Cell Differentiation, 5 (1976) 69--81 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

CULTURES

OF NORMAL

HUMAN MAMMARY

69

CELLS *

Edwin V. G A F F N E Y , Frank P. POLANOWSKI, Susan E. BLACKBURN, James T. LAMBIASE, Robert E. BURKE

Department of Biology, Pennsylvania State University, University Park, Penn. 16802, U.S.A. Accepted 28 April 1976 This paper describes a method of obtaining epithelial cells from large quantities of normal human breast tissue and the response of these cells in culture to lactogenic hormones. Suspensions of single cells and clusters of cells resembling normal ductal and alveolar structures were obtained by mechanical disaggregation and subsequent (3 h) incubation of tissue fragments in 0.5 mg/ml collagenase. Cells rapidly attached to glass or plastic surfaces within 48 h and grew to form large colonies which maintained their epithelial appearance throughout 2 months of observation. Cell cycling as monitored by DNA syn.thesis was enhanced by insulin, hydrocortisone, or ovine prolactin (in concentrations of 5.0 /lg/ml each) at respectively 2, 3 and 5 days of incubation. These results were observed in cultures derived from 3 premenopause samples of mammary tissue maintained in medium with 1% fetal calf serum. Prolactin at a concentration of 5 pg/ml induced phosphoprotein synthesis 8-fold over control values. In addition, prolactin induced morphological changes in cells including the development of distended endoplasmic reticulum, large microvilli, and the deposition of glycogen granules. These initial results led to the tentative conclusion that prolactin was sufficient to initiate some of the characteristics in cultured cells normally associated with lactating tissues.

A l t h o u g h a f e w s t u d i e s h a v e e x a m i n e d t h e in v i t r o g r o w t h o f e x p l a n t s o r i s o l a t e d c e l l s o f t h e n o r m a l h u m a n m a m m a r y g l a n d , l i t t l e i n f o r m a t i o n is a v a i l a b l e w h i c h d e a l s s p e c i f i c a l l y w i t h t h e r e s p o n s e o f cell m o n o l a y e r s t o h o r m o n e t r e a t m e n t ( C e r i a n i e t al., 1 9 7 2 ; F l a x m a n e t al., 1 9 7 2 ; F l a x m a n , 1 9 7 3 , 1 9 7 4 ) . S u c h s t u d i e s h a v e b e e n r e s t r i c t e d b e c a u s e 1) t h e m o s t f r e q u e n t ly available tissue specimens are obtained from patients who have undergone treatment for some form of cancer and thus may not represent normal material, 2) s u r g i c a l s p e c i m e n s a r e u s u a l l y s m a l l a n d in t h e c a s e o f n o n - l a c t a t i n g tissue contain a relatively small proportion of epithelial cells compared to t o t a l o r g a n m a s s , a n d 3) a p r o c e d u r e f o r t h e s u c c e s s f u l d i s s o c i a t i o n o f l a r g e quantities of tissue and the subsequent maintenance of cells has not been described. T e c h n i q u e s w e r e d e v e l o p e d f o r t h e p r e s e n t s t u d y w h i c h p e r m i t t e d t h e isol a t i o n o f l a r g e n u m b e r s o f s i n g l e cells a n d c e l l c l u s t e r s f r o m s e v e r a l h u n d r e d * This work was supported by a contract from the U.S. National Cancer Institute CB33898.

70 grams of tissue obtained through reduction mammoplasty or autopsy. The use of tissue derived from benign or malignant breast samples was purposely avoided. Cultures could be established routinely and consisted of predominantly epithelial-like cell patches. Monolayer cultures have been used in this laboratory as one approach to understanding the prerequisites for human m a m m a r y cell differentiation and for exploring the influence of hormones on the predisposition of mammary cells to malignant alteration. The response to insulin, hydrocortisone and/or prolactin, monitored in terms of DNA and phosphoprotein synthesis and modifications in cell ultrastructure, differed from previous studies in organ culture systems. MATERIALS AND METHODS Cell preparations Specimens of normal human m a m m a r y tissue were obtained through reduction mammoplasty or autopsy. Breast tissue was cut into pieces weighing approx. 100 g and placed into a sterile jar containing 500 ml McCoy's 5a medium for transportation to the laboratory. Tissues were then finely minced with scissors into fragments measuring approx. 8 mm 3. These fragments were washed three times by centrifugation through Hank's Balanced Salt Solution. Pieces containing excess fat material remained floating and were discarded: Pellets were resuspended in serum-free McCoy's medium 5a (Gibco, Inc.) containing collagenase (Sigma, Inc.) at a final concentration of 0.5 mg/ml. Lipids associated with a large proportion of the d ssociated tissue appeared to impede collagenase activity and this was circumvented by the repeated washing procedure. Tissue fragments were then maintained stationary for 1 h followed by gently swirling for an additional hour at 37°C. The collagenase supernate was removed and the single cells and cell clusters resembling normal duct and alveolar structures were collected by centrifugation at 500 g. This procedure was repeated with the addition of fresh collagenase to the remaining fragments. Cells were maintained in medium supplemented with 10% fetal calf serum and 50 pg/ml gentamicin. Appropriate culture containers were inoculated with a cell concentration of 2 X 10S/ml. The final volume of digested tissue represented less than 20% of the starting breast material. Initial efforts had shown that incubation in collagenase extended periods resulted in a loss of cell viability and a severe decrease in the proportion of cell populations capable of attaching to culture flasks. In addition, prolonged exposure to the enzyme increased digestion of the stroma resulting in fibroblasts which contaminated the epithelial cell populations. Hormone treatment Monolayer cultures were successfully established from all of the tissue samples, with a cellular attachment rate in each of approx. 40%. Cultures

71 were fluid changed and treated with hormones as early as 48 h after seeding depending on the experiment. Insulin, hydrocortisone, and prolactin singly or in combination were added in concentrations up to 5.0 pg/ml. Cultures were maintained in medium containing 1% serum and supernatant fluids were renewed daily t h r o u g h o u t the h o r m o n e incubation period. Prolactin was not detectable in the bovine serum used in these experiments as measured by radioimmunoassay using rat, ovine and human standards. DNA synthesis in epithelial cells was monitored by radioautography, a technique permitting the visual selection of cell types. Triplicate cultures for each hormone variable were incubated in 1.0 pCi/ml 3H-thymidine for 4 h. Cultures were fixed in methanol--acetic acid, dipped in Kodak liquid emulsion (NTB-2) and exposed for two days prior to developing in D-19. A minim u m number of 1500 cells per culture were counted to determine the proportion of cells in S phase. Cultures established in 25 cm 2 plastic flasks (5 ml/flask) were used for measuring the synthesis of phosphoproteins. Triplicate cultures for each hormone combination were each incubated for 4 h in 15 pCi 33Pi/ml. Rennin precipitable phosphoproteins were then extracted in the presence of carrier bovine casein from the 100,000 g supernates of cell homogenates according to the methods of Juergens et al. (1965). Cell protein c o n t e n t was measured by the Lowry m e t h o d (Lowry et al., 1951). The recovery of carrier casein and rennin calculated on the basis of optical density at 260 and 280 nm was consistently greater than 100%. This difference was attributed to cellular protein. Cell morphology Ultrastructural changes were studied by electron microscopy. Control cultures and cultures treated f o r 9 days with hormones were scraped with a rubber policeman and suspended in cacodylate buffer and 1.5% glutaraldehyde. Cells were rinsed in buffer, fixed in a 1.5% solution of osmium tetroxide, dehydrated through ethanol and embedded in Spurt resin (Spurr, 1969). The material was sectioned with a diamond knife in a Porter--Blum MT-2 microtome and sequentially stained with uranyl acetate and lead citrate (Venable et al., 1965). Sections were examined in a Hitachi HU-11E-1 microscope. RESULTS Cell culture characteristics A major technical problem was the dissociation of large amounts of normal breast material to yield sufficient numbers of epithelial cells for culture. Ten specimens of tissue were obtained through reduction mammoplasty or autopsy on women ranging in age from 19 to 67 years. Two specimens were removed as late as 24 h after death and the period between death and prepa-

72 ration o f the final cell suspension approached 60 h in these experiments. Viability of cells derived from autopsy specimens as measured by t r y p a n blue exclusion was always in excess of 90%, with 35% to 40% of the cells attaching to plastic or glass flasks within 24 h. The subsequent use of autopsy tissue in long term experiments was prohibited, however, by the rapid contamination of cell cultures reflecting a lack of sterile conditions during tissue removal. Th e six specimens obtained through cosmetic breast surgery remained free o f c o n t a m i n a t i o n and eventually became the preferred cell source. Cell yields o f 2--3 X 106/g of fat-free tissue were routinely obtained using the procedure described in Materials and Methods. A 3-h incubation period in 0.5% collagenase was appropriate t o maintain a high p r o p o r t i o n of epithelial cells in the resulting suspension and avoided excessive digestion of the stroma. This t r e a t m e n t interval yielded b o t h viable single cells and a considerable n u m b e r o f intact alveolar and ductal elements (Fig. 1A and B). Cells isolated by this p r o c e d u r e were characterized by a viability in excess of 95% and an attachm e n t rate exceeding 40%. This was i n d e p e n d e n t of patient age. Within 48 h following seeding, cultures contained num erous islands or clumps of cells at-

Fig. 1. Clusters of cells derived from fragments of normal human mammary tissue digested with 0.5 mg]ml collagenase for 3 h. A) Duct-like element: x 1180. B) Alveolar-like structure. Original x 2285.

73 tached to flask surfaces. These gradually enlarged through spreading and cell division (Fig. 2). When cells were plated at high densities (5 X 10 s cells/ml) patches of cells eventually coalesced to form confluent sheets. These cells demonstrated a polygonal pattern, typical of cultured epithelium. Domes, which have been frequently observed in mouse m a m m a r y cell cultures, were n o t seen with human cells. However, large clumps of cells presumably derived as part of the alveolar system failed to grow out completely on flat surfaces and maintained a hemispheric structure in the center of some colonies. Although the presence of individual fibroblasts was apparent in one week old cultures, the majority of cells were epithelial-like in appearance throughout the 2 m o n t h s of observation. Attempts to transfer one week or one m o n t h old cultures by exposure to trypsin (0.25%) met with limited success. Only 40% of the cells attached to new flasks on transfer from primary cultures and subsequent observations on subcultures revealed t h a t cell divisions were infrequent. Autoradiographic studies on the synthesis of DNA were conducted with cultures established from 3 of these tissue samples. The results in medium with 1% serum revealed that the proportion of epithelial cells participating in DNA synthesis following 4 h incubation in 1.0 pCi/ml (3H-TdR) declined from approx. 10% at 2 days to 1% by 10 days after seeding and remained at that level t h r o u g h o u t one m o n t h of observation.

Fig. 2. A p p e a r a n c e o f c o l o n y o f normal h u m a n m a m m a r y cells in primary culture for o n e w e e k . Original x 6 8 0 .

74

Response to hormones All experiments in which h o r m o n e s were included in the growth media were c o n d u c t e d with cultures established from tissues of three premenopause nulliparous women, 19, 21 or 23 years of age. Autoradiographic studies o f DNA synthesis in cultured m a m m a r y cells required a visual selection o f cell types for counting labeled nuclei. Only cells which were found in colonies similar t o t hat shown in Fig. 2 were c o u n t e d in these studies. The affinity o f the epithelial cell cytoplasm for the eosin stain as well as cell m o r p h o l o g y at the light microscopic level aided in identification. Preliminary studies revealed that the synthesis of DNA was enhanced in the presence o f certain h o r m o n e s and that this effect may be c o n c e n t r a t i o n d e p e n d e n t . Multiwell slide cultures obtained from the tissues of either the 21 or 23 year old patient were fluid changed at 2 days after seeding with medium containing 1% serum and insulin or prolactin in concentrations up to 5 #g/ml. H o r m o n e supplemented media were renewed daily for 4 days and triplicate cultures incubated for 4 h in 1.0 pCi/ml 3H-TdR. The results from two experiments as illustrated in Fig. 3 are based on counts of 1500 cells per slide for each h o r m o n e concent r a t i on. T he p r o p o r t i o n of cells synthesizing DNA increased slightly with prolactin and dramatically with insulin in concentrations above the 0.05 pg/ml level. It appears that the higher h o r m o n e

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HORMONE CONCENTRATION (ng/ml) Fig. 3. H o r m o n e c o n c e n t r a t i o n d e p e n d e n t D N A s y n t h e s i s in one w e e k old p r i m a r y cultures o f n o r m a l epithelial m a m m a r y cells e s t a b l i s h e d f r o m tissues o f a 21 and a 23 year old p a t i e n t . Cells m a i n t a i n e d for 4 days in m e d i a s u p p l e m e n t e d w i t h 1% fcs and insulin or ovine p r o l a c t i n in c o n c e n t r a t i o n s up to 5 p g / m l . I n c u b a t e d o n day 4 with 1.0 p C i / m l 3Ht h y m i d i n e for 4 h. P e r c e n t cells in S phase based o n c o u n t s o f 1500 cells in t h r e e s a m p l e s o f each tissue sample.

75

concentrations, although in excess of normal physiological values, are necessary to elicit a mitogenic response in vitro. Additional experiments were conducted to reveal the temporal relationship between hormonal t r e a t m e n t and the enhancement of DNA synthesis. Two day old cultures established from reduction mammoplasty tissue of the 23 year old patient were fluid changed with medium containing 1% serum or media supplemented with 5 pg/ml insulin, hydrocortisone, and ovine prolactin singly or in combination. Culture fluids were renewed daily and each day triplicate cultures for each variable were incubated in 1 gCi/ml 3HTdR for 4 h. Fig. 4 shows that DNA synthesis was enhanced by insulin at 48 h, followed by hydrocortisone at 72 h and hydrocortisone in combination with either prolactin or insulin by 96 h of incubation. A slight increase was also noted at 72 h in cultures incubated with all three hormones. The pattern of DNA synthesis was also followed in primary cultures derived from the tissue of a 19 year old who had undergone cosmetic surgery. As in the previous experiment, cells were fluid changed 48 h after seeding and supernatant fluids renewed daily with the hormone mixtures. Triplicate samples were incubated each day for 4 h in 1.0 pCi/ml 3H-thymidine. The sequence and timing of insulin and hydrocortisone stimulated DNA synthesis were the same t h r o u g h o u t the first four days of the experiment as those previously observed in cells derived from tissues of the 23 year old patient. On day five all hormones or hormone combinations enhanced DNA synthesis in

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Fig. 4. One w e e k old p r i m a r y c u l t u r e s g r o w n in m e d i a s u p p l e m e n t e d w i t h 1% fcs a n d 5 m g / m l insulin, h y d r o c o r t i s o n e a n d ovine p r o l a c t i n singly or in c o m b i n a t i o n . C u l t u r e fluids were r e n e w e d daily a n d t r i p l i c a t e s a m p l e s of e a c h h o r m o n e c o m b i n a t i o n i n c u b a t e d for 4 h e a c h d a y in 1.0 p C i / m l 3 H - t h y m i d i n e . P r o p o r t i o n o f cells s y n t h e s i z i n g D N A det e r m i n e d f r o m c o u n t s o n 1 0 0 0 cells in e a c h sample.

76

the epithelial cell populations but from day 6 the number of cells synthesizing DNA in the hormone treated cultures was not significantly different from the controls. The specificity of the mitogenic response to hormones was investigated with fibroblasts and epithelial cells from other tissue sources. Experiments were conducted with four day old contact inhibited cultures of human embryonic lung fibroblasts (HEL} and ten day old primary cultures of epithelial h u m a n amnion cells (HA). Less than 0.1% of the cells were in mitosis at the time the experiment was begun. Leighton tube cultures of each cell type were maintained with a daily fluid change of media supplemented with 1% serum and 5 pg/ml insulin, prolactin or a mixture of insulin, prolactin and hydrocortisone (IHP). Triplicate cultures of HEL were incubated for 1 h and HA for 4 h with 1.0 pCi/ml 3H-thymidine. The number of cells synthesizing DNA was determined from observations on 1500 cells per culture. The proportion of fibroblast populations synthesizing DNA in prolactin and insulin was not significantly different from control values during the first three days of incubation. However, t r e a t m e n t with IHP resulted in 30% fewer cells in S-phase t h r o u g h o u t the experiment. Insulin enhanced DNA synthesis by 25%

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77

in HA cells after two days incubation. In contrast, insulin and IHP inhibited synthesis by day 4 of treatment (Fig. 5). Prolactin was not observed to induce a mitogenic effect in either cell type regardless of the time of incubation. Cultures established in 25 cm 2 plastic flasks from the tissue specimens of the 19 year old patient were incubated for four days in the above h o r m o n e combinations and then exposed to 75 pCi per culture of 33Pi for 4 h. Total soluble cell protein was determined in both the precipitated phosphoprotein fractions and in the supernatant fractions obtained following rennin treatm e n t and h o t TCA extraction. The data expressed as radioactivity per mg of rennin precipitable cell phosphoprotein and non-precipitable cell protein are shown in Fig. 6. Normal h u m a n m a m m a r y cells grown as monolayer cultures appeared capable of synthesizing phosphoprotein in response to hormonal stimulation. The synthesis of rennin precipitable phosphoprotein was increased 8-fold in the presence of prolactin and 5-fold in the presence of insulin and insulin in combination with hydrocortisone as compared to controls. Fluctuations in total radioactivity of supernatant fractions correlated with changes found in the phosphoprotein fractions.

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Fig. 6. P h o s p h o p r o t e i n s y n t h e s i s in p r i m a r y epithelial m a m m a r y cells. C u l t u r e s maint a i n e d in m e d i a w i t h 1% fcs and 5 m g / m l insulin, h y d r o c o r t i s o n e and ovine p r o l a c t i n singly or in c o m b i n a t i o n for 4 days. Media were r e n e w e d daily and cultures i n c u b a t e d in 75 pCi 33pi per culture flask for 4 h o n day 4. Data r e p r e s e n t average o f t h r e e cultures. Resuits e x p r e s s e d as c p m in e i t h e r t h e r e n n i n p r e c i p i t a t e d f r a c t i o n or s u p e r n a t a n t f r a c t i o n per mg cell p r o t e i n .

78

Cell morphology Cultured ceils from tissues of both the 19 and 23 year old patients were examined with the electron microscope in order to establish their epithelial origin and to determine whether hormones induced any dramatic changes in morphology. Cells were grown in 25 cm 2 plastic flasks and treated with hormones for 9 days. Populations of control cells exhibited a well developed series of desmosomes between plasma membranes, short microvilli, and an undistended rough endoplasmic reticulum. Numerous but irregular mitochondria were also noted. Although a distinct Golgi apparatus was not consistently apparent, lysosomes and lipid deposits were frequently observed (Fig. 7). Hormones or combinations thereof did not induce structural alterations, except for prolactin which induced several distinct cell structure changes.

Fig. 7. E l e c t r o n m i c r o g r a p h o f n o r m a l m a m m a r y cell m a i n t a i n e d for 9 days in m e d i u m a n d 1% serum. A r r o w s r e f e r t o p r o m i n e n t d e s m o s o m e s b e t w e e n cell m e m b r a n e s , x 14,612. Fig. 8. U l t r a s t r u c t u r a l a p p e a r a n c e o f m a m m a r y cell c u l t u r e d in m e d i u m w i t h 1% s e r u m a n d o v i n e p r o l a c t i n for 9 days. Cell is c h a r a c t e r i z e d b y irregularly s h a p e d nucleus, larger microvilli a n d t h e d e p o s i t i o n o f d a r k g l y c o g e n granules in the c y t o p l a s m . These cells are c o n s i d e r a b l y larger t h a n c o n t r o l s . Original x 8 0 4 6 .

79

Fig. 9. Deposition of glycogen granules near periphery of cultured mammary cell treated with prolactin. Original X 10,532.

For example, nuclei which appeared generally oval in control cultures were highly irregular in the prolactin treated cells with numerous indentations. The endoplasmic reticulum was more highly developed and appeared distended with dense material. Cell surfaces were characterized by elongated microvilli many times the size of those observed with untreated cells (Fig. 8). In addition, 50% of the cells treated with prolactin were seen to contain large depositions of granular material resembling glycogen (Figs. 8 and 9). Vesicles containing granular products resembling the casein material described by Bargman et al. (1969) were observed in only 6% of the cells and larger casein micelles associated with the plasma membrane were observed in only 3% of the cells. Glycogen granules were also present in cultures treated with insulin, hydrocortisone and prolactin. However, these cells were distinguished by an extensive vacuolization of the cytoplasm implying that the IHP h o r m o n e combination was not optimal for cell maintenance in culture. DISCUSSION A simple technique involving extensive dissection of breast tissue samples coupled with limited enzyme dissociation has resulted in the recovery of large numbers of normal epithelium for in vitro study. The success of this approach depends on several factors. First, reduction mammoplasty served as the most useful source of tissue due to the large sample size and the sterility measures employed during tissue removal. Second, a short incubation in col-

80 lagenase enriched the epithelial cell population while limiting the digestion of stromal material. The selection of this enzyme for m a m m a r y tissue was based on a previous report (Lasfarques, 1957). Lasfarques et al. (1971) emphasized that collagenase should be used in those situations where epithelium is embedded in a collagen matrix. Third, our initial observations revealed t h a t excess fat material inhibits breast tissue dissociation by collagenase. Therefore, selection of fat-free tissue fragments by extensive washing in a balanced salt solution is highly recommended. Light microscopic observations on the growth of cells and cell changes in primary cultures revealed that the initial inocula grew to form colonies of polygonal cells. The morphological characteristics of these colonies fulfilled the criteria previously used to identify epithelial-like m a m m a r y cells in monolayer culture (Whitescarver et al., 1968). In addition, these colonies resembled the normal epithelial cultures established from human milk and postweaning breast fluids (Buehring, 1971; Russo et al., 1975). The qualitative and quantitative results on h o r m o n e responsiveness described herein are difficult to compare with those of previous studies for the following reasons. First, previous work was conducted with tissues obtained from patients of different ages who had undergone mastectomy for some cancerous lesion. Second, most studies were conducted with organ cultured specimens rather than with cells grown in monolayer. Third, the length of time cells or tissues were maintained in vitro prior to h o r m o n e treatment differed. Our studies showed that insulin and hydrocortisone induced a temporary enhancement of DNA synthesis in the epithelial proportion of m a m m a r y cell populations at 24 and 48 h of incubation. Prolactin elicited a mitogenic response in cells following a 5 day period of incubation and was sufficient to elicit the synthesis of phosphoproteins. These results demonstrated that a mitogenic response to insulin and hydrocortisone was not prerequisite to the induction of phosphoprotein synthesis by prolactin. The data agrees with that of Barker et al. (1969) who showed that the alveolar epithelium in tissue slices of the m a m m a r y gland was maintained in an apparently healthy condition up to 6 days in defined medium. The addition of insulin induced an epithelial hyperplasia indicating a mitogenic response. Similarly, Flaxman (1974) showed that both insulin and prolactin doubled the rate of DNA synthesis in 7 day old organ cultures of normal m a m m a r y epithelium from postmenopause women. In contrast, insulin and prolactin did not enhance proliferation in 10 day old cell cultures. Human cells treated with prolactin consistently demonstrated certain significant ultrastructural changes when compared to controls. These included the unexpected deposition glycogen granules and an accumulation of a large quantity of material within the rough endoplasmic reticulum. These changes implied t h a t prolactin would induce some of the ultrastructural characteristics normally associated with lactating human cells (Tobon et al., 1974). Unlike previous studies with organ cultures derived from midpregnant mouse glands, insulin, hydrocortisone and prolactin in combination were not suffi-

81 cient t o i n d u c e milk synthesis in n o r m a l h u m a n cells derived f r o m n o n l a c t a t i n g tissues. I n s t e a d t h e e l e c t r o n m i c r o s c o p i c results revealed t h a t this h o r m o n e c o m b i n a t i o n led t o s t r u c t u r a l a b n o r m a l i t i e s such as an extensive c y t o p l a s m i c v a c u o l i z a t i o n w h i c h was a c c o m p a n i e d b y an a c c u m u l a t i o n o f lipids. Thus, a d d i t i o n a l h o r m o n e f a c t o r s m a y be r e q u i r e d t o i n d u c e l a c t a t i o n . It is i m p o r t a n t to n o t e t h a t all h o r m o n e studies were c o n d u c t e d with prim a r y cultures established f r o m tissues o f y o u n g p r e m e n o p a u s e n u l l i p a r o u s females. A d d i t i o n a l studies with cells c u l t u r e d f r o m samples o f t h e postm e n o p a u s e stage will provide an o p p o r t u n i t y t o e x a m i n e the relationship bet w e e n p a t i e n t age and h o r m o n e responsiveness.

REFERENCES Bargmann, W. and U. Walsh: eds. M. Reynolds and S.T. Foley, (University of Pennsylvania Press, Philadelphia) pp. 43--52 (1969). Barker, B.E., H. Fanger and P. Farnes: Exp. Cell. Res. 35, 437--448 (1969). Buehring, G.: Culture of human mammary epithelial cells: keeping abreast with a new method. J. Nat. Cancer Inst. 49, 1433--1434 (1972). Ceriani, R.L., G.P. Contesso and B.M. Notaf: Cancer Res. 32, 2190--2196 (1972). Elias, J.J.: Proc. Soc. Exp. Biol. Med. 101,500--502 (1959). Flaxman, B.A.: J. Invest. Dermatol. 61, 67--71 (1973). Flaxman, B.A.: Dermatology 63, 48--57 (1974). Flaxman, B.A. and E.J. VanScott: Cancer Res. 32, 2407--2412 (1972). Juergens, W.G., F.E. Stockdale, Y.J. Topper and J.J. Elias: Proc. Nat. Acad. Sci. U.S.A. 54, 629--634 (1965). Lasfarques, E.Y.: Anat. Rec. 127, 117--129 (1957). Lasfarques, E.Y. and D.H. Moore: In Vitro 7, 21--25 (1971). Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall: Biol. Chem. 193, 265--275 (1951). Norgren, A.: Acta Univ. Lund. Sect. II 11, 1--24 (1967). Russo, J., P. Furmanski and M.A. Rich: An ultrastructural study of normal human mammary cells in culture. Amer. J. Anat. 142,221--232 (1975). Spurr, A.R.: J. Ultrastruct. Res. 36, 21--46 (1969). Stockdale, F.E., W.G. Juergens and Y.J. Topper: Develop. Biol. 13, 266--281 (1966). Tobon, H. and H. Salazar: Ultrastructure of the human mammary gland. II. Postpartum lactogenesis. J. Clin. Endocr. 40,834--844 (1975). Venable, J.H. and R. Coggeshall: J. Cell. Biol. 25,407--425 (1965). Whitescarver, J., L. Recher, J.A. Sykes and L. Briggs: Problems involved in culturing human breast tissue. Tex. Rep. Biol. Med. 26,613--628 (1968).

Cultures of normal human mammary cells.

Cell Differentiation, 5 (1976) 69--81 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands CULTURES OF NORMAL HUMAN MAMMARY...
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