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Human mammary slices in organ culture
Erperimental
Cell Research 35, 437-448 (1964)
HUMAN
MAMMARY
I. METHOD
437
SLICES IN ORGAN
OF CULTURE
AND
PRELIMINARY
OBSERVATIONS ON THE OF INSULIN1 B. E. BARKER, Department
of Pathology,
H. FANGER
Rhode Island Hospital,
CULTURE
EFFECT
and P. FARNES Providence,
Rhode Island,
U.S.A.
Received June 10, 1963 2
use of organ culture techniques has been extensive in the study of development of embryonic tissues, and in the evaluation of the effects of chemical agents and hormones on embryonic and adult tissues [9, 161. Elias has pointed out the possible value of organ explantation of adult tissues in the study of effects of hormones, particularly in the demonstration of direct hormone action on isolated target tissues [4]. Such techniques have been utilized by a number of investigators in the study of hormone action on animal breast tissue [5, 6, 7, 14, 15, ‘LO, 21, X2]. In assessing the individual and combined roles of hormones on human breast tissue, organ culture seemed a reasonable approach. In addition to information concerning the specific actions of individual hormones on breast as a target tissue, such studies might provide useful tools for evaluating therapeutic roles of hormones in malignancy of the breast. The effects of insulin were studied initially because insulin has been found to be essential for maximal survival of breast tissue of mice and rats in organ culture [l, 6, 8, 22, 231. In addition, several investigators have reported that insulin stimulates growth in a variety of explanted tissues [3, 10, 11, 12, 171. The present report includes data showing maintenance of adult human breast tissue slices in organ culture without hormonal supplement, and consistent hyperplasia of ductal epithelium produced in vitro by the addition of insulin.
THE
MATERIALS
AND
METHODS
Sterile breast tissue was obtained from 30 patients at the time of surgical biopsy of the breast. An attempt was made to obtain the tissue as far distant from the site 1 This investigation was supported by Research Public Health Service. 2 Revised version received January 27, 1964. 29 - 641805
Grant
Ca-O“YZ&07
PHA
Experimcntul
from
the U.S.
Cell liesearch 35
438
B. E. Barker, H. Fanger and P. Fames
of pathology as possible. Table I shows the distribution of age and pathology found in the breast from the surgical specimen. No gross pathology was seen in the tissues used for culture in this series although the possibility of submicroscopic changes could not be ruled out. Bellco jars with sidearms and a capacity of 5 ml were used as the culture vessels. Breast tissue was thinly sliced, using either a razor blade (Fig. 1) or a Stadie-Riggs TABLE
I. Years of age
Diagnosis
‘20-29
Normal breast Fibrocystic disease Fibroadenoma Carcinoma Paget’s disease of nipple
30-39
40-49
50-59
2 1
9
3
3
2 1
1 I 1
60-69
70+
1 3
2
microtome. Each slice measured approximately 5 x 5 x I mm and was placed on a tantalum gauze circle with its edges dipped in Vaseline-paraffin. This raft was floated on 5 ml of medium in the jar and the cover sealed in place with Vaseline-paraffin (Fig. 2). Representative slices of fresh tissue were fixed in Helly’s fluid, and prepared and stained with H & E. Several media were used to test the survival of the tissue (Table II). Glucose supplement of 400 mg was added to NCTC 109 to raise the glucose level to that of Waymouth’s medium (500 mg per cent). Incubation periods varied from 1-12 days during which time the medium was not changed. In the insulin experiments every other slice of tissue was placed on medium containing 1.6 units of U40 Lilly clinical insulin per 5 ml. The medium was not changed during the experiment and no additional insulin was added. At time of sacrifice pH and glucose concentration of the media were determined. Tissues were fixed in Helly’s fluid and prepared and stained with H & E. Fig. I.--Sterile xl mm. Fig. 2.-Culture medium. Fig. 3.-Breast views showing
human breast tissue being sliced for culture. ready
for incubation
with
Slices are approximately
slice of tissue on tantalum
raft floating
tissue of 40.year-old woman previous to culture: (a) low power, well organized lobule with alveolar ducts. (a) x 50, (h) x 200.
Fig. 4.-Same as Fig. 3. 12 days in culture, 280 mg%. The lobule is very well organized normal. (a) x 50, (b) x 200.
5 mm x 5 mm on 5 ml of
(0) high power
Waymouth’s 80 %, serum 20 %, pH 6.6, glucose and the morphology of the tissue components is
Fig. B.--Same case as Figs. 3, 4. 12 days in culture. Waymouth’s 80 %, serum 20 %, pH 7.2, glucose 380 mg %. The lobule is intact although it does not appear as well maintained as in Fig. 3. (a) x 50, (6) x 2ou. Experimental
Cell Research 35
Human mammary slices in organ culture
Experimentnl
439
Cell Research 35
440
B. E. Barker, H. Fanger and P. Fames EXPERIMENTAL
RESULTS
Contamination.-Although only usual precautions were taken to prevent or exclude contaminants during preparation of the tissue and no antibiotics were included in the media, only 2 of the 285 cultures were grossly contaminated. PH.-The pH of the media was not adjusted during incubation, and the ‘IIABLE
II. No. of cultures
YIedia NCTC
38
109
serum,a insulinb insulin glucoseC NCTC 109 I serum, glucose glucose, insulin serum, glucose, insulin Waymouth’s Waymouth’s, serum Waymouth’s, serum, insulin
43 6 4 17 2 2 16 59 27
I
a All serum added 20 per cent human pooled male serum. b All insulin added 1.6 units Lilly U40 per 5 ml. c 400 mg glucose per 100 ml added.
Fig. B.-Breast tissue 7 days in Waymouth’s rosis of all tissue components. x 200.
80 ~6, serum 20 %, pH 7.4, glucose 370 mg 7;. Nec-
Fig. 7.-Breast tissue of 5l-year-old woman 6 days in NCTC 109 80 96, serum 20 96, pH 6.7, glucose 26 mg %. Most of the tissue components are necrotic although the peripheral zone still is intact. (a) x 50, (b) x 200. Fig. S.-Same case as in Fig. 7. 6 days in NCTC 109 80 %, serum 20 %, glucose added 400 mg 7; on day 1. pH 6.8, glucose 348 mg %. Well organized lobules with healthy ductal epithelium. (a) x 50, (b) x 200. Fig. 9.-Breast tissue of 22-year-old and stroma. x 200.
woman
previous
to culture
showing
lobule-alveolar
ducts
Fig. lO.-Same case as in Fig. 9. 6 days in NCTC 109 80 96, serum 20 ?J,,pH 6.8, glucose 34 mg I:&. Widespread necrosis. x 200. Fig. Il.-Same case as in Figs. 9, 10. 6 days in Waymouth’s. Ductal epithelium and surrounding stroma well maintained x 200.
No strum. pH 7.0, glucose 460 mg :t. although glucose utilization was low.
Fig. 12.-Same case as in Figs. 9, 10, 11. 6 days in Waymouth’s 80 %, serum 20 Y,, pH 6.8, glucose 354 mg %. Well maintained columnar epithelium in ductal structures. x 200. Experimental
Cell Research 35
Human mammary slices in organ crdfure
Experimental
Cell Research 35
442
B. E. Barker,
H. Fanger
and P. Fames
range of pH at time of sacrifice was 7.8-6.1. Occasionally the vaselineparaffin seal on the jar leaked, accounting for a pH above 7.2. Good survival of the tissue was obtained from pH 6.1-7.2 (Figs. 3, 4, 3) and no consistent distinction could be made in morphological characteristics of cultures within this range. Necrosis was prevalent when tissue was maintained at a pH above 7.2 (Fig. 6). Glucose.--Glucose concentration was the single most important factor in the survival of breast tissue. Using NCTC 109 with or without 20 per cent serum, the initial glucose concentration was 100-95 mg per cent and by the fourth to sixth day in culture it had dropped to less than 50 mg per cent and necrosis was prevalent (Fig. 7). Supplementing the medium to 500 mg per cent glucose initially or using Waymouth’s medium produced very satisfactory survival (Fig. 8). Most cultures utilized 150-200 mg per cent glucose by the sixth day. Cultures with serum additive utilized more glucose (Figs. 9, 10, 11, 12). Extremely high glucose concentration was not detrimental (Figs. 12, 13). General survival of tissue with or without insulin additive.--In the initial study reported here the tissues remained l-12 days in vitro. If the glucose concentration and pH were in an optimal range the 1%day cultures had very little necrosis and appeared equally as viable as S-day cultures (Figs. 4 and 5). Although there was great variability in the age of the patients and hence in the amount of epithelium present in the breast, there was consistency in the behavior of the tissue in vitro. Since the initial study, 60 additional cases have shown similar behavior with good maintenance up to 17 days in culture. Explants with abundant ductal tissue often had necrotic central zones by Fig. iY.-Same case as Figs. 9, 10, 11, 12. 6 days in NCTC 109 80 76, serum 20 %, glucose added at day 1. pH 6.8 glucose 3780 mg %. Well maintained tissue at extremely high glucose level. x zoo. Fig. 14.-Breast tissue of 29.year-old pregnant woman 5 days in Waymouth’s 80 per cent, serum 20 %, pH 6.7, glucose 316. Large complex lobule shown in low-power view (a). The outer zones of these lobules were fairly well maintained (b), although the central portions were necrotic (c). (a) x 50, (b) x 300, (c) x 300. Fig. 15.-Breast tissue of 23-year-old woman 6 days in NCTC 109 80 %, serum 20 %, pH 6.8. Typical surface growth is shown resulting from migration of ductal cells out of a duct cut in sectioning. x 75. Fig. 16.-Breast tissue of 50-year-old woman 5 days in NCTC 109 without serum. Excellent maintenance of lobular integrity (a) as well as normal orientation of lobulo-alveolar ductal cells (b). (a) x 75, (b) x 600. Fig. 17.-Breast tissue of Syear-old woman 9 days in Waymouth’s showing well maintained endothelium and smooth muscle layers. Experimental
Cell &search
35
80 %, serum 20 94. Arteriole ): 200.
Human
mammary
slices in organ culture
Experimental
443
Cell Research
35
444
B. E. Barker, H. Fanger and P. Fames
6 days while the peripheral areas were well maintained or proliferative (Fig. 14). As more cases were prepared the amount of necrosis lessened, indicating that a major cause of necrosis in our system was injury to the tissue before it was put into culture. Overhandling of the tissue with forceps, compression with the slide or microtome in cutting the slices, or drying the slices by prolonged preparation time seemed to be the major items in subsequent necrosis of the tissue. There was appreciable necrosis in 18 per cent of the explants in this series. Surface growth was observed in most cultures by the 4th day and was formed from epithelium of cut ducts as well as bp fibroblastic proliferation (Fig. 15). Medirr.-If initial glucose concentration was in the range of 500 mg per cent the breast slices were well maintained in either NCTC 109 or \Vaymouth’s medium. While serum was not essential for short-term survival there was an impression of more consistent maintenance in the serum. Waymouth’s medium supplemented with 20 per cent human pooled male serum has been selected for further studies.
SPECIFIC
RESULTS
components of the slice were well maintained for at least 7 days without marked deviation from the normal morphology. Occasional mitoses were present in ductal epithelium and connective tissue fibroblasts. Smooth muscle cells and endothelium were hypertrophic as compared to fresh tissue not placed in nutrient media. Incubation of fresh tissue in media for t-2 hr produced cells which appeared swollen; this was most likely due to imbibition of fluid in osmotic adjustment. Ductal and myoepithelial cells remained well delineated in vitro (Fig. 16) and mast cells and plasma cells were prominent throughout the culture period. Blood vessels remained well oriented in the tissue for 5-7 days after which there was a variable tendency for vessel components to migrate through the stroma (Fig. 17). Survival
of breast
components
in medium
without
insulin
added.-All
Fig. lb.--Same case as shown in Figs. 9, IO, 11, 12, 13. 6 days in Waymouth’s 80 % serum 20 %, insulin 1.6 units/5 ml. The ducts are lined with hyperplastic epithelium. These ductal cells are large with abundant cytoplasm and mitotic figures are readily seen (arrow). (a) x 100, (b) x 300. Fig. lg.-Breast tissue of 4%year-old woman 12 days in Waymouth’s 80 “/o, serum 20 %, insulin 1.6 units/5 ml. The ductal lumina are completely occluded by the proliferating cell mass (a). A high-power view shows the large cells which resemble those seen in areas of squamous metaplasia (b). (a) x 125, (b) Y 350. Experimental
Cell Research 35
Human mammary slices in organ culture
.
445
.
t
18b
Experimental
Cell Research 3.5
446
B. E. Barker,
H. Fanger
and P. Farnes
Survival of breast components in medium with insulin added.-Organ cultures of human breast tissue cultured in the presence of insulin showed consistent morphologic changes as illustrated in Fig 18 (compare with Fig. 13). Insulin treated cultures showed invariable hyperplasia of ductal cells. In older cultures the proliferation \vas so profuse as to occlude the ductal lumina (Fig. 19a). In addition, morphology of the ductal cells in insulin cultures was markedly altered from the controls. These cells xvere large, with pale-staining cytoplasm, and foci suggestive of squamous metaplasia were seen (Fig. 19 b). Changes in the other components of breast tissue were less striking than the change in the ductal cells. Plasma cells, mast cells, stroma and blood vessels showed no consistent insulin effect although a suggestion of increased hypertrophy and hyperplasia in smooth muscle cells of arterioles was noted in several cultures. DISCUSSION
Certain disadvantages are intrinsic in the use of human tissue for in vitro studies, and in the selection of tissue for this investigation, a number of unavoidable variables occurred. Age differences of patients, hormonal status, and unknown factors which may influence proliferation and/or involutional changes in human mammary tissue could not be controlled in the material available. Nevertheless, studies were restricted to histologically normal tissues, and remarkably consistent behavior of these heterogeneous samples was observed in this system. Human adult mammary tissue survives under these conditions without hormone supplement. Human pooled serum from male donors was used in an efiort to minimize any inconstant hormone levels. Slices cultured with serum showed minimal differences from slices cultured in serum-free medium and this suggests that little if any demonstrable hormonal effect occurs with the use of human pooled serum. The morphologic changes observed with insulin in this culture system are not without precedent. Gey [l l] studying effects of insulin on chick embryo fibroblasts noted that the hormone provoked increase in proliferative activity. Insulin has been shown to stimulate mitosis, as well as glycogen deposition in a variety of other tissues [2, 13, 241. The exact mode of action of insulin in our cultures has not been determined. Its action may depend, however, on its effect in increasing glucose up-take in cells [19, 251 and in increasing incorporation of amino acids into protein. Further studies are underway, using tritiated thymidine, to determine the parent cell of the Experimental
Cell Research 35
Human mammary slices in organ culture
447
hyperplasia in the ducts. This parent line may be epithelial, myoepithelial, or a combination of both cell types. The present technique is a convenient and reproducible system for study of effects of hormones and other substances on human mammary tissue in vitro. Hormones such as insulin may produce dramatic and consistent changes in morphology of tissue in this system.
SUMMARY
A technique for culture of human mammary slices has been devised, which permits survival and limited proliferation of all elements for periods up to 12 days in vitro. Slices survive in a variety of media with or without serum, providing that glucose concentrations are maintained above critical levels. In these organ cultures, addition of insulin to the medium results in marked morphological changes of ductal epithelium including proliferation, hypertrophy, and foci resembling squamous metaplasia.
ADDENDUM
Since this article went to press, Rivera (Proc. Sot. 735, 1963) has demonstrated a hyperplastic response ducts cultivated for 12 days in the presence of insulin. peared to be stimulated more than the luminal cells, that this response to insulin represents an augmentation feration encountered in this cell type in Go.
Exptl Biol. Med. 114, of mouse mammary The basal cells apand it was postulated of the normal proli-
REFERENCES 1. 2. 3. 4. .5. 6. 7. 8. 9. 10. 11. 12. 13.
BERX, H. A. and RIVERA, E. M., Proc. Am. Ass. Cancer Res. 3, 94 (1960). BULLOUGH, UT. S., Exptl Cell Res. 7, 176 (1954). CREN, J. M., J. Physiol. 125, 148 (1954). ELIAS, J. J., Henry Ford Hospital International Symposium: Biological Interactions in Normal and Neoplastic Growth, p. 355. Little, Brown and Company, Boston, 1962. ~ Science 126, 842 (1957). ~ Proc. Sot. Exptl Biol. Med. 101, 500 (1959). ELIAS, J. J. and RIVERA, E. M., Cancer Res. 19, 505 (1959). ~ Ancd. Rec. 139, 224 (1961). FELL, H. B., Synthesis of Molecular and Cellular Structure. p. 139. The Ronald Press Comnanv. New York. 1961. FRANKS, k: M., Exptl bell Res. 22, 56 (1961). GEY, G. 0. and THALHEIMER, W., J. Am. Med. Ass. 82, 1609 (1924). VOX HAAM, E. and CAPPEL, L., Am. J. Cancer 39, 354 (1940). KRAHL, M. E., The Action of Insulin on Cells. Academic Press, New York, 1961. Experimental
Cell Research 35
B. E. Barker, H. Fanger and P. Fames 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24. 25.
LASFARGUES, E. Y., Compt. rend. sot. biol. 154, 1720 (1960). LASFARGUES, E. Y. and MURRAY, M. R., Deu. Biol. 1, 413 (1959). LASNITZKI, I., Jnt. Reu. Cytol. 7, 79 (1958). LATTA, J. S. and BUCHOLZ, D. J., Z. Zellforsch. 23, 152 (1939). MAXCHESTER, I<. L. and YOUNG, F. G., The Mechanism of Action of Insulin, p. 113. Blackwell’s Scientific Publications, Oxford, 1960. MORETTI, R. L. and DE OME, I<. B., .J. Xnft. Cancer Inst. 29, 321 (1962). PKOP, F. J., Xature 184, 379 (1959). ~ Exptl Cell Res. 20, 256 (1960). Tenth Cong. Internat. Biol. Cell, p. 154, 1960. RIVER.%, I’. &I. and BERK, H. A., Endocrinot. 69, 340 (1961). SII>X%N, R. L., Anaf. Rec. 124, 723 (1956). VERR.ER, J. V., JR., BLACKARD, W. G. and ESGEL, F. L., Endocrinol. 70, 420 (1962).
Experimental
Cell Research 35
Cell Research 35, 437-448 (1964)
HUMAN
MAMMARY
I. METHOD
437
SLICES IN ORGAN
OF CULTURE
AND
PRELIMINARY
OBSERVATIONS ON THE OF INSULIN1 B. E. BARKER, Department
of Pathology,
H. FANGER
Rhode Island Hospital,
CULTURE
EFFECT
and P. FARNES Providence,
Rhode Island,
U.S.A.
Received June 10, 1963 2
use of organ culture techniques has been extensive in the study of development of embryonic tissues, and in the evaluation of the effects of chemical agents and hormones on embryonic and adult tissues [9, 161. Elias has pointed out the possible value of organ explantation of adult tissues in the study of effects of hormones, particularly in the demonstration of direct hormone action on isolated target tissues [4]. Such techniques have been utilized by a number of investigators in the study of hormone action on animal breast tissue [5, 6, 7, 14, 15, ‘LO, 21, X2]. In assessing the individual and combined roles of hormones on human breast tissue, organ culture seemed a reasonable approach. In addition to information concerning the specific actions of individual hormones on breast as a target tissue, such studies might provide useful tools for evaluating therapeutic roles of hormones in malignancy of the breast. The effects of insulin were studied initially because insulin has been found to be essential for maximal survival of breast tissue of mice and rats in organ culture [l, 6, 8, 22, 231. In addition, several investigators have reported that insulin stimulates growth in a variety of explanted tissues [3, 10, 11, 12, 171. The present report includes data showing maintenance of adult human breast tissue slices in organ culture without hormonal supplement, and consistent hyperplasia of ductal epithelium produced in vitro by the addition of insulin.
THE
MATERIALS
AND
METHODS
Sterile breast tissue was obtained from 30 patients at the time of surgical biopsy of the breast. An attempt was made to obtain the tissue as far distant from the site 1 This investigation was supported by Research Public Health Service. 2 Revised version received January 27, 1964. 29 - 641805
Grant
Ca-O“YZ&07
PHA
Experimcntul
from
the U.S.
Cell liesearch 35
438
B. E. Barker, H. Fanger and P. Fames
of pathology as possible. Table I shows the distribution of age and pathology found in the breast from the surgical specimen. No gross pathology was seen in the tissues used for culture in this series although the possibility of submicroscopic changes could not be ruled out. Bellco jars with sidearms and a capacity of 5 ml were used as the culture vessels. Breast tissue was thinly sliced, using either a razor blade (Fig. 1) or a Stadie-Riggs TABLE
I. Years of age
Diagnosis
‘20-29
Normal breast Fibrocystic disease Fibroadenoma Carcinoma Paget’s disease of nipple
30-39
40-49
50-59
2 1
9
3
3
2 1
1 I 1
60-69
70+
1 3
2
microtome. Each slice measured approximately 5 x 5 x I mm and was placed on a tantalum gauze circle with its edges dipped in Vaseline-paraffin. This raft was floated on 5 ml of medium in the jar and the cover sealed in place with Vaseline-paraffin (Fig. 2). Representative slices of fresh tissue were fixed in Helly’s fluid, and prepared and stained with H & E. Several media were used to test the survival of the tissue (Table II). Glucose supplement of 400 mg was added to NCTC 109 to raise the glucose level to that of Waymouth’s medium (500 mg per cent). Incubation periods varied from 1-12 days during which time the medium was not changed. In the insulin experiments every other slice of tissue was placed on medium containing 1.6 units of U40 Lilly clinical insulin per 5 ml. The medium was not changed during the experiment and no additional insulin was added. At time of sacrifice pH and glucose concentration of the media were determined. Tissues were fixed in Helly’s fluid and prepared and stained with H & E. Fig. I.--Sterile xl mm. Fig. 2.-Culture medium. Fig. 3.-Breast views showing
human breast tissue being sliced for culture. ready
for incubation
with
Slices are approximately
slice of tissue on tantalum
raft floating
tissue of 40.year-old woman previous to culture: (a) low power, well organized lobule with alveolar ducts. (a) x 50, (h) x 200.
Fig. 4.-Same as Fig. 3. 12 days in culture, 280 mg%. The lobule is very well organized normal. (a) x 50, (b) x 200.
5 mm x 5 mm on 5 ml of
(0) high power
Waymouth’s 80 %, serum 20 %, pH 6.6, glucose and the morphology of the tissue components is
Fig. B.--Same case as Figs. 3, 4. 12 days in culture. Waymouth’s 80 %, serum 20 %, pH 7.2, glucose 380 mg %. The lobule is intact although it does not appear as well maintained as in Fig. 3. (a) x 50, (6) x 2ou. Experimental
Cell Research 35
Human mammary slices in organ culture
Experimentnl
439
Cell Research 35
440
B. E. Barker, H. Fanger and P. Fames EXPERIMENTAL
RESULTS
Contamination.-Although only usual precautions were taken to prevent or exclude contaminants during preparation of the tissue and no antibiotics were included in the media, only 2 of the 285 cultures were grossly contaminated. PH.-The pH of the media was not adjusted during incubation, and the ‘IIABLE
II. No. of cultures
YIedia NCTC
38
109
serum,a insulinb insulin glucoseC NCTC 109 I serum, glucose glucose, insulin serum, glucose, insulin Waymouth’s Waymouth’s, serum Waymouth’s, serum, insulin
43 6 4 17 2 2 16 59 27
I
a All serum added 20 per cent human pooled male serum. b All insulin added 1.6 units Lilly U40 per 5 ml. c 400 mg glucose per 100 ml added.
Fig. B.-Breast tissue 7 days in Waymouth’s rosis of all tissue components. x 200.
80 ~6, serum 20 %, pH 7.4, glucose 370 mg 7;. Nec-
Fig. 7.-Breast tissue of 5l-year-old woman 6 days in NCTC 109 80 96, serum 20 96, pH 6.7, glucose 26 mg %. Most of the tissue components are necrotic although the peripheral zone still is intact. (a) x 50, (b) x 200. Fig. S.-Same case as in Fig. 7. 6 days in NCTC 109 80 %, serum 20 %, glucose added 400 mg 7; on day 1. pH 6.8, glucose 348 mg %. Well organized lobules with healthy ductal epithelium. (a) x 50, (b) x 200. Fig. 9.-Breast tissue of 22-year-old and stroma. x 200.
woman
previous
to culture
showing
lobule-alveolar
ducts
Fig. lO.-Same case as in Fig. 9. 6 days in NCTC 109 80 96, serum 20 ?J,,pH 6.8, glucose 34 mg I:&. Widespread necrosis. x 200. Fig. Il.-Same case as in Figs. 9, 10. 6 days in Waymouth’s. Ductal epithelium and surrounding stroma well maintained x 200.
No strum. pH 7.0, glucose 460 mg :t. although glucose utilization was low.
Fig. 12.-Same case as in Figs. 9, 10, 11. 6 days in Waymouth’s 80 %, serum 20 Y,, pH 6.8, glucose 354 mg %. Well maintained columnar epithelium in ductal structures. x 200. Experimental
Cell Research 35
Human mammary slices in organ crdfure
Experimental
Cell Research 35
442
B. E. Barker,
H. Fanger
and P. Fames
range of pH at time of sacrifice was 7.8-6.1. Occasionally the vaselineparaffin seal on the jar leaked, accounting for a pH above 7.2. Good survival of the tissue was obtained from pH 6.1-7.2 (Figs. 3, 4, 3) and no consistent distinction could be made in morphological characteristics of cultures within this range. Necrosis was prevalent when tissue was maintained at a pH above 7.2 (Fig. 6). Glucose.--Glucose concentration was the single most important factor in the survival of breast tissue. Using NCTC 109 with or without 20 per cent serum, the initial glucose concentration was 100-95 mg per cent and by the fourth to sixth day in culture it had dropped to less than 50 mg per cent and necrosis was prevalent (Fig. 7). Supplementing the medium to 500 mg per cent glucose initially or using Waymouth’s medium produced very satisfactory survival (Fig. 8). Most cultures utilized 150-200 mg per cent glucose by the sixth day. Cultures with serum additive utilized more glucose (Figs. 9, 10, 11, 12). Extremely high glucose concentration was not detrimental (Figs. 12, 13). General survival of tissue with or without insulin additive.--In the initial study reported here the tissues remained l-12 days in vitro. If the glucose concentration and pH were in an optimal range the 1%day cultures had very little necrosis and appeared equally as viable as S-day cultures (Figs. 4 and 5). Although there was great variability in the age of the patients and hence in the amount of epithelium present in the breast, there was consistency in the behavior of the tissue in vitro. Since the initial study, 60 additional cases have shown similar behavior with good maintenance up to 17 days in culture. Explants with abundant ductal tissue often had necrotic central zones by Fig. iY.-Same case as Figs. 9, 10, 11, 12. 6 days in NCTC 109 80 76, serum 20 %, glucose added at day 1. pH 6.8 glucose 3780 mg %. Well maintained tissue at extremely high glucose level. x zoo. Fig. 14.-Breast tissue of 29.year-old pregnant woman 5 days in Waymouth’s 80 per cent, serum 20 %, pH 6.7, glucose 316. Large complex lobule shown in low-power view (a). The outer zones of these lobules were fairly well maintained (b), although the central portions were necrotic (c). (a) x 50, (b) x 300, (c) x 300. Fig. 15.-Breast tissue of 23-year-old woman 6 days in NCTC 109 80 %, serum 20 %, pH 6.8. Typical surface growth is shown resulting from migration of ductal cells out of a duct cut in sectioning. x 75. Fig. 16.-Breast tissue of 50-year-old woman 5 days in NCTC 109 without serum. Excellent maintenance of lobular integrity (a) as well as normal orientation of lobulo-alveolar ductal cells (b). (a) x 75, (b) x 600. Fig. 17.-Breast tissue of Syear-old woman 9 days in Waymouth’s showing well maintained endothelium and smooth muscle layers. Experimental
Cell &search
35
80 %, serum 20 94. Arteriole ): 200.
Human
mammary
slices in organ culture
Experimental
443
Cell Research
35
444
B. E. Barker, H. Fanger and P. Fames
6 days while the peripheral areas were well maintained or proliferative (Fig. 14). As more cases were prepared the amount of necrosis lessened, indicating that a major cause of necrosis in our system was injury to the tissue before it was put into culture. Overhandling of the tissue with forceps, compression with the slide or microtome in cutting the slices, or drying the slices by prolonged preparation time seemed to be the major items in subsequent necrosis of the tissue. There was appreciable necrosis in 18 per cent of the explants in this series. Surface growth was observed in most cultures by the 4th day and was formed from epithelium of cut ducts as well as bp fibroblastic proliferation (Fig. 15). Medirr.-If initial glucose concentration was in the range of 500 mg per cent the breast slices were well maintained in either NCTC 109 or \Vaymouth’s medium. While serum was not essential for short-term survival there was an impression of more consistent maintenance in the serum. Waymouth’s medium supplemented with 20 per cent human pooled male serum has been selected for further studies.
SPECIFIC
RESULTS
components of the slice were well maintained for at least 7 days without marked deviation from the normal morphology. Occasional mitoses were present in ductal epithelium and connective tissue fibroblasts. Smooth muscle cells and endothelium were hypertrophic as compared to fresh tissue not placed in nutrient media. Incubation of fresh tissue in media for t-2 hr produced cells which appeared swollen; this was most likely due to imbibition of fluid in osmotic adjustment. Ductal and myoepithelial cells remained well delineated in vitro (Fig. 16) and mast cells and plasma cells were prominent throughout the culture period. Blood vessels remained well oriented in the tissue for 5-7 days after which there was a variable tendency for vessel components to migrate through the stroma (Fig. 17). Survival
of breast
components
in medium
without
insulin
added.-All
Fig. lb.--Same case as shown in Figs. 9, IO, 11, 12, 13. 6 days in Waymouth’s 80 % serum 20 %, insulin 1.6 units/5 ml. The ducts are lined with hyperplastic epithelium. These ductal cells are large with abundant cytoplasm and mitotic figures are readily seen (arrow). (a) x 100, (b) x 300. Fig. lg.-Breast tissue of 4%year-old woman 12 days in Waymouth’s 80 “/o, serum 20 %, insulin 1.6 units/5 ml. The ductal lumina are completely occluded by the proliferating cell mass (a). A high-power view shows the large cells which resemble those seen in areas of squamous metaplasia (b). (a) x 125, (b) Y 350. Experimental
Cell Research 35
Human mammary slices in organ culture
.
445
.
t
18b
Experimental
Cell Research 3.5
446
B. E. Barker,
H. Fanger
and P. Farnes
Survival of breast components in medium with insulin added.-Organ cultures of human breast tissue cultured in the presence of insulin showed consistent morphologic changes as illustrated in Fig 18 (compare with Fig. 13). Insulin treated cultures showed invariable hyperplasia of ductal cells. In older cultures the proliferation \vas so profuse as to occlude the ductal lumina (Fig. 19a). In addition, morphology of the ductal cells in insulin cultures was markedly altered from the controls. These cells xvere large, with pale-staining cytoplasm, and foci suggestive of squamous metaplasia were seen (Fig. 19 b). Changes in the other components of breast tissue were less striking than the change in the ductal cells. Plasma cells, mast cells, stroma and blood vessels showed no consistent insulin effect although a suggestion of increased hypertrophy and hyperplasia in smooth muscle cells of arterioles was noted in several cultures. DISCUSSION
Certain disadvantages are intrinsic in the use of human tissue for in vitro studies, and in the selection of tissue for this investigation, a number of unavoidable variables occurred. Age differences of patients, hormonal status, and unknown factors which may influence proliferation and/or involutional changes in human mammary tissue could not be controlled in the material available. Nevertheless, studies were restricted to histologically normal tissues, and remarkably consistent behavior of these heterogeneous samples was observed in this system. Human adult mammary tissue survives under these conditions without hormone supplement. Human pooled serum from male donors was used in an efiort to minimize any inconstant hormone levels. Slices cultured with serum showed minimal differences from slices cultured in serum-free medium and this suggests that little if any demonstrable hormonal effect occurs with the use of human pooled serum. The morphologic changes observed with insulin in this culture system are not without precedent. Gey [l l] studying effects of insulin on chick embryo fibroblasts noted that the hormone provoked increase in proliferative activity. Insulin has been shown to stimulate mitosis, as well as glycogen deposition in a variety of other tissues [2, 13, 241. The exact mode of action of insulin in our cultures has not been determined. Its action may depend, however, on its effect in increasing glucose up-take in cells [19, 251 and in increasing incorporation of amino acids into protein. Further studies are underway, using tritiated thymidine, to determine the parent cell of the Experimental
Cell Research 35
Human mammary slices in organ culture
447
hyperplasia in the ducts. This parent line may be epithelial, myoepithelial, or a combination of both cell types. The present technique is a convenient and reproducible system for study of effects of hormones and other substances on human mammary tissue in vitro. Hormones such as insulin may produce dramatic and consistent changes in morphology of tissue in this system.
SUMMARY
A technique for culture of human mammary slices has been devised, which permits survival and limited proliferation of all elements for periods up to 12 days in vitro. Slices survive in a variety of media with or without serum, providing that glucose concentrations are maintained above critical levels. In these organ cultures, addition of insulin to the medium results in marked morphological changes of ductal epithelium including proliferation, hypertrophy, and foci resembling squamous metaplasia.
ADDENDUM
Since this article went to press, Rivera (Proc. Sot. 735, 1963) has demonstrated a hyperplastic response ducts cultivated for 12 days in the presence of insulin. peared to be stimulated more than the luminal cells, that this response to insulin represents an augmentation feration encountered in this cell type in Go.
Exptl Biol. Med. 114, of mouse mammary The basal cells apand it was postulated of the normal proli-
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