriate for conserva-

  mature hepatocytes (see Figure 1). In the presence of

  PB, the growth of unpigmented epiphyseal cells

  thelial, mentioned above, is scarcely observed (cf.

Figures 2 to 4).

  Table 1 represents the hepatocyte numbers

  survivors treated with PB at various concentrations

for 6 days.

TABLE 1

  Effect of PB concentration on surviving hepatocytes

in the primary culture.

  PB PB Culture duration: Number of hepatocytes * conc. treatment age in 4 (mM) (days) days (x 104 / box)

0.75 6 7 43.3 + 1.3

1.5 6 7 48.1 + 0.6

3.0 6 7 92.4 3.5

  * Seeding size: 140 x 104 cells / 60 mm box / 4 ml of DI medium

  average + SD of 3 different boxes.

  In order to study the effect of PB concentration on hepatocyte survival in the primary culture, the cultures are treated with 0, 75, 1.5 and 3 mM PB in the basal medium for 6 days, after a period of

  1 day fixation in the DI medium. The environments contain

  PB mantle are replaced every other day. The numbers of surviving hepatocytes in the presence of 0.75 and 1.5 mM

  of PB are about half of those who survive in pre-

  3 mM PB (Table 1). In addition, surviving hepatocytes in media containing 0.75 and 1.5 mM PB exhibit morphological degenerations which are very

  similar to those observed in the absence of PB.

  Moreover, we can not obtain any effect of

  When PB is persistently present

(see Figure 5).

  (2) Preservation effect of cellular functions

lai-res Albumin secretion

  Surviving hepatocytes, in the presence of 3 mM PB,

  have a remarkable secretion of albumin in the middle

  cultivation sites, for at least 35 days, during cultivation.

  primary education. Although these hepatocyte secretions decrease as time passes in the basic DI, PB, and PBDI media, those of the PB and PBDI media do not change significantly for at least 28 days after the 7th day of primary culture. , showing a relatively high level of several gg / ml / 2 days (Table 2). In the absence of PB, the combined intake of dexamethasone and insulin significantly increases the secretion of albumin, while in the presence of 3 mM PB these hormones do not affect the secretion

of albumin by hepatocytes. -

TABLE 2

  Duration of albumin production of hepatocytes in culture

  primary, in the presence or absence of PB.

  days Secreted albumin (gg / ml) of base crop DI PB PBDI

0 - 1 15.6 + 2.3

* 1 - 3 17.0 + 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 + 0.6

  - 7 0.8 + 0.2 2.0 + 0.7 4.7 + 0.3 6.6 + 0.7

  12 - 14 0.2 + 0.03 0.4 + 0.2 3.0 + 0.5 3.9 + 0.2

19 - 21 3.1 + 0.3 5.1 + 0.6

26 - 28 5.1 + 0.6 4.8 + 0.1

33 - 35 6.0 0.1 4.2 0.5

  Average + SD from three different determinations.

  Frankly isolated hepatocytes are seeded at 1.4 x 106 cells / 60 mm dish containing 4 ml of medium.

  DI, are incubated for 24 hours and then

  in the basal medium, DI medium, PB medium or medium

  PBDI. The secretion of albumin is expressed in gg / ml of half

  place / 2 days of culture, and the values are expressed

  like the average + SD of three different boxes.

  Activities of G6 Pase and TAT G6Pase activities, in cultures conserved in the four different environments above, hardly differ.

  from each other (Table 3), and they decrease rapidly

  in all cases as time passes. In all these cases, only about 15% of G6Pase activity in the liver homogenate is observed at the 7th day of primary culture.

rat (53 + 5 mU / mg protein).

  Table 3 (on page 14) shows the result.

TABLE 3

  Duration of G6Pase activities in cultured hepatocytes

  primary, in the presence or absence of PB.

  Days G6Pase (mU / mg protein) Basic Culture 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

  14.2 + 2.7 12.6 + 0.7 14.3 + 2.4 14.9 + 3.0

  7 8.1 + 0.9 7.0 + 1.7 8.3 + 0.6 9.8 + 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

  Average + SD of three different experiments.

  Liver homogenate: 53 + 5 mU / mg protein.

  Freshly isolated hepatocytes are seeded at 3.5 x 10 6 cells / 100 mm bales containing 10 ml of medium.

  DI, are incubated for 24 hours and then

  as indicated in the base or DI medium,

  PB, or PBDI. The cultured cells are collected from

  shot of 4 boots and are homogenized. The results are

  expressed as mean + SD from three experiences

different rations.

  The TAT activities in the cultures, kept in the basic medium and the DI medium, decrease only as the culture lasts, and these cultures, on the 7th day of primary culture, respectively indicate 10% and 15% of the activity. TAT in the supernatant part of rat liver (44 + 7 mU / mg protein) (Table 4). On the other hand, the TAT activities in the cultures conserved in the PB and PBDI media, also decrease gradually, as the culture lasts, but they hardly change after the 7th day of primary culture. The cultures conserved in PB and PBDI contain respectively 27 and 57% of the TAT activity in the supernatant part of rat liver, even at day 35 of the primary culture (Table 4).

Table 4 shows the result.

TABLE 4

  Duration of TAT activities in hepatocytes in primary culture, in the presence and absence of PB culture days TAT (mU / mg protein) base culture 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 4 3.4 34.5 + 8.3 51.3 + 6.4

  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

  1.9 + 0.7 3.5 + 0.3 9.5 + 0.6 20.2 + 0.8

  2514 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

11.9 + 1.9 25.1 + 12.9

  Mean + SD of three different experiments.

  Supernatant part of liver: 44 + 7 mU / mg protein.

  The freshly isolated hepatocytes are inoculated

  at 3.5 x 10 6 cells / 10-100 mm bale, containing 10 ml of DI medium, are incubated for 24 hours and then stored as indicated in basal or oral medium.

  DI, PB or PBDI medium. Cultured cells are collected

  are bound from the boxes and are homogenized. The results

  States are expressed as mean + SD from 3 different experiments.

  A well-accepted expression of a different function

  Liver cell is the ability of hepatocytes to induce liver-specific enzymes in response to hormones. As a result, this parameter is also used.

  as a marker for the preservation of different functions

  in surviving hepatocytes in the presence of 3 mM PB. The cultures preserved in the PB medium are put

  incubate with dexamethasone (10 gM) for 18 hours.

  before harvesting the cells. The addition of dexamethasone

  sone results in a triplicate triplicate induction of TAT activity, but does not induce induction of G6Pase activity (Table 5). Surviving hepatocytes in the PB medium

  are able to respond to the dexamethazone of this

  at least until the 28th day of primary cultivation.

Table 5 shows the result.

TABLE 5

  Induction by dexamethasone of TAT and G6Pase activities in hepatocytes in primary culture in PB medium G6Pase Days (mU / mg TAT (mU / mg protein) of PB PB + Dex PB PB + Dex culture *

  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

  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

  Average + SD from three different experiments.

  * Dexamethasone (10 kM) is added 18 hours before harvest.

cells.

  Cytotoxic action of 3-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) on hepatocytes cultured in the presence or absence of PB It is well known that PB induces, in animals, liver enzymes metabolizing microsomal drugs.

  [R. Synder et al. "Hepatic Cytochrome P-45 Mono

  oxygenase System ", 227, Pergamon Press, Oxford (1982) j.

  Also, have studies been conducted on the effect of PB treatment on the cytotoxicity of 3'-Me-DAB on hepatocytes

in primary culture.

  Freshly isolated hepatocytes are fixed in

  in the DI medium for 24 hours. Then, the cul-

  are continued, kept in the same medium, or

  in PBDI medium for 2 to 10 additional days.

  These cultures are exposed to 0.24 mM 3'-Me-DAB from

  day 1, 2 or 3 of the primary crop. The number of hepatitis

  surviving tocytes is much higher in the PBDI medium

  only in the DI medium (Figure 6). In other words, the sensi-

  Hepatocytes at 3'-Me-DAB are remarkably well

  duit by treatment with PB. In addition, the longer the period of BP treatment prior to 3'-Me-DAB exposure becomes longer, the more the hepatocytes become insensitive to 3'-Me-DAB. Figure 6 shows the citotoxic effect of 3'-Me-DAB

  on hepatocytes in primary culture, in the presence or ab-

PB.

Cytochrome P-450 contents

  Biochemical determinations of the total levels of cyto-

  chromium P-450, and the immunochemical determinations of

  P-450pB and P-450MC are produced using hepatocyte homogenates in primary culture, treated with

or without 3 mM PB, or 10 AM MC.

  Total content of cytochrome P-450, in hepatocytes

  grown in DI medium for 24 hours after planting.

  67.8% of freshly isolated hepatocytes (Tables 6 and 7). At this time of the culture, PB and MC are added. The medium containing PB is renewed every two days. Although PB treatment effectively decreases the rate of fall of the total cytochrome P-450 content of hepatocytes in primary culture,

  could not induce P-450pB in the cells (Table 6).

  On the other hand, because of the treatment with MC, the content of

  total cytochrome P-450 tends to increase in hepatocellular

  in primary culture (Table 7). In addition,

  by MC, induces evidence of P-450MC in the cells

(Table 7)

TABLE 6

  Effect of PB on cytochrome P-450 levels in

hepatocytes in primary culture.

  days Total content of P-450 (pmol / mg homogenate of protein) culture None base DI PB PBDI

0 146 (7.2)

1 99 (3.0)

  3 40 (0.07) 50 (0) 70 (3.0) 69 (1.9)

n / a N / A 49 56 7 N / A N / A 46 36

  Numbers in parentheses indicate the specific contents

  in P-450pB in pmol / mg protein homogenate.

nd = not detectable.

  The freshly isolated hepatocytes are inoculated at 3.5 x 10 6 cells / 100 mm dish containing 10 ml of medium.

  DI, are incubated for 24 hours and then

  as indicated in the basal medium or the DI medium,

  PB or PBDI. The numbers in parentheses indicate the

  P-450pB in pmol / mg protein homogenate.

TABLE 7

  MC effect on levels of cytochrome P-450 in

hepatocytes in primary culture.

  days Total content of P-450 (pmol / mg protein homogenate) base culture Nil base * u ueate4C * DI-MC * PB + MC * PBDI + MC *

0 146 (8.0)

1 99 (7.0) **

  93 (40.1) -106 (38.8) 115 (30.5) 146 (12.9)

  Numbers in parentheses indicate the specific contents

  in P-450MC, pmol / mg of protein homogenate.

  * MC (104M) is added to cultures 24 hours after

commencement.

** -MC.

  MC is dissolved in DMSO, and is added to culture media.

  to give a final concentration of 10 lM, giving

  thus a final DMSO concentration of 0.5%.

  Freshly isolated tocytes are sown at 3.5 x 106

  lules of 100 mm containing 10 ml of DI medium, and are incubated for 24 hours, then

  in the medium, base or medium DI, PB or PBDI, ad-

  packaged with 10 g MC, as indicated. Numbers in parentheses indicate P-450 ™ contents in pmol / mg

of protein homogenate.

  Effect of MC on Hepatocyte Survival 1, 10 or 100 μM MC is added to primary hepatocyte cultures after a 1-day fixation period,

  to examine whether MC can be active or not to

  hepatocytes in primary culture. The number of

  surviving patocytes, which are grown for 6 days

  in the basal medium containing 1 and 10 MC MR, represented

  53.7% and 50.8%, respectively, of those cultured during the same period in PB (Table 8). In the basal medium, MC at 100 gM is very toxic to hepatocytes. Similarly, in the DI medium containing MC at 1, 10 and 100 μM, the numbers of surviving hepatocytes at day 7 of the primary culture are about half of those in the PBDI medium (Table 8). During the following culture week, the number of hepatocytes further decreased in both basal medium and DI medium containing MC. In

  In addition, the surviving hepatocytes in these two cases

  appear morphologically to those found in the MC-free middle and DI-free medium, presenting

  degeneration characteristics such as degranulation

  cytoplasm and multinucleation. Non-cells

  pigmented epithelial cells and

  broblast, proliferate in media containing MC, but do not proliferate in PB medium and PBDI medium.

TABLE 8

  Effect of MC on survival of hepatocytes in primary culture. PB MC Number of hepatocytes (x 104 / bunch) conc. conc. basic DI (mM) (gM) day 7 day 14 day 7 day 14

  250 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 + 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 + 1.9 10.0 + 0.9

  3 0 89.8 + 2.0 65.8 + 4.7 92.3 + 5.0 68.2 + 2.3

  -1 4 c Seeding size: 140 x 10 cells / box

  of 60 mm containing 4 ml of DI medium.

  Average + SD of 3 different boots.

* 0.5% DMSO.

  The freshly isolated hepatocytes are seeded at 1.4 x 10 6 cells / 60 mm bale, containing 4 ml of DI medium, are incubated for 24 hours and then stored in basal media or DI medium, added with MC. at 1, 10 or 100 FM. Control cultures are preserved

  in base medium containing 0.5% DMSO, or half

  place PB, after the fixation period of 24 hours. In the 7th

  and on the 14th day of primary culture, the number of

  surviving tocytes is determined by trypan blue removal, in a hemocytometer. The results are expressed

  like the average + SD of 3 different boots.

  Figure 7 shows the condition of hepatocytes in primary culture, at the seventh day of culture (x 100); in this figure E refers to those treated with 10 μM MC one day after seeding, while F refers to those treated with 3 mM PB for 6 days. These data suggest that the conservation effect of PB on hepatocyte seas may not be caused by

  increased detoxification.

  4) Preservation effects on cells of initiation

  other hepatocarcinogens

  PB is known as a hepatocarcinogenic initiator.

  sequence, we have examined BHT and DD, which are well known

  hepatocarcinogenic initiators, as well as DDE, which is a

  analogue of DDT, to know whether they have

to that of PB.

  As can be seen in the following Table 9, each

  any of these compounds has cytotoxicity, but does not

  of conservation effect on March hepatocytes.

TABLE 9

  Effect of different chemicals on the survival of hepatocytes in primary culture Reagent Concentrate-Number of hepatocytes Secreted albumin tion4 tone (x104 / box) (g / ml) (nM) 7 days 14 days 5-7 days 12-14 days

BHT 3 0 +0 0 +0

1.5 0 + 0 0 + 0

0.75 0 + 0 0 + 0

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

0.5 12.2 + 2.1 0 +. 0

0.1 26.2 + 2.0 1.8 T 0.8

0.01 24.2 + 2.7 1.9 + 0.4

0.001 23.8 + 2.9 1.7 + 0.5

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 ô + 0 O + 0

1.5 2.8 T 0.8 0 O 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 + 0.1.9 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

  ut CO TABLE 9 (continued) Reagent Concentration Number of hepatocytes Secreted albumin (x104 / box) (ggml) 7 days 14 days 5-7 days 12-14 days Indo 2 0 + 0 O + 0

  1 76.4 + 6.7 22.5 + 1.1 4.06 + 0.08 2.40 + 0.05

  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 O + 0

0.5 1.0 + 0.5 O O

0.1 3.4 + 1.0 O

0.05 2.2 + 0.3 0 + O0

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

  Nil - 47.4 + 4.1 4.9 + 0.4 0.85 + 0.23 0.21 + 0.03 Seeding size: 140 x 104 cells / 60 mm dish filled with 4 ml of

DI medium.

  Each reagent is added 24 hours after the start of the culture.

  Average + SD of 3 different boxes. As previously stated, amobarbital, which is a derivative of PB, does not promote fC carcinogenesis. Peraino et al. Cancer Res., 2884 (1977). In a study of the conservation effect of the cell, amobarbital, it was found that it exhibits a preservative effect of mature hepatocytes, similar to that of PB, at a concentration of 0.75 mM Table 10 shows this result

TABLE 10

  Amobarbital cell conservation effect Concentra ReagentPercentage of live cells at day 7 to day 14 of amobarbital culture 0.75 mm 80.2 + 4.0 36.3 + 5.7

PB 3 77.5 + 4.7 50.3 + 8.7

  (control trial) - 38.2 + 3.7 4.3 0.4 Seeding size: 140 x 104 cells / bunch

60 mm / 4ml.

  These data suggest that the conservation effect on

  mature hepatocytes is not common to all initiators

  patocarcinogènes. On the other hand, pentobarbital, which is a derivative of PB and is referred to as "Pento" in Table 9, shows, at a

  concentration of 0.75 mM, a liver

ripe tocytes (see Table 9).

  ) Cell conservation effect with erythromembrane stabilizers Indomethacin and chlorpromazine, which are known as

  erythromembrane stabilizers, are studied in such a way

  Milaire. Chlorpromazine is highly cytotoxic, and has no effect of conservation on mature hepatocytes,

  while indomethacin actually inhibits the degeneration

  morphological decline of mature hepatocytes at a concentration

1 mM, while being cytotoxic. As a result, chlorpromazine can not retain cells for

  long period, unlike PB (see Table 9).

  These data suggest that the stabilization of functions

  cell membranes can lead to conserva-

  prolonged hepatocytes by PB. II.Effect of preservation of the cell by barbituric acid derivatives: As amobarbital and pentobarbital have

  proved to have a cell-conserving effect, if-

  compared to PB, it was investigated whether other derivatives

  barbituric acid also had this effect.

  Each reagent was used at concentrations of 3 mM,

1.5 mM and 0.75 mM.

  Each medium is introduced into a petri dish (0 60 mm) and seeded with 140 x 104 hepatocytes

  ripe rat. After 24 hours of cultivation, bringing the concentration

  cellular treatment at i106 + 13.6 x 104 cells / box,

  adds to this medium each reagent at one of the concentra-

  above. Hexobarbital and mephobarbital

  bital are added as 0.5% DMSO solutions, while the other reagents are added directly to

each medium.

  Live cell levels are assayed 7 and 14 days a-

  near seeding. Table 11 shows the results.

TABLE 11

  Reagent Concen- Percentage of live cells (n = 3) (MM) 7 days after 14 days after seeding Amobarbital seeding 3 0.3 + 0.3 0 + 0

1.5 3.4 + 1.4 2.6 + 0.6

0.75 80.2 T 4.0 36.3 + 5.7

  hexobarbital 3 88.1 + 2.4 43.8 + 1.3

1.5 53.8 + 4.3 30.2 + 5.0

0.75 40.7 T 1.2 12.6 T 1.7

  mephobarbital 3 45.5 + 4.5 4.7 + 1.0

1.5 70.2 T 1.8 23.1 T 2.3

0.75 51.9 T 6.8 19.1 + 4.6

  TABLE 11 (cont.) Reagent Concen- Percentage of live cells (n = 3) (mM) (5 days after 14 days after seeding pentobarbital seeding 3 0.1 + 0.1 0 + 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

  dimethyl 3 65.5 + 4.0 16.7 + 1.4 -1.3 barbituri- 1.5 56.5 + 3.6 3.8 + 0.8 0.75 43.1 + 5, 6 3,5 + 0,8 uramil acid N, N-diacetic 3 0 + 0 0 + 0

1.5 69.8 + 7.8 36.6 + 4.4

0.75 52.5 + 3.3 25.2 T 1.1

acid barbitu-

3 38.0 + 1.8 5.5 + 1.4

1.5 37.8 + 2.6 4.4 + 1.1

0.75 35.8 + 2.2 3.5 + 0.7

  Phenobarbital 3 77.5 + 4.7 50.3 + 8.7

environment

  mant DMSO 7Q, 4 38.8 + 11.5 3.2 + 0.5

medium does not

closing no

DMSO - 38.2 + 3.7 4.3 + 0.4

  According to this table, barbituric acid has no effect, whereas amobarbital,

  hexobarbital, mephobarbital, cyclobarbital,

  1,3-dimethyl barbiturate, pentobarbital, uramil N, Ndiacetic acid, and phenobarbital,

  effect of cell conservation, although the concentration

  The optimum amount varies depending on the reagent. It is confirmed therefore

  that the derivatives of barbituric acid prove to be effective

  these for cell conservation.

Claims (3)

claims   1. A method for prolonging epileptic cell culture   theliales, characterized in that one adds to the   culture place a derivative of barbituric acid.   2. Method according to claim 1, characterized in that the normal epithelial cells are cells primaries from the liver.   3. Method according to one of claims 1 or 2, characterized   characterized in that the barbituric acid derivative is a compound of the general formula:  R3 RI / R R1 t N R2 NOT R   wherein R1 and R2 each represent a hydrogen atom   gene; an alkyl group; a linear alkyl group or   mified, optionally substituted and having 1 to 6 carbon atoms; a cyclic hydrocarbon group, saturated or not   saturated; or a substituted amino group; and R3 and R4 represent   each feel an atom of hydrogen; an alkyl group;   or a substituted alkyl group having 1 or 2 carbon atoms bone.