An Ultrastructural Study of Normal Human Mammary
Epithelial Cells in Culture '
JOSE RUSSO, PHILIP FURMANSKI AND M A R V I N A. RICH
Division of Biological Sciences, Michigan Cancer Foundation,
110 East W a r r e n A v e n u e , Detroit, Michigan 48201
ABSTRACT
The ultrastructure o f normal human mammary cells cultured
from post-weaning breast fluids is described. Cells from confluent monolayers in
two week old cultures were studied. The epithelial nature of these cells was
established by the demonstration of a well developed system of cell-to-cell interdigitation and numerous desmosomes. These cells also share with breast epithelial cells in vivo, polarity, with blunt short microvilli on the apical surface
and an oriented arrangement of organelles in the basal and apical portions of
the cells. The Golgi apparatus, which is the most highly developed organelle, is
localized in the apical pole and contains substantial quantities of secretory material in the cisternae and vesicles. A variegated palisade of finely granular material mixed with tonofilaments is seen in the basal portion of the cells; many
of these tonofilaments end in the terminal web of the desmosomes.
The regular occurrence of these cells in breast fluids during the terminal
phases of lactation suggests that th9ir separation is a part of normal breast
involution.
Many methods have been used i n attempts to isolate and cultivate epithelial
cells from human mammary glands and
mammary tumors (Buehring, '72; Feller,
Stewart and Kantor, '72; Lasfargues et al.,
'72). Recently, we reported that milk obtained from human donors contains few
cells (generally less than lo4 cells per
sample of 30-160 ml); these cells did not
grow in culture. The concentration and
total number of cells increase dramatically
at weaning (Furmanski et al., '74a,b).
These cells can be placed in culture and,
in the presence of autologous serum, grow
with a generation time of 60-72 hours.
Such cultures of normal human mammary epithelial cells, free from contamination by fibroblasts, provide a n important
source of cells for both comparison with
mammary tumor cells in a variety of experimental studies and as a substrate for
infection with candidate human tumor
viruses (Keydar et al., '73; Moore et al.,
'69; Schlom et al., '72).
We therefore initiated detailed studies
on the morphological characterization of
these cells, the establishment of their epiAM. J. ANAT., 142: 221-232.
thelial nature and a comparison of their
morphological characteristics with those
described for normal human mammary
epithelium i n vivo.
M A T E R I A L S A N D METHODS
Breast fluid obtained from human donors after weaning was centrifuged at low
speed (200 X g). The cells collected were
diluted in culture medium and counted in
a Hemacytometer. The culture medium
consisted of Dulbecco's modified Eagle's
medium containing autologous serum
(15% v/v), insulin (10 pg/ml) and antibiotics, a s described previously (Furmanski et al.,'74a,b). The cells rapidly attached
and grew, with a doubling time of 60-72
hours. The cultures were used for morphological studies when they had formed
confluent monolayers, approximately two
weeks after plating. Cells from five different donors were used in the studies described here.
1 This work was supported i n part by Contract NIHNCI-E-71-2421 and grant CA 14100 from the National
Cancer Institute and an Institutional Grant to the
Michigan Cance; Foundation from the United Foundation of Detroit, Michigan.
22 1
222
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
Cultures were fixed in place using a
glutaraldehyde-osmium-uranyl acetate sequence (Hirsch and Fedorko, '68). After
dehydration, the cells were removed from
the flask with propylene oxide and rinsed
three times in order to remove plastic residue from the layer of cells. The material
was embedded in Epon (Luft, '61) and
sectioned with a diamond knife in an LKBultratome. The sections were stained with
lead citrate (Reynolds, ' 6 3 ) and examined
in a Siemens Elmiskop IA.
RESULTS
Cells in culture, from each of the five
donors studied, exhibited a homogenous
pattern consisting of a single cell type
(figs. 1, 2). Monolayers formed by these
cells showed some degree of overlapping.
The shape of the cells, while variable,
could generally be described as cuboidal.
The cells were joined by a well developed
system of cytoplasmic interdigitations and
numerous desmosomes (fig. 5). More than
one desmosome was frequently observed
to connect two cells. In some instances,
chains of three to four desmosomes were
observed.
The other characteristics which these
cells shared with normal mammary epithelium are a distinct cellular polarity, surface specialization in the form of projecting short, blunt microvilli (fig. 2 ) , and the
arrangement of organelles within the cell.
The latter is especially evident for Golgi
apparatus, secretory material and centrioles in the apical pole of the cell (figs. 2,
7, 9 ) .
The basal portions of the cells were filled
with condensed, finely granular material,
distributed as a variegated palisade adjacent to the cell membrane where it was
attached to the culture vessel (figs. 2, 3 , 5).
This granular material was mixed with
longitudinal and cross sections of tonofilaments (fig. 5).
The condensed granular material had a
diameter of 80 to 90 A. Similar material
was observed inside of vesicular structures
in the basal portions of the cells (figs. 3 ,
4). These vesicles were surrounded by a
unit membrane, and some opened into the
cytoplasm.
The presence of this granular material
i n the basal portion of the cell may be
considered abortive hemidesmosome formation, and may be related to the strong
adhesion of these cells to the culture substrate. Tonofilaments were also distributed
throughout the remainder of the cytoplasm, some of which were in the form of
bundles of fibrils connected to the desmosomes, forming a terminal web (fig. 5).
A polymorphic population of mitochondria, differing in size and shape, was found
throughout the cytoplasm of human mammary epithelial cells i n culture. In some
cells they were more concentrated in the
apical and lateral areas (figs. 2, 8). All
the mitochondria possessed numerous cristae and a dense matrix with one or two
small dense granules (fig. 8).
Rough endoplasmic reticulum was distributed randomly in the cytoplasm. This
organelle was not well developed in these
cells, but was more evident than smooth
endoplasmic reticulum, which was very
scarce (fig. 8). Free ribosomes and polyribosomes were observed more frequently
in the basal portion of the cells (fig. 5).
The Golgi apparatus was the most highly
developed organelle in these cells. I t consisted of numerous flat cisternae and vesicles filled with granular material of differing electron density (fig. 9 ) , in various
stages of packaging as secretory products,
with a n approximate diameter of 25-50 A.
It is of interest to note, that this is the
diameter reported for casein (Bargmann
and Welsh, '69).
Occasional dense lysosomes were seen in
the cytoplasm. The centriolar apparatus
(fig. 7 ) was generally localized in the
apical portion of the cell among the Golgi
vesicles and lipid inclusions. The concentration of lipid droplets varied greatly from
one cell to another. Some cells were extremely rich in lipid droplets while others
possessed few droplets (figs. 7, 8). The
lipids did not have the characteristics of
secretory material and we have not observed secretion of this material from the
cells.
The nucleus of these cells was generally
ovoid (figs. 1, 2 ) , but cells with spherical
or elongated nuclei were also observed. In
some cells the spherical nucleus was indented. Heterochromatin was distributed
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
around the periphery of the nucleus in contact with the inner layer of the nuclear envelope, Usually single, rounded nucleoli
were observed, in contact with the nuclear
envelope.
DISCUSSION
The observations presented here clearly
show that these cultured cells isolated
from post-weaning human breast fluids are
epithelial. An extensive system of cell junctions, cellular polarity, surface differentiation and secretory activity, all features of
mammary epithelium, are prominent in
these cells.
Finely granular material found in the
Golgi vesicles, very similar to that described in the Golgi vesicles of the lactating mammary gland in mice (Bargmann
and Welsh, '69; Wellings, '69), may represent the product of synthesis and secretion of milk protein. While it is difficult to
establish the exact size of this granular
material, our estimate of particles with
diameters of 25-50 A is in accord with
previous reports on milk casein (Bargmann and Welsh, '69; Waugh, '67; Wellings, '69). Biochemical analysis for the
synthesis of casein and a-lactalbumin, currently in progress, will clarify this point.
The finely granular material in the basal
portion of the cells may be synthesized in
the endoplasmic reticulum and released
into the cytoplasm from vesicles in which
it was also seen. The relationship between
this granular material and tonofilaments is
not yet clear; both have a diameter of 8090 A and are seen in close proximity along
the cell membrane attached to the culture
vessel.
The topographic origin of these cells in
the mammary gland is of great interest.
The mammary gland is a secretory organ
covered by epithelium along the ducts and
ductules (Sykes et al., '68; Tannenbaum
et al., '69; Toker, '67; Waugh and van der
Haeven, '62). Two types of cells are found
in the glandular epithelium. One is the
epithelial cell that surrounds the lumen
into which milk is secreted. The other is
the myoepithelial cell, which is located in
the basal portion of ducts and ductules,
between the luminal epithelial layer and
the basal membrane. The ultrastructural
223
characteristics of cells cultured from postweaning fluids are consistent with those of
luminal epithelial cells. This conclusion is
in agreement with previous observations
that epithelial cells in lactating milk arise
from the ductal epithelium (Papanicolaou
et al., '58). Histochemical and immunological studies in progress are aimed at
establishing the specific topographical 10calization of these cells.
The tremendous increase in the concentration and total number of cells secreted
at or following weaning may be due to random detachment of breast epithelium, or
may be a consequence of a specific physiological mechanism involved in the involution of the gland at the end of lactation.'
In milk, epithelial cells, fibroblasts, macrophages and a large quantity of cellular
debris are observed (Papanicolaou et al.,
'58). Attempts to culture cells from this
fluid were consistently unsuccessful (Furmanski et al., '74a). Postweaning fluids,
on the other hand, contain a very much
higher concentration of epithelial cells,
which retain a capacity to grow in short
term cultures (Furmanski et al., '74a,b).
These cells, therefore, may represent breast
secretory epithelium which is released during the process of breast involution after
weaning, a time in which the levels of
hypophyseal hormones that maintain secretion and the tissues of the breast during
lactation, decrease (Bargmann and Welsh,
'69; Cross and Findlay, '69; Meites et al.,
'69). This detachment of epithelial cells
from the ductal lining may represent a
mechanism for the involution of the breast
at the end of lactation, alternative or in
addition to the mechanisms proposed by
other authors (Helminen and Ericsson,
'68; Mumford, '63; Richards and Benson,
771).
ACKNOWLEDGMENTS
We acknowledge the excellent technical
assistance of Mr. Ronald H. Bradley,
Robert Kaspark, Clifford Longley and the
assistance of Ms. Ruth Rich in the acquisition of biological resources.
LITERATURE CITED
Bargmann, W., and U . Welsh 1969 On the
ultrastructure of the mammary gland. In:
2
Furmanski et al., manuscript in progress.
224
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
“Lactogenesis, The Initiation of Milk Secretion
and Parturition.” M. Reynolds and S. J. Foley,
eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 43-52.
Buehring, G. C. 1972 Culture of human mammary epithelial cell: Keeping abreast with a
new method. J. Natl. Cancer Inst., 49: 14331434.
Cross, B. A., and A. L. R. Findlay 1969 Comparative and sensory aspects of milk ejection.
In: “Lactogenesis, The Initiation of Milk Secretion and Parturition.” M. Reynolds and S . J.
Foley, eds. University of Pennsylvania Press,
Philadelphia, Pennsylvania, pp. 245-252.
Feller, W. F., S. E. Stewart and J. Kantor 1972
Primary tissue culture explants of human
breast cancer. J. Natl. Cancer Inst., 48: 11171120.
Furmanski, P., C. Longley, D. Fouchey, R. Rich
and M. A. Rich 1974a Normal human mammary cells in culture: Evidence for 0ncornav:rus-like particles. J. Natl. Cancer Inst., 52:
975-977.
- 197413 Oncornavirus-like particles in
human mammary cells in culture. Fed. Proc.,
33: 753 (abstract).
Helminen, H . J., and J. L. E. Ericsson 1968
Studies on mammary gland involution. I. On
the ultrastructure of the lactating mammary
gland. J. Ultrast. Res., 25: 193-213.
Hirsch, J. G., and M. E. Fedorko 1968 Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium
tetroxide and postfixation in uranyl acetate.
J. Cell Biol., 38: 615-627.
Keydar, J., Z. Gilead, S. Karby and E. Hare1
1973 Production of virus by embryonic cultures co-cultivated with breast tumor cells or
infected with milk from breast cancer patients.
Nature New Biol., 242 : 49-52.
Lasfargues, E. Y., W. G. Coutinho and D. H.
Moore 1972 Pitfalls in the isolation of a
human breast carcinoma virus in tissue culture.
J. Natl. Cancer Inst., 48: 1101-1105.
Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem.
Cytol., 9 : 409-414.
Meites, J., M. Sar and J. L. Voogt 1969 Effect
of suckling and pituitary release of prolactin,
ACTH, GH and TSH in the rat. In: ‘Lactogenesis, The Initiation of Milk Secretion and
Parturition,” M. Reynolds and S. J. Foley,
eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 171-179.
Moore, D. H., J. Charney, B. Kramarsky, E. Y.
Lasfargues. N. H. Sarkas, M. J. Brennan, J. H .
Burrows, S . M. Sirsat, J. C. Paymaster and
A. B. Vaidya 1969 Search for a human
breast oncornavirus. Nature (London), 229:
611-614.
Munford, R. E. 1963 Changes in the mammary
glsnds of rats and mice during pregnancy,
1actat:on and involution: I. Histological structure. J. Endocrinol., 28: 37-45.
Papanicolaou, G. N., D. G. Holmquist, G. H.
Bader and E. A. Falk 1958 Exfoliative cytology of the human mammary gland and its
value in the diagnosis of cancer and other
diseases of the breast. Cancer, 11: 3 7 7 4 0 9 .
Reynolds, E. S. 1963 The use of lead citrate
at high pH as a n electron opaque stain in
electron microscopy. J. Cell Biol., 17: 208-212.
Richards, K. C., and G. K. Benson 1971 Ultrastructural changes accompanying involution of
the mammary gland in the albino rat. J. Endocrinol., 51: 127-132.
Schlom, J., S. Spiegelman and D. H. Moore 1972
Reverse transcriptase and high molecular
weight RNA in particles from mouse and human milk. J. Natl. Cancer Inst., 48: 1197-1203.
Sykes, J. A., L. Recher, P. H. Jernstrom and
J. Whitescarver 1968 Morphological investigation of human breast cancer. J. Natl. Cancer
Inst., 40: 195-223.
Tannenbaum, M., M. Weiss and A. J. Marx 1969
Ultrastructure of the human mammary ductule.
Cancer, 23: 958-978.
Taker, C. 1967 Observations of the ultrastructure of a mammary ductule. J. Ultrastruct. Res.,
21: 9-25.
Waugh, D. F. 1967 The interaction of a’, p
and k-caseins in micelle formation. Disc. Faraday SOC.,25: 186-192.
Waugh, D. F., and E. van der Haevea 1962
Fine structure of the human adult female
breast. Lab. Invest., 11: 220-228.
Wellings, S. R. 1969 Ultrastructural basis of
lactogenesis. In: “Lactogenesis, The Initiation
of Milk Secretion and Parturition,” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp.
5-25.
PLATES
PLATE 1
EXPLANATION OF FIGURES
226
1
One micron section of human epithelial cells from post-weaning fluid
in culture. x 850.
2
Human mammary epithelial cells from post-weaning fluid after two
weeks in culture. Microvilli M, desmosomes D, mitoch-ndria m,
filaments F, Golgi apparatus G, lysosome L, granular material in the
basal portion of the cell GM. x 7,500.
3
Basal portion of the epithelial cells, with the granular material GM
attached to the membrane, forming a variegated palisade; vesicles V
with granular material of the same characteristics. x 28,000.
4
High magnification of a vesicle V apparently releasing granular material GM into the basal portion of the cell. x 44,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 1
227
PLATE 2
EXPLANATION OF FIGURES
228
5
TWOcells joined by desmosomes in the apical and basal poles of the
cells. Numerous tonofilaments T are present, some confluent, forming
tonofibrils ending i n the terminal web of the desmosomes. Free ribosomes R and polyribosomes PR are i n the cytoplasm. x 34,500. The
upper inset shows a desmosome. x 60,000.
6
Cytoplasmic interdigitation (arrows) between the epithelial cells
from the post-weaning fluid. The granular material GM is apparently attached to the cell membrane. >; 18,200.
7
Golgi apparatus G and vesicles with secretory material S surrounding the centrioles C. Upper left corner and lower right corner, lipid
droplets Lip. x 18,200.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 2
229
PLATE 3
EXPLANATION OF FIGURES
230
8
Portion of the cytoplasm showing mitochondria m with different size
and shape, granular endoplasmic reticulum ger, lysosomes L, lipid
droplets Lip, tonofilaments T. x 15,700.
9
Putative secretory material S and its relation to Golgi apparatus G ,
lipid droplets Lip and mitochondria rn. x 15,700. Inset: High magnification of the finely granular material in a secretory vesicle.
x 72,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 3
231