In Vitro Cell. Dev. Biol. 26:1186-1194, December 1990 9 1990 Tissue Culture Association 0883-8364/90 $01.50+0.00
ROUTINE CULTURING OF NORMAL, DYSPLASTIC AND MALIGNANT HUMAN MAMMARY EPITHELIAL CELLS FROM SMALL TISSUE SAMPLES JOANNE T. EMERMAN AND DARCY A. WILKINSON Department of Anatomy, Universityof British Columbia, 2177 WesbrookMall, Vancouver, British Columbia, V6T 1IV5 Canada (Accepted 25 September 1990)
SUMMARY We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and scrum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely produce sufficient numbers of HMEC from small tissue Samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. Key words: human mammary epithelial cells; primary culture; serum-free medium. strated (14). Likewise, the necessity of including normal controls in order to discern aberrant patterns of gene expression in tumor ceils has been demonstrated (34). It has been demonstrated that HMEC in primary culture maintain their phenotype on collagen-coated dishes (22). We have shown that malignant HMEC grow well on dehydrated collagen and this culture system can be used to study hormone and drug effects on hu man mammary neoplasms (9,11). With regard to medium components, we have reported good growth of both nonmalignant and malignant cells in 5% human serum in short term primary cultures (9). However, human serum also encourages the growth of fibroblasts with time in culture (20, this study). We have grown HMEC from both normal and malignant origin in the serum-free medium described by tlammond et al. (15), but the cultures do not reach tile cell density achieved with human serum when growth ceases (9). This finding has recently been reported by others (3). In this report we demonstrate that culture conditions utilizing a serum-free medium described by Yang and co-workers (2,32) and collagencoated dishes consistently select for and stimulate the growth of HMEC from normal, dysplastic and malignant tissue. Cultures were terminated in 3 weeks to avoid genetic aberrations that may be induced with time in culture.
INTRODUCTION
There has been considerable progress in developing cell culture conditions to study the biology of the human breast. Conditions in culture have been established that permit the passage of cells and development of cell lines from cells derived from normal (3,15), fibrocystic (5) and primary tumor tissue (3,23) as well as ceils obtained from pleural effusions (12). However, to accomplish this, the cells must undergo a selection process and only a small number of the original cell population continues to grow (3,30,31). In order to avoid cell selection as well as olher artifacts that may be induced by long term culture, conditions for primary cultures of human mammary epithelial ceils (HMEC) have been improved (4,9,17,18,20,31-33). The use of extracellular matrices other than plastic, such as endothelial cell extraccllular matrix (4), dishes coated with dehydrated collagen (2,9,11,20,22,32) and collagen gels (9,32,33) greatly improve HMEC attachment and growth. While the medium component fetal bovine serum allows attachment but restrains the growth of these cells (9,15,20), human serum promotes both attachment and growth (9,18,20). A number of serum-free media have been developed that selectively encourage the growth of HMEC (4,15,22,32). Despite these improvements, however, the use of primary cuhure systems for studies of HMEC requiring large numbers of cells has been limited due to the small tissue samples available for research, the poor epithelial cell yield from tissue dissociation and their limited prohferativc capacity in vitro. We have concentrated our efforts on identifying primary cell cuture conditions that will enable us to routinely grow sufficient numbers of nonmalignant and malignant HMEC for molecular biological investigations. The importance of using primary cell cultures as well as cell lines for studying genetic anomalies has been demon-
MATERIALS AND METHODS
Cell culture. Normal mammary tissue was obtained from reduction mammoplasties and from areas adjacent to malignancies. Fibrocystic tissue, fibroadenomas and carcinomas were obtained from biopsies and mastectomics. The tissues were trimmed of fat, minced and stored until needed in liquid N2 in medium composed of 50% Dulbecco's modified Eagle's medium (1)ME; Terry Fox Laboratory, Vancouver, B.C.), 44% calf serum (Grand Island Biol. Co. 1186
1187
ItUMAN MAMMARY EPITHELIAL CELLS TABLE 1
160O
EPITHELIAL CELLS OBTAINED FROM DISSOCIATED NORMAL, DYSPLASTIC AND MALIGNANT HUMAN MAMMARY TISSUE Specimen
Cell Number Tissue (g)
Tissue (g)
Normal9 (n = 4) Normalb (n = 7) Fibroadenoma (n = 16) Fibrocystic (n = 13) Carcinoma (n = 20)
6.20 0.51 0.84 0.81 0.87
+ + + + +
1.67 ~ 0.09 0.10 0.16 0.23
0.42 X 1.05 X 1.41X 0.67 • 1.05 X
+ + 106 • 10 6 • 106 + 10 6 10 6
2.35 X 3.14 • 5.01X 1.41 • 2.29 X
l0 s 10 s 10 s l0 s l0 s
o--t
~ 1200 o
X
800
9 Reduction mammoplasties. t, Areas adjacent to malignant tissue. = SEM.
0
[GIBCO], Burlington, Ontario) and 6% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO) (Dr. M. Stampfer, personal communication). As previously described (9), the tissue was dissociated in a 1:1 mixture of Ham's F12:DME (Terry Fox Lab) containing 10 mM HEPES buffer (Sigma), 20 m g / m l bovine serum albumin (BSA, Fraction V, GIBCO), 5/.tg/ml insulin, 300 U/ml collagenase and 100 U/ml hyaluronidase (Sigma) at 37 ~ C for 1 6 - 1 8 h. After dissociation, the cell suspension was centrifuged at 40 • for 30 sec. The pellet, consisting of epithelial organoids, was washed twice with F12:DME and resuspended in plating medium consisting of F12:DME, 10 mM HEPES, 5 g g / m l insulin and 5% human serum and seeded onto collagen-coated 35 mm tissue culture dishes. Although we did not obtain a cell number from the organoids, they were cultured to maximize the number of epithelial cells obtained
400
_oj o o
30O
iSi,:'::;.:
X
~
o 100 ~i~. ~~
-r-
S
SF -> S SF Media
ferent media. Cultures were seeded at 5 X 104 cells/cm2 onto collagencoated dishes in medium coasisting of FI2:DME (1:1), 10 mM HEPES, 5 /.tg/ml insulin and 5% human serum (ltuS). After 24 h, some of the cultures were switched to sermn-free medium composed of FI 2:DME, l0 mM HEPES, 5 mg/ml BSA, 10 ng/ml cholera toxin, 10/.tg/ml insulin and 0.5 ,ug/ml cortisol; these were the serum-flee cultures. Cells were cultured in medium with serum (S) for 16 d (solid bar) or transferred from serum-flee to medium with serum (SF S) on d 7 (white striped bar). Cells were cultured in serum-free medium (SE) for 16 d or transferred from serum to serumfree medium (S SF) on d 7 (black striped bar). Growth was determined by a histometric method described in Materials and Methods and expressed as a percentage increase in the histometric growth index (IIGI) on d 16 compared to its initial value on d 2 (IIGIo), the first d of cell counting.
5
10 15 Days in culture
20
25
FIG. 2. Growth curves of human mammary epithelial cells (HMEC) cultured in serum-free medium. Cultures were seeded at 2.5-5 X 104 cells/ cm2 in medium containing 5% HuS; at 24 h, they were switched to serumfree medium as described in Fig. 1. Growth was determined by a histometric method described in Materials and Methods and Fig. 1. Cells from normal tissue, - - 0 - - ; cells from fibrocystic tissue, - - A - - ; cells from 2 malignant specimens, - - ! 1 - - and --IZI--.
from each tissue sample. The supernatant, consisting of single cells and small aggregates of cells, was centrifuged at 100 Xg for 4 min. The pellet, consisting of an enriched population of epithelial cells, was washed twice and viable cells, determined by trypan blue exclusion, were counted on a hemacytometer. The supernatant, consisting of stromal cells, was cultured in some experiments. The epithelial cells were resuspended in culture medium and seeded at 2 . 5 - 5 X 104 cells/cm 2 onto collagen-coated dishes. Cultures were incubated at 37 ~ C in 95% air:5% CO2. After 24 h, the medium on most of the cultures was removed, the dishes washed twice and serum-free medium (32) consisting of F12:DME, 10 mM HEPES, 5 mg/ml BSA, 10 ng/ml cholera toxin, 10 # g / m l insulin and 0.5 ttg/ml cortisol (Sigma) was added to the cultures. In some experiments, the ionic Ca concentration of the culture medium was reduced from 1.05 mM Ca ++ to 0.06 mM Ca ++ (29) on different days. The collagen solution was obtained from rat tail collagen as described previously (10). About 1 ml of collagen solution was placed in each dish, spread evenly to coat the dish, the excess aspirated and the dish allowed to air dry for 189 h.
S --> SF
FIc. 1. Growth of epithelial cells from a fihroadenoma cultured in dif-
0
TABLE 2 HISTOMETRIC GROWTH INDEX OF HUMAN MAMMARY EPITHELIAL CELLS AND FIBROBLASTS IN SERUM-FREE MEDIUM Specimen
Epilhelial Cells
Fibroblasts
HGI (%)9 1 2
790 355
0 0
9 HGI - HGIo X 100 = % as described in Materials and Methods. The HGI0 HGI, measurement on d 12 in culture; the HGIo, the initial value on d 6, first d of cell counting.
1188
EMERMAN AND WILKINSON a grid of 2 mm 2 squares etched onto the underside of the dish were used in these experiments. On different days, the percent of a given area of a dish that was covered with epithelial cells was determined as follows. A 10 mm 2 area was marked offin the center of the grid. When the dish was viewed at 125 • magnification through a phase contrast microscope, a graticulc grid (10 • 10 squares) that was inserted in the microscope eyepiece was superimposed onto 1 of each 2 mm 2 square of the dish. The area of the square in the graticule grid covered with epithelial cells was recorded on graph paper that had been divided to duplicate the view seen through the microscope. Maps were drawn that duplicated the outline of epithelial patches seen on the dish. This provided a histometric growth index (HGI) and the results were expressed as a percentage increase in the HGI as compared to its initial value at day 0 (HGIo), the first day of cell mapping (18). HGI - ttGIo X 100 = % HGIo In some experiments, cell growth was also determined by measuring the DNA content of the cultures at termination. Cultures were scraped with a Teflon policeman into ice cold resuspension buffer consisting of 0.1 M NaCI, 10 mM Tris and 10 mM EDTA, pelleted and stored at - 7 0 ~ C until needed. The DNA was then extracted with phenol/chloroform (19). The DNA was quantified using a UV spoctrophotomctcr at 260 and 280 nm. There was a good correlation between the histometric growth index and the I)NA assay, confirming the validity of the histomctric method of cell counting. DNA content was converted to cell number, assuming 7 pg DNA/cell (9). In 2 experiments that indicated an 8 fold increase in cell number by the HGI, the cell count was 5 X 106 cells as determined by DNA content. In 2 experiments that indicated a 4
Fie,. 3. Phase contrast micrographs of cultures of ItMEC in serum-free medium. A) 9 d culture of cells from normal breast tissue. Small epithelial cells formed foci in a lawn of larger cells. B) 16 d culture of cells from fibrocystic tissue. Still larger, elongated epithelial cells were found along the periphery of small clumps of tissues. X192.
Human serum was pooled from blood collected in the mornings from donors who fasted the previous 8 - 1 2 h (9). Donors included patients undergoing reduction mammoplasties and breast biopsies for benign breast conditions. Cell growth. Cell growth was determined by a histomctric method on the same cultures that were used for morphological studies as there were not enough cells per tissue sample to sacrifice cultures for cell counting. Tissue culture dishes (Lux; 35 mm) with
Ft(;. 4. Phase contrast micrograph of epithelial cells from a fibroadenoma cultured in serum-free medium. Domes (arrowhead) were observed in the epithelial nmnolayer. X192.
1189
IIUMAN MAMMARY EPITHELIAL CEI,LS RESULTS
Cell growth. Although it was possible to obtain a cell number from the epithelial organoids by dissociating the organoids to single cells with trypsin, this was avoided in order to maximize cell yields. However, the number of single epithelial cells dissociated from the tissues was determined. Tile cell yield per gram of norm'd, dysplastic and malignant tissue is listed in Table l. Except for tissue obtained from reduction mammoplasties, less than a gram of tissue was received from each patient. The number of cells obtained from dissociation of these ranged from approximately 0.5 X 106 for normal tissue obtained from reduction mammoplasties to 1.5 X 106 for fibroadenomas. Cells were seeded as small organoids or at 2.5 to 5 X 104 cells/ cm 2 onto collagen-coated dishes in medium containing 5% human serum. After 24 h, the medium of some of the cultures was changed to serum-free medium. On d 7, some of the cultures incubated in serum were switched to serum-free medium and visa versa. The growth rates of epithelial cells in all cultures examined (n=5), regardless of tissue source, were approximately 2 - 4 fold higher in the presence of serum-free medium than in serum-containing medium. A representative experiment is illustrated in Fig. 1, which shows the differences in growth of epithelial cells from a fibroadenoma maintained in medium with and without serum. Cells cultured in serum-free medium or transferred from serum-containing to serum-free medium had significantly higher percent increases in their HGI than sister cultures in serum-containing medium or switched from serum-free to serum-containing medium. Although the growth rates of epithelial cells from different specimens varied, cells from all tissue sources grew well and generally approached the Fit;. 5. Fluorescent mierograph of a 14 d culture of epithelial cells from a mammary carcinoma in serum-free medium. Positive immunofluorescent staining with antikeratin identified the cells as epithelial. Fibroblast cultures were negative for keratin filaments. •
fold increase in cell number by the HGI, the cell number was 2.3 X 106 cells as determined by DNA content. Light Microscopy. Culture morphology was documented by photographing living cells under phase contrast optics in a I,eitz inverted microscope. To confirm that the cells were epithelial by light microscopy, cultures were stained for keratin using as the primary antibody pooled AE1 and AE3 monoclonal antibodies to human epithelial keratin (Hybriteeh Inc., San Diego, CA) and as the secondary antibody rhodamine-labeled goat anti-mouse immunoglobulins (HyClone, Logan, Utah). Cells were fixed in methanol, permeablized in methanohacetone (1:1) and washed in PBS. Cells were exposed to primary antibody for 1 h at 37 ~ C, washed with PBS , exposed to secondary antibody for 1 h at 37 ~ C, washed in PBS and mounted in gelvatol (21). Slides were viewed with a Zeiss Photomicroscope III. The film used was Kodak T-max pushed to 1600 ASA and developed in T-max developer. To determine if the prominent vacuoles observed in the epithelial cells were fat droplets, cultures were fixed with 10% formalin and stained with Sudan Red and hematoxylin (8). Electron microscopy. Cultures were fixed in 1.5% paraformaldebyde, 1.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.3), postfixed in 1% OsO 4 and embedded in Epon using standard techniques (9). Sections were stained with uranyl acetate and lead citrate and observed in a Philips 300 electron microscope.
FIc. 6. Uhrastrueture of epithelial cells from fibroeystic tissue in serum-free medium, l,ateral surfaces were joined by desmosomes. In addition to those associated with desmosomes, small bundles of intermediate filaments with random orientation were abundant in the cytoplasm. • 70O.
1190
EMERMAN AND WILKINSON
stationary phase of growth between 15-21 d (Fig. 2). In medium with serum, fibroblasts began to dominate the cuhures around 10 d in culture. In contrast, the number of fibroblasts decreased or remained stationary in serum-free medium. The HGIs of epithelial cells and fibroblasts from cultures derived from 2 malignant specimens in given in Table 2. Successful culture were defined as ones in which the epithelial cells neared confluence around 21 d in vitro and very few fibroblasts were present. In this regard, the epithelial cells grew better from small organoids than from isolated cells. Although their growth properties were comparable, the number of successful cultures obtained from cell organoids was greater than that from single ceils. Interestingly, despite the fact that the cells from all tissue sources grew well in serum-free medium, there was considerable variation in the number of successful cultures per tissue type. Approximately 8 0 - 9 0 % of the cultures seeded with ceils from normal tissue, fibrocystic tissue and fibroadenomas were successful. On the other hand, only 65% of cultures from malignant tissue obtained from biopies and 40% of those obtained from mastectomies were successful. An obvious difference between the groups was the average age of the patients. The mean age of patients with fibroadenomas was the lowest, 35.8 years (SEM + 3.8) and that of patients with malignancies was the highest, 56.3 years (SEM + 3.2) and 65.0 years (SEM -4- 1.4) for biopsies and mastectomies respectively. There was no correlation between the successful culturing of malignant tissue and their estrogen receptor status. (The estrogen receptor data were obtained from the pathology reports). Cells from the same specimen cultured at different times yielded similar results, suggesting that the success of the cultures was a feature of the tissue and not due to variations in culture procedures. Culture morphology. After 3 - 5 d in vitro, different morphologies were evident between cultures incubated in serum-containing and serum-free medium. Cuboidal epithelial cells of relatively uniform size formed cobblestone patches in the presence of serum. With time in culture, the epithelial patches were often infiltrated with fibroblasts and lost their organization. The fibroblasts were bipolar, spindle-shaped cells generally oriented parallel to one another. In serum-free medium, the epithelial islands were generally free of fibroblasts and 3 sizes of epithelial cells formed the cobblestone appearance of these islands. Small epithelial cells clustered together to form isolated patches or formed small foci in a lawn of larger cells (Fig. 3 A). Still larger epithelial cells that were somewhat elongated grew along the periphery of the islands or small clumps of tissue (Fig. 3 B). Domes or hemicysts were frequently observed in the epithelial patches (Fig. 4). The few fibroblasts that were present were large, fiat, pale cells, some of which contained very noticeable stress fibers in their cytoplasm. Cell morphology. In cultures of enriched epithelial cell populations incubated with and without serum, cells were identified as epithelial by the presence of keratin filaments observed by fluorescent microscopy (Fig. 5). Fibroblasts cultured from the same tissue samples were negative for keratin filaments. Epithelial cells were further identified by their ultrastructural characteristics. Cells had numerous microvilli at their apical surfaces and adjacent cells were joined by tight junctions and desmosomes. Intermediate filaments were not only found in association with desmosomes, but were also prominent throughout the cytoplasm (Fig. 6), as has been previously described (16,23,24). At approximately 10 days, most cultures, regardless of the tissue
Fit;. 7. Phase contrast micrograph of a 20 d culture of epithelial cells from a fibroadenoma ill serum-free medium. A) Many epithelial cells developed single large intracytoplasmic vacuoles around 10 d in culture. B) In older cultures, large, fiat cells containing many small vacuoles were also present. •
source, included epithelial cells that developed single large intracytoplasmic vacuoles in their perinuclear cytoplasm (Fig. 7 A). This morphology differed from that observed in older cultures, when large, fiat cells containing many small vacuoles began to appear (Fig. 7 B). Viewed in the electron microscope, most of the vacuoles were identified as inclusions that were not membrane-bounded (Fig. 8 A); occasionally they were identified as intracytoplasmic lumen
1191
tIUMAN MAMMARY EPITHELIAL CELLS
dium and visa versa (Fig. 9 A,B,C,D). Serum-free medium selected for epithelial cell growth, whereas serum-containing medium supported both epithelial cell and fibroblast growth. Growth in low Ca ++ medium. By 21 d in vitro, the epithelial cells generally reached the stationary phase of growth in serum-free medium. Soule and McGrath (29) have demonstrated that primary cultures of HMEC maintained in serum-containing medium with a low Ca ++ concentration continue to grow, producing viable floating cells up to 6 months in culture. The floating cells could subsequently be subcultured. To determine if the yield of epithelial cells in the serum-free medium could be increased, the Ca ++ concentration was reduced form 1.05 mM (high Ca ++) to 0.06 mM (low Ca ++) (n=6). Cultures were switched to low Ca ++ medium at 1, 6, 10, 13 or 17 d in vitro. The effect of low Ca ++ medium on the growth of fibroblasts isolated from the same tissue samples was also monitored. The morphology of the epithelial cell patches changed after 1-2 d in low Ca++; the cells became rounder with phase-bright borders (Fig. 10 A). After several days, the cells formed a monolayer of flattened cells (larger than those in high Ca ++) and round, floating cells (floaters) appeared in the medium (Fig. 10 B). Since the cells appeared larger than those in medium with high Ca ++, we were not confident that the histometric growth indeces were comparable. However, DNA determinations indicated that the final cell densities were the same. Fibroblasts and fat cells did not undergo changes in morphology and they did not produce floaters. A representative experiment measuring growth of attached and floating cells is illustrated in Fig. 11. Although cultures grown in low Ca ++ did product: floaters, viability was low and we were not successful in generating subcultures from these floaters. Our best attempts yielded slow-growing secondary cultures when floaters, produced after 10 d in low Ca ++ medium (viability was highest at this time, Fig. ] 1 ), were subcultured onto collagen-coated dishes in medium with 5% human serum and 1.05 mM Ca ++. DISCUSSION
FIG. 8. Electron mierographsofttMECcuhvredinscrum-freemcdium. A) A cell from fibrocystic tissue containing a non-memb,'ane bounded inclusion in the perinuclear cytoplasm. • B) A cell from a mammary carcinoma containing an intracytoplasmic lumen lined with irregular microvilli and surrounded by a network intermediate filaments. )
ROUTINE CULTURING OF NORMAL, DYSPLASTIC AND MALIGNANT HUMAN MAMMARY EPITHELIAL CELLS FROM SMALL TISSUE SAMPLES JOANNE T. EMERMAN AND DARCY A. WILKINSON Department of Anatomy, Universityof British Columbia, 2177 WesbrookMall, Vancouver, British Columbia, V6T 1IV5 Canada (Accepted 25 September 1990)
SUMMARY We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and scrum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely produce sufficient numbers of HMEC from small tissue Samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. Key words: human mammary epithelial cells; primary culture; serum-free medium. strated (14). Likewise, the necessity of including normal controls in order to discern aberrant patterns of gene expression in tumor ceils has been demonstrated (34). It has been demonstrated that HMEC in primary culture maintain their phenotype on collagen-coated dishes (22). We have shown that malignant HMEC grow well on dehydrated collagen and this culture system can be used to study hormone and drug effects on hu man mammary neoplasms (9,11). With regard to medium components, we have reported good growth of both nonmalignant and malignant cells in 5% human serum in short term primary cultures (9). However, human serum also encourages the growth of fibroblasts with time in culture (20, this study). We have grown HMEC from both normal and malignant origin in the serum-free medium described by tlammond et al. (15), but the cultures do not reach tile cell density achieved with human serum when growth ceases (9). This finding has recently been reported by others (3). In this report we demonstrate that culture conditions utilizing a serum-free medium described by Yang and co-workers (2,32) and collagencoated dishes consistently select for and stimulate the growth of HMEC from normal, dysplastic and malignant tissue. Cultures were terminated in 3 weeks to avoid genetic aberrations that may be induced with time in culture.
INTRODUCTION
There has been considerable progress in developing cell culture conditions to study the biology of the human breast. Conditions in culture have been established that permit the passage of cells and development of cell lines from cells derived from normal (3,15), fibrocystic (5) and primary tumor tissue (3,23) as well as ceils obtained from pleural effusions (12). However, to accomplish this, the cells must undergo a selection process and only a small number of the original cell population continues to grow (3,30,31). In order to avoid cell selection as well as olher artifacts that may be induced by long term culture, conditions for primary cultures of human mammary epithelial ceils (HMEC) have been improved (4,9,17,18,20,31-33). The use of extracellular matrices other than plastic, such as endothelial cell extraccllular matrix (4), dishes coated with dehydrated collagen (2,9,11,20,22,32) and collagen gels (9,32,33) greatly improve HMEC attachment and growth. While the medium component fetal bovine serum allows attachment but restrains the growth of these cells (9,15,20), human serum promotes both attachment and growth (9,18,20). A number of serum-free media have been developed that selectively encourage the growth of HMEC (4,15,22,32). Despite these improvements, however, the use of primary cuhure systems for studies of HMEC requiring large numbers of cells has been limited due to the small tissue samples available for research, the poor epithelial cell yield from tissue dissociation and their limited prohferativc capacity in vitro. We have concentrated our efforts on identifying primary cell cuture conditions that will enable us to routinely grow sufficient numbers of nonmalignant and malignant HMEC for molecular biological investigations. The importance of using primary cell cultures as well as cell lines for studying genetic anomalies has been demon-
MATERIALS AND METHODS
Cell culture. Normal mammary tissue was obtained from reduction mammoplasties and from areas adjacent to malignancies. Fibrocystic tissue, fibroadenomas and carcinomas were obtained from biopsies and mastectomics. The tissues were trimmed of fat, minced and stored until needed in liquid N2 in medium composed of 50% Dulbecco's modified Eagle's medium (1)ME; Terry Fox Laboratory, Vancouver, B.C.), 44% calf serum (Grand Island Biol. Co. 1186
1187
ItUMAN MAMMARY EPITHELIAL CELLS TABLE 1
160O
EPITHELIAL CELLS OBTAINED FROM DISSOCIATED NORMAL, DYSPLASTIC AND MALIGNANT HUMAN MAMMARY TISSUE Specimen
Cell Number Tissue (g)
Tissue (g)
Normal9 (n = 4) Normalb (n = 7) Fibroadenoma (n = 16) Fibrocystic (n = 13) Carcinoma (n = 20)
6.20 0.51 0.84 0.81 0.87
+ + + + +
1.67 ~ 0.09 0.10 0.16 0.23
0.42 X 1.05 X 1.41X 0.67 • 1.05 X
+ + 106 • 10 6 • 106 + 10 6 10 6
2.35 X 3.14 • 5.01X 1.41 • 2.29 X
l0 s 10 s 10 s l0 s l0 s
o--t
~ 1200 o
X
800
9 Reduction mammoplasties. t, Areas adjacent to malignant tissue. = SEM.
0
[GIBCO], Burlington, Ontario) and 6% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO) (Dr. M. Stampfer, personal communication). As previously described (9), the tissue was dissociated in a 1:1 mixture of Ham's F12:DME (Terry Fox Lab) containing 10 mM HEPES buffer (Sigma), 20 m g / m l bovine serum albumin (BSA, Fraction V, GIBCO), 5/.tg/ml insulin, 300 U/ml collagenase and 100 U/ml hyaluronidase (Sigma) at 37 ~ C for 1 6 - 1 8 h. After dissociation, the cell suspension was centrifuged at 40 • for 30 sec. The pellet, consisting of epithelial organoids, was washed twice with F12:DME and resuspended in plating medium consisting of F12:DME, 10 mM HEPES, 5 g g / m l insulin and 5% human serum and seeded onto collagen-coated 35 mm tissue culture dishes. Although we did not obtain a cell number from the organoids, they were cultured to maximize the number of epithelial cells obtained
400
_oj o o
30O
iSi,:'::;.:
X
~
o 100 ~i~. ~~
-r-
S
SF -> S SF Media
ferent media. Cultures were seeded at 5 X 104 cells/cm2 onto collagencoated dishes in medium coasisting of FI2:DME (1:1), 10 mM HEPES, 5 /.tg/ml insulin and 5% human serum (ltuS). After 24 h, some of the cultures were switched to sermn-free medium composed of FI 2:DME, l0 mM HEPES, 5 mg/ml BSA, 10 ng/ml cholera toxin, 10/.tg/ml insulin and 0.5 ,ug/ml cortisol; these were the serum-flee cultures. Cells were cultured in medium with serum (S) for 16 d (solid bar) or transferred from serum-flee to medium with serum (SF S) on d 7 (white striped bar). Cells were cultured in serum-free medium (SE) for 16 d or transferred from serum to serumfree medium (S SF) on d 7 (black striped bar). Growth was determined by a histometric method described in Materials and Methods and expressed as a percentage increase in the histometric growth index (IIGI) on d 16 compared to its initial value on d 2 (IIGIo), the first d of cell counting.
5
10 15 Days in culture
20
25
FIG. 2. Growth curves of human mammary epithelial cells (HMEC) cultured in serum-free medium. Cultures were seeded at 2.5-5 X 104 cells/ cm2 in medium containing 5% HuS; at 24 h, they were switched to serumfree medium as described in Fig. 1. Growth was determined by a histometric method described in Materials and Methods and Fig. 1. Cells from normal tissue, - - 0 - - ; cells from fibrocystic tissue, - - A - - ; cells from 2 malignant specimens, - - ! 1 - - and --IZI--.
from each tissue sample. The supernatant, consisting of single cells and small aggregates of cells, was centrifuged at 100 Xg for 4 min. The pellet, consisting of an enriched population of epithelial cells, was washed twice and viable cells, determined by trypan blue exclusion, were counted on a hemacytometer. The supernatant, consisting of stromal cells, was cultured in some experiments. The epithelial cells were resuspended in culture medium and seeded at 2 . 5 - 5 X 104 cells/cm 2 onto collagen-coated dishes. Cultures were incubated at 37 ~ C in 95% air:5% CO2. After 24 h, the medium on most of the cultures was removed, the dishes washed twice and serum-free medium (32) consisting of F12:DME, 10 mM HEPES, 5 mg/ml BSA, 10 ng/ml cholera toxin, 10 # g / m l insulin and 0.5 ttg/ml cortisol (Sigma) was added to the cultures. In some experiments, the ionic Ca concentration of the culture medium was reduced from 1.05 mM Ca ++ to 0.06 mM Ca ++ (29) on different days. The collagen solution was obtained from rat tail collagen as described previously (10). About 1 ml of collagen solution was placed in each dish, spread evenly to coat the dish, the excess aspirated and the dish allowed to air dry for 189 h.
S --> SF
FIc. 1. Growth of epithelial cells from a fihroadenoma cultured in dif-
0
TABLE 2 HISTOMETRIC GROWTH INDEX OF HUMAN MAMMARY EPITHELIAL CELLS AND FIBROBLASTS IN SERUM-FREE MEDIUM Specimen
Epilhelial Cells
Fibroblasts
HGI (%)9 1 2
790 355
0 0
9 HGI - HGIo X 100 = % as described in Materials and Methods. The HGI0 HGI, measurement on d 12 in culture; the HGIo, the initial value on d 6, first d of cell counting.
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EMERMAN AND WILKINSON a grid of 2 mm 2 squares etched onto the underside of the dish were used in these experiments. On different days, the percent of a given area of a dish that was covered with epithelial cells was determined as follows. A 10 mm 2 area was marked offin the center of the grid. When the dish was viewed at 125 • magnification through a phase contrast microscope, a graticulc grid (10 • 10 squares) that was inserted in the microscope eyepiece was superimposed onto 1 of each 2 mm 2 square of the dish. The area of the square in the graticule grid covered with epithelial cells was recorded on graph paper that had been divided to duplicate the view seen through the microscope. Maps were drawn that duplicated the outline of epithelial patches seen on the dish. This provided a histometric growth index (HGI) and the results were expressed as a percentage increase in the HGI as compared to its initial value at day 0 (HGIo), the first day of cell mapping (18). HGI - ttGIo X 100 = % HGIo In some experiments, cell growth was also determined by measuring the DNA content of the cultures at termination. Cultures were scraped with a Teflon policeman into ice cold resuspension buffer consisting of 0.1 M NaCI, 10 mM Tris and 10 mM EDTA, pelleted and stored at - 7 0 ~ C until needed. The DNA was then extracted with phenol/chloroform (19). The DNA was quantified using a UV spoctrophotomctcr at 260 and 280 nm. There was a good correlation between the histometric growth index and the I)NA assay, confirming the validity of the histomctric method of cell counting. DNA content was converted to cell number, assuming 7 pg DNA/cell (9). In 2 experiments that indicated an 8 fold increase in cell number by the HGI, the cell count was 5 X 106 cells as determined by DNA content. In 2 experiments that indicated a 4
Fie,. 3. Phase contrast micrographs of cultures of ItMEC in serum-free medium. A) 9 d culture of cells from normal breast tissue. Small epithelial cells formed foci in a lawn of larger cells. B) 16 d culture of cells from fibrocystic tissue. Still larger, elongated epithelial cells were found along the periphery of small clumps of tissues. X192.
Human serum was pooled from blood collected in the mornings from donors who fasted the previous 8 - 1 2 h (9). Donors included patients undergoing reduction mammoplasties and breast biopsies for benign breast conditions. Cell growth. Cell growth was determined by a histomctric method on the same cultures that were used for morphological studies as there were not enough cells per tissue sample to sacrifice cultures for cell counting. Tissue culture dishes (Lux; 35 mm) with
Ft(;. 4. Phase contrast micrograph of epithelial cells from a fibroadenoma cultured in serum-free medium. Domes (arrowhead) were observed in the epithelial nmnolayer. X192.
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Cell growth. Although it was possible to obtain a cell number from the epithelial organoids by dissociating the organoids to single cells with trypsin, this was avoided in order to maximize cell yields. However, the number of single epithelial cells dissociated from the tissues was determined. Tile cell yield per gram of norm'd, dysplastic and malignant tissue is listed in Table l. Except for tissue obtained from reduction mammoplasties, less than a gram of tissue was received from each patient. The number of cells obtained from dissociation of these ranged from approximately 0.5 X 106 for normal tissue obtained from reduction mammoplasties to 1.5 X 106 for fibroadenomas. Cells were seeded as small organoids or at 2.5 to 5 X 104 cells/ cm 2 onto collagen-coated dishes in medium containing 5% human serum. After 24 h, the medium of some of the cultures was changed to serum-free medium. On d 7, some of the cultures incubated in serum were switched to serum-free medium and visa versa. The growth rates of epithelial cells in all cultures examined (n=5), regardless of tissue source, were approximately 2 - 4 fold higher in the presence of serum-free medium than in serum-containing medium. A representative experiment is illustrated in Fig. 1, which shows the differences in growth of epithelial cells from a fibroadenoma maintained in medium with and without serum. Cells cultured in serum-free medium or transferred from serum-containing to serum-free medium had significantly higher percent increases in their HGI than sister cultures in serum-containing medium or switched from serum-free to serum-containing medium. Although the growth rates of epithelial cells from different specimens varied, cells from all tissue sources grew well and generally approached the Fit;. 5. Fluorescent mierograph of a 14 d culture of epithelial cells from a mammary carcinoma in serum-free medium. Positive immunofluorescent staining with antikeratin identified the cells as epithelial. Fibroblast cultures were negative for keratin filaments. •
fold increase in cell number by the HGI, the cell number was 2.3 X 106 cells as determined by DNA content. Light Microscopy. Culture morphology was documented by photographing living cells under phase contrast optics in a I,eitz inverted microscope. To confirm that the cells were epithelial by light microscopy, cultures were stained for keratin using as the primary antibody pooled AE1 and AE3 monoclonal antibodies to human epithelial keratin (Hybriteeh Inc., San Diego, CA) and as the secondary antibody rhodamine-labeled goat anti-mouse immunoglobulins (HyClone, Logan, Utah). Cells were fixed in methanol, permeablized in methanohacetone (1:1) and washed in PBS. Cells were exposed to primary antibody for 1 h at 37 ~ C, washed with PBS , exposed to secondary antibody for 1 h at 37 ~ C, washed in PBS and mounted in gelvatol (21). Slides were viewed with a Zeiss Photomicroscope III. The film used was Kodak T-max pushed to 1600 ASA and developed in T-max developer. To determine if the prominent vacuoles observed in the epithelial cells were fat droplets, cultures were fixed with 10% formalin and stained with Sudan Red and hematoxylin (8). Electron microscopy. Cultures were fixed in 1.5% paraformaldebyde, 1.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.3), postfixed in 1% OsO 4 and embedded in Epon using standard techniques (9). Sections were stained with uranyl acetate and lead citrate and observed in a Philips 300 electron microscope.
FIc. 6. Uhrastrueture of epithelial cells from fibroeystic tissue in serum-free medium, l,ateral surfaces were joined by desmosomes. In addition to those associated with desmosomes, small bundles of intermediate filaments with random orientation were abundant in the cytoplasm. • 70O.
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stationary phase of growth between 15-21 d (Fig. 2). In medium with serum, fibroblasts began to dominate the cuhures around 10 d in culture. In contrast, the number of fibroblasts decreased or remained stationary in serum-free medium. The HGIs of epithelial cells and fibroblasts from cultures derived from 2 malignant specimens in given in Table 2. Successful culture were defined as ones in which the epithelial cells neared confluence around 21 d in vitro and very few fibroblasts were present. In this regard, the epithelial cells grew better from small organoids than from isolated cells. Although their growth properties were comparable, the number of successful cultures obtained from cell organoids was greater than that from single ceils. Interestingly, despite the fact that the cells from all tissue sources grew well in serum-free medium, there was considerable variation in the number of successful cultures per tissue type. Approximately 8 0 - 9 0 % of the cultures seeded with ceils from normal tissue, fibrocystic tissue and fibroadenomas were successful. On the other hand, only 65% of cultures from malignant tissue obtained from biopies and 40% of those obtained from mastectomies were successful. An obvious difference between the groups was the average age of the patients. The mean age of patients with fibroadenomas was the lowest, 35.8 years (SEM + 3.8) and that of patients with malignancies was the highest, 56.3 years (SEM + 3.2) and 65.0 years (SEM -4- 1.4) for biopsies and mastectomies respectively. There was no correlation between the successful culturing of malignant tissue and their estrogen receptor status. (The estrogen receptor data were obtained from the pathology reports). Cells from the same specimen cultured at different times yielded similar results, suggesting that the success of the cultures was a feature of the tissue and not due to variations in culture procedures. Culture morphology. After 3 - 5 d in vitro, different morphologies were evident between cultures incubated in serum-containing and serum-free medium. Cuboidal epithelial cells of relatively uniform size formed cobblestone patches in the presence of serum. With time in culture, the epithelial patches were often infiltrated with fibroblasts and lost their organization. The fibroblasts were bipolar, spindle-shaped cells generally oriented parallel to one another. In serum-free medium, the epithelial islands were generally free of fibroblasts and 3 sizes of epithelial cells formed the cobblestone appearance of these islands. Small epithelial cells clustered together to form isolated patches or formed small foci in a lawn of larger cells (Fig. 3 A). Still larger epithelial cells that were somewhat elongated grew along the periphery of the islands or small clumps of tissue (Fig. 3 B). Domes or hemicysts were frequently observed in the epithelial patches (Fig. 4). The few fibroblasts that were present were large, fiat, pale cells, some of which contained very noticeable stress fibers in their cytoplasm. Cell morphology. In cultures of enriched epithelial cell populations incubated with and without serum, cells were identified as epithelial by the presence of keratin filaments observed by fluorescent microscopy (Fig. 5). Fibroblasts cultured from the same tissue samples were negative for keratin filaments. Epithelial cells were further identified by their ultrastructural characteristics. Cells had numerous microvilli at their apical surfaces and adjacent cells were joined by tight junctions and desmosomes. Intermediate filaments were not only found in association with desmosomes, but were also prominent throughout the cytoplasm (Fig. 6), as has been previously described (16,23,24). At approximately 10 days, most cultures, regardless of the tissue
Fit;. 7. Phase contrast micrograph of a 20 d culture of epithelial cells from a fibroadenoma ill serum-free medium. A) Many epithelial cells developed single large intracytoplasmic vacuoles around 10 d in culture. B) In older cultures, large, fiat cells containing many small vacuoles were also present. •
source, included epithelial cells that developed single large intracytoplasmic vacuoles in their perinuclear cytoplasm (Fig. 7 A). This morphology differed from that observed in older cultures, when large, fiat cells containing many small vacuoles began to appear (Fig. 7 B). Viewed in the electron microscope, most of the vacuoles were identified as inclusions that were not membrane-bounded (Fig. 8 A); occasionally they were identified as intracytoplasmic lumen
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dium and visa versa (Fig. 9 A,B,C,D). Serum-free medium selected for epithelial cell growth, whereas serum-containing medium supported both epithelial cell and fibroblast growth. Growth in low Ca ++ medium. By 21 d in vitro, the epithelial cells generally reached the stationary phase of growth in serum-free medium. Soule and McGrath (29) have demonstrated that primary cultures of HMEC maintained in serum-containing medium with a low Ca ++ concentration continue to grow, producing viable floating cells up to 6 months in culture. The floating cells could subsequently be subcultured. To determine if the yield of epithelial cells in the serum-free medium could be increased, the Ca ++ concentration was reduced form 1.05 mM (high Ca ++) to 0.06 mM (low Ca ++) (n=6). Cultures were switched to low Ca ++ medium at 1, 6, 10, 13 or 17 d in vitro. The effect of low Ca ++ medium on the growth of fibroblasts isolated from the same tissue samples was also monitored. The morphology of the epithelial cell patches changed after 1-2 d in low Ca++; the cells became rounder with phase-bright borders (Fig. 10 A). After several days, the cells formed a monolayer of flattened cells (larger than those in high Ca ++) and round, floating cells (floaters) appeared in the medium (Fig. 10 B). Since the cells appeared larger than those in medium with high Ca ++, we were not confident that the histometric growth indeces were comparable. However, DNA determinations indicated that the final cell densities were the same. Fibroblasts and fat cells did not undergo changes in morphology and they did not produce floaters. A representative experiment measuring growth of attached and floating cells is illustrated in Fig. 11. Although cultures grown in low Ca ++ did product: floaters, viability was low and we were not successful in generating subcultures from these floaters. Our best attempts yielded slow-growing secondary cultures when floaters, produced after 10 d in low Ca ++ medium (viability was highest at this time, Fig. ] 1 ), were subcultured onto collagen-coated dishes in medium with 5% human serum and 1.05 mM Ca ++. DISCUSSION
FIG. 8. Electron mierographsofttMECcuhvredinscrum-freemcdium. A) A cell from fibrocystic tissue containing a non-memb,'ane bounded inclusion in the perinuclear cytoplasm. • B) A cell from a mammary carcinoma containing an intracytoplasmic lumen lined with irregular microvilli and surrounded by a network intermediate filaments. )
