A model for in vitro proliferation of undifferentiated bovine mammary epithelial cells

Cell Biology

A

International

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Vol. 10, No. 12, December

1986

MODEL FOR IN VITRO PROLIFERATION OF UNDIFFERENTIATED EPITHELIAL CELLS

923

BOVINE MAMMARY

A. Shamay and A. Gertler Department of Agricultural Biochemistry, Hebrew University of Jerusalem, P.O.Box

Faculty of 12, Rehovot,

Agriculture, 76100, Israel.

The

ABSTRACT A method for culturing bovine undifferentiated mammary epithelial cells embedded in collagen gels is described. Cell growth was quantitated by incorporation of tritiated thymidine. Maximal rate of incorporation was achieved in media supplemented with sera, however considerable growth was observed in serum-free medium supplemented with insulin, transferrin and selenium. The growth promoting effect of insulin was maximally expressed only at high nonphysiological concentrations. INTRODUCTION One of the major factors that affect milk production in dairy cows is the growth and development of mammary secretory cells prior to parturition. Identification and isolation of mammary growth factors (MGF2.1 requires an appropriate testing system, and its absence is an obstacle to progress. In vivo experiments are time consuming, extremely expensive and require large amounts of materials. The problem is even more severe in view of species specificity which requires these tests to be done in tissue from heifers or female calves, rather than laboratory animals. We have tried to address this problem by developing suitable in vitro systems for measurement of cell growth, such as cultures on biomatrix derived from endothelial cells (Vlodawsky et al. 1980) and from bovine mammary gland (Wicha et al. 19811, or on attached and floating collagen gels (Emerman and Pitelka 1977). All these efforts were met with little success. On the other hand, successful growth of bovine mammary cells from differentiated tissue, on attached collagen gels, as estimated by colony size was reported by Mackenzie et al. (1982). We have recently found that the most promising approach is a culture embedded in collagen gels (Richards et al. 1983). Adaptation of this method to bovine mammary undifferentiated epithelial cells and development of a serum free system is the subject of the present communication. MATERIALS ANDMETHODS Materials. M-199 medium powder (Gibco Co., Grand Island, NY. USA) was dissolved in glass-distilled water containing 1OmM Hepes, pH 7.4, sterilized by vacuum filtration through 0.2 pm filters and kept at 4OC 03OS-1651/86/12092~07/$03.0010 G1986Academic Presslnc. (London)Ltd.

924

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1986

up to 3 weeks. Aa antibiotic-antimycotic mixture consisting of 5~10~ U. pencillin, 5x10 ug streptomycin and 125 pg fungizone/ml (Biolab, Jerusalem, Israel) was kept frozen and added to the medium (3 ml/l) before use. Collagenase (type II, 135 U/mg) was obtained from (Freehold, NJ, USA) Deoxyribonuclease Worthington Biochemical Corp. type I, hyaluronidase (type 1-S 300 U/mg) bovine insulin, bovine serum albumin (RIA-grade), soybean trypsin inhibitor, reduced glutathione, and selenic acid were purchased from Sigma Co. (St. Louis, MO, USA). Nitex filters of various sizes were obtained from Tetko Co. (Elmsford, NY, USA), fetal calf serum (FCS) from Sera-Lab Ltd (Sussex, England) and tissue-culture grade transferrin from Mannheim-Boehringer Co. (Mannheim, West Germany). Male calf serum (9 months old) and pregnant (3 months) cow serum were prepared in our lab and sterilized by filtration through 0.2 pm filters. Basal medium was prepared from M-199 medium by addition of 0.25% bovine serum albumin, transferrin (5 pg/ml), bovine insulin (5 pg/ml), selenic acid (1 ng selenium/ml), soybean trypsin inhibitor (100 U/ml) and reduced glutathione (1 gels were prepared according to Richards et al. pg/ml 1. Collagen (1983). Tritiated thymidine (L3H]-TdR) was purchased from Amersham England) and scintillation liquid was prepared as (Buckinghamshire, described previously (Gertler et al. 1982). Mammary tissue from healthy Israeli-Friesian Tissue preparation. 4-6 months old female calves, were excised aseptically immediately slaughter and put into 100 ml Medium 199 supplemented with after antibiotic-antimycotic mixture. The tissue was transferred to the laboratory, cut into small pieces, and placed in a 500 ml Erlenmeyer flask containing M-199 medium supplemented with collagenase (lmg/ml), hyaluronidase (lmg/ml) and bovine insulin (1 pg/ml), in a ratio of 10 ml medium per 1 g tissue. The flask was swirled on a qyratory water bath at 100 rpm at 37OC for 3-4 h. During this period ,tissue fragments were further broken by occasional passage through a 10 ml pipette with a large orifice. One hour prior to termination of the enzymatic digestion, 0.5 ml of 0.04% DNase per 100 ml of digest was added. At the end of incubation, the suspension was passed through a Nitex filter (200 ~1 and the orqanoids were collected by centrifugation (4 min at 3OOxq). The orqanoids were washed 3-5 times with M-199 medium containing 0.04% DNAse and the pellet was suspended in 10 ml medium. The suspension was passed through a Nitex cloth (100 The clumps collected from the filter were resuspended in fresh urn). medium until Clumps of and kept on ice embedded in collagen. epithelial cells in minimal volume of M-199 medium, were added to ths cold collagen solution, yielding a final concentration of 4-8 x 10 cells/ml. Cell number was determined after digestion with citric acid, staining with Crystal Violet and counting the nuclei. The collagen-cell suspension (0.5 ml) was then overlaid on 0.3 ml pregelled collagen, in each well of 24-multiwell plates and allowed to gel at room temperature. As soon as the layer gelled,‘ 1 ml of M-199 medium con aininq the appropriate additives was added. All media 5 contained [ HI-TdR (1 pCi/mlA in order to determine DNA synthesis. The cells were cultured at 37 C in air : CO2 (95:5), and the media were changed every 48h.

Cell Biology

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1986

925

Thymidine incorporation. In order to determine the L3H]-TdR incorporation the medium was aspirated off the wells, gels were lifted carefully with broad tipped forceps and placed in 1.5ml Eppendorf tubes. Fifty pl of 4.3 M acetic acid were added to each tube and the tubes were incubated for 20 minutes at 37OC to dissolve the gels. The cells were recovered by centrifugation (5 min at 5OOxg) and washed consecutively with 4% perchloric acid (PCA), 80% EtOH, 100% EtOH and 4% PCA. Then the cells were hydrolysed in 1 ml 6% PCA (80°C, lh). After cooling aliquots of 0.5ml were withrawn, mixed with lOm1 of scintillation liquid and counted in a Beckman B-scintillation counter. method

DNA determination. DNA was estimated by a fluorimetric according to Downs and Wilfinger (1983).

sensitive

RESULTS AND DISCUSSION Results presented in Table 1 clearly indicate that clumps of bovine mammary epithelial cells embedded in collagen gel can synthesize DNA and proliferate under the appropriate conditions. Supplementation of the medium with insulin only, was insufficient, but w en as little as 1% of FCS (Exp. no. 1) was added, incorporation of 5 [ HI-TdR occurred. Unlike the insulin-supplemented M-199 medium, the basal medium, which included also transferrin, selenium, reduced glutathione, trypsin inhibitor and bovine serum albumin, induced DNA synthesis. However the response was far below maximal. This is concluded from the findings, that in M-199 + insulin (Exp. no. 2) and in the basal medium supplemented with 5% of either male calf serum or pregnant cow serum (Exp. no. 31, the rate of DNA synthesis aqd cell proliferation was higher then in the serum-free basal medium. [ HI-TdR incorporation was accompanied by a parallel increase in the total amount of DNA (not shown). Since the DNA was not determined at all time-points but mgstly after 7 or 8 days the results are expressed as incorporation of [ HI-TdR per well rather then per pg of DNA. We have no doubt that the incorporation represents true growth as evidenced by an increasein total DNA and microscopical analysis (Fig 1). Although the described experiments are not sufficient to reach a final conclusion, it also seems that addition of the sera shortened the la3 period required for the initiation of the DNA synthesis. Thus, in addition to growth promoting factors in the basal medium, both sera probably contained other growth factors. In an additional experiment (results not shown), we have tested effect of the 'nsulin concentrations in the basal medium (0.025 - 100.0 ]1g/ml) on [ 3 HI-TdR incorporation. At low insulin concentrations (0.025 - 0.1 ~g/ml),no growth promoting effect was observed, while at 1.6-100 pg/ml the effect reached its maximal magnitude. These results implicate that the growth promoting effect of insulin may be mediated through insulin-like growth factors receptors, which exhibit low affinity for insulin (Nissley et al. 1985).

Cell Biology

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Table

ExP.

1

1

International

Incorporation

of

in collagen

gels.

Medium

2

3

M-199+Ins4

2

4 5 6

into

cells

cultured

of

3H-TdR

for

1986

O-8 days

(dpm/wellxlO

-3)2

after:

2d

4d

6d

7or8d3

0.720.1

0.620.1

0.9LO.l

1.520.1

FCS

0.220.1

0.220.1

0.220.1

0,2+0.1

M-199+Ins+l.O%

FCS

1.520.6

17.2f2.3*

31.1+6.0*

23.9+2.8*

M-199+Ins+lO.O%FCS

3.3+0.5*

61.1+9.1*

46.225.6*

73.0+1.2*

M-199+Ins4

6.721.3

5.721.4

3.221.2

4.421.2

Basal

5.320.6

34.023.9*

104.7+11.4*

253.5+15.9*

255.2+14.1*

365.6+7.7* 447.0+24.4*

Medium

M-199+5%

cs6

3.120.4

101.5+3.3*

M-199+5%

PCS6

8.620.7

42.1+1.3*

335.2+6.0*

1.520.1

0.5+0.1

0.620.1

1.120.4

M-199+Ins4 Medium

Basal

Medium+5% CS6

Basal

Medium+S%

1.220.1

3.6+0.7*

21.6+1.8*

70.9+22.5*

10.0+1.3*

44.2+7.9*

72.5+5.5*

214.3+10.8*

2.9+0.3*

48.3+7.6*

122.7+24.0*

PCS6

The mammary tissue

from

visual

more developed

than

and 2, n=3;

Exp.3,

inspection,

Mean 2 SFM, marked

3

Vol. 10, No. 12, December

M-199+Ins+O.l%

Basal

1

r3H]-TdR

Incorporation

no. 1

Reports,

*

with

Exp.1 are

the

significantly

containing

M-199 + Ins.

7d in Exp.

1, 8d in Exp.

calf

used

in Exp.

no 2, was,

in calves n=4.

different

used

(n = no.

(P
2 and 3.

Not determined. calf

serum,

PCS - 3 month pregnant

cow serum.

as judged Exp.

of wells).

from

5 p/ml. CS - male

in

ND5

the

by

1 and 3. All

controls

means

Cell Biology

Fig.

1..

international

Reports,

Vol. 10, No. 12, December

1986

927

Phas ;e micrograph of female calf mammary epithelial ce !llS cu 1tur ,ed in collagen gels for 4 days in the presence of M-.199 + insu .lin and (top) or M-199 + insulin + 10% FCS (center bc '&to!? I). Magnification x 160.

928

Fig.

Cell Biology

2.

International

Reports,

Vol. 10, No. 72, December

Histological section of female calf mammary epithelial cultured in collagen gel under conditions described legend to Fig. 2. Magnification x 160.

1986

in

cells the

It hould be noted that in all 4 experiments the extent of hormonal treatments (i.e. overall [ 3 HI-TdR incorporation at similar basal medium or M-199 supplemented with insulin and FCS), was quite likely from the different variable. This variability resulted most of differentiation of the calf tissue or from genetic stages differences. Further studies with larger samples of animals are required to elucidate this question and our present model provides the It should be mentioned that in other experiments in necessary tool. which we have used shorter pulse periods (18 h) the results were very similar. Microscopical analysis of the cells grown in the presence of 10% FCS revealed two visually different features of outgrowth, similar to that reported by Yang et al. (1980) and McGrath et al. (1985). One type of growth is characterized by a needle-like (Fig 1, center), while the other (Fig 1, bottom) by a star-like appearance. Both types of outgrowth appeared in all experimental treatments, except in wells (Fig 1 treated with M-199 + insulin, in which no outgrowth occured In all experiments where growth occured, confluency was followed top). by contraction of the collagen gel and formation of a dense bulk of Histological examination (Fig. 2) revealed that the the cells. which is characterized by a outgrowths have an alveoli-like structure, lumen surrounded by epithelial cells. method for culturing undifferentiated In summary the present for bovine mammary gland cells in vitro, provides a convenient model studying the growth process and assaying the biological activity of mammary growth factors that may be specific to bovine mammary tissue. The importance of the present model is further emphasized by the similar experiments in vivo in large unfeasibility of performing also that the present method has at least two animals. It seems

Cell Biology

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Vol. 10, No. 12, December

1986

929

advantages over that published by Mackienzie et al. (1982): (a) cells are not washed out due to the lack of attachment and therefore serum-free medium can be used through all experimental stages, (b) incorporation of 13H]-TdR as an index for cell growth is more sensitive and less time consuming. Acknowledgment. We thank Dr G. Brenner and Dr D. Hochman of the Marbek for their help at the slaughter house, and Mrs C. Neufeld Co.Israel, for her secretarial assistance. This investigation was supported by a grant from the National Council for Research and Development, (Israel) grant no. 2446-2-86 and by the grant from the United States-Israel Binational Agricultural Research and Development Fund (BARD) grant no. US-962-85. REFERENCES Downs

T.R. and Wilfinger W.W. (1983) Fluorimetric quantification of DNA in cells and tissue. Analytical Biochemistry 131,538-547. and induction of Emerman J.T. and Pitelka D.R. (1977) Maintenance morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13,316-328. Weil A. and Cohen N. (1982) Hormonal control of casein Gertler A., synthesis in organ culture of bovine lactating mammary gland. Journal of Dairy Research 9,387-398. McGrath M., Palmer S. and Nandi S. (1985) Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF. Journal of Cellular Physiology 125,182-191. Mackenzie D.D.S., Forsyth, I.A., Brooker B.E. and Turvey E. (1982) Culture of bovine mammary epithelial cells on collagen gels. Tissue & Cell 14,231-241. Nissley S.P., Haskell J.F., Sasaki N., DeVroede M.A. and Rechler M.M. (1985) Insulin-like growth factor receptors. Journal of Cell Science (Supplement) 3,39-51. Richards J., Larson L., Guzman R., Tomooka Y., Osborn R., Imagawa W., and Nandi S. (1983) Methods for culturing mammary epithelial cells in a rat tail collagen gel matrix. Journal of Tissue Culture Methods 8,31-36. Vlodawsky I., Liu G.M. and Gospodarowich D. (1980) Morphological appearance, growth-behavior and migratory activity of human-tumor cells maintained on extracellular matrix versus plastic. Cell 19,607-616. Wicha M.S., Lowrie G., Kohn E., Bagavandoss P. and Mahn T. (1981) Extracellular matrix promotes mammary epithelial growth and differentiation in vitro. Proceedings of the National Academy of Sciences (USA), 79,3213-3217. J., Imagawa W. and Nandi S. (1980) Sustained growth Yang J., Richards in primary culture of normal mammary epithelial cells embedded in collagen gels. Proceedings of the National Academy of Sciences (USA) 77,2088-2092. Received

12.8.86

Accepted

20.10.86