- Email: [email protected]
Cyto-Dynamic Properties of Urinary Neoplasms: I. Cultivation in Vitro of Transitional Cell Carcinoma of the Ureter
THE JOURNAL OF UROLOGY
Vol. 64, No. 5, November 1950 Printed in U.S.A.
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS: I. CULTIVATION IN VITRO OF TRANSITIONAL CELL CARCINOMA OF THE URETER RAYMOND G. BUNGE
AND
ROBERT J. STEIN
Frain the Departments of Urology and of Obstetrics and Gynecology, College of Medicine, State University of Iowa, Iowa City, Iowa
The valuable contributions of histologists have, on the basis of nonviable, stained sections of tissue, provided classifications, static growth patterns, cellular details, etc. Recently, more attention has been paid to living cells, and particularly to their relationships and environment. This was Schwann's broad concept of cell study: And it has been restated by Carrel :1 "A cell depends as strictly upon its medium as the nucleus upon the cytoplasm." New avenues of investigation, new techniques, new knowledge in allied fields augur well for more complete understanding of this relationship. The investigation of disease processes has been confined, for the most part, within the boundaries of Virchow's basic formulations, and it was not until 1907 that Ross Harrison demonstrated that tissue could be grown outside of the body. 2 Since Harrison's demonstration of tissue culture, a large related and comprehensive literature has arisen, but application to human tissues, and particularly human neoplasms, has been limited, especially in urinary neoplasia.3 The technique provides an excellent opportunity to study cells in the living state and their metabolism, as well as changes which may occur due to controlled environmental alterations. It is the purpose of this and subsequent papers to describe the behavior of genito-urinary neoplasms cultivated in vitro. METHODS
There are essentially six methods for the cultivation of tissue in vitro: 1) The hanging drop method developed by Lewis and Lewis, 4 Carrel, 5 and Maximow, 6 which enables one to study cells over a short period. 2) The open watch glass method of Fell which permits the cultivation of a large quantity of tissue, but does not permit cells to be observed and gaseous exchange controlled. 7 3) The flask method of Carrel provides cultivation of tissue over a long period of time and control of the medium as well as gas tension. 5 4) The roller tube of Gey employs an ordinary test tube and retains all the features of the flask technique except that, due to the curvature of the tube, high powered microscopic observation is not possible. 8 Carrel, A.: Science, 73: 297, 1931. Harrison, R. G.: Proc. Soc. Exp. Biol. & Med., 4: 140, 1906-07. Fischer, A.: Gewebuzuchtung. Munich: Muller and Steinicke, 1930, 3rd ed. 4 Lewis, M. R. and Lewis, W. H.: Anat. Rec., 5: 277, 1911. 5 Carrel, A.: J. Exp. Med., 17: 14, 1913. 6 Matimow, A.: Carnegie Inst. Wash., Contrib. to Embryol., 16: 47, 1925. 7 Fell, H. B : Arch. Exp. Zellforsch, 7: 69, 1928. 8 Gey, G. 0.: Amer. J. Cancer, 17: 752, 1933. 646 1
2
3
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
647
Frn. 1. Paraffin section of original tumor of ureter. (H & E stain.)
Frn. 2. Papillary outgrowth of ureteral tumor cells from original explant in usual routine medium of unknown chemical composition.
648
RAYMOND G. BUNGE AND ROBERT J. STEIN
FIG. 3. Tumor cells growing in synthetic medium. Compare morphology with figure 4. Note typical "tadpole" cells as seen in urinary sediments.
FIG. 4. Cells taken directly from original tumor. Note similarity to cells growing in issue culture. (H & E stain.)
CY'.!.'0-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
649
5) The fluid cultivation method of Parker enables one to maintain tissue in a functional state without growth in a fluid medium. 9 6) The organ culture methods of Carrel and Lindbergh, 10 and Werthesson11 permit one to keep excised organs in a viable state. In this :investigation, the roller tube technique has been employed because of its simplicity and the ability to maintain the tissue in a living state for a long period. Since a fluid phase is used as the nutrient, it may be removed at any time and examined chemically. Tissue was obtained asepticaUy from a surgical specimen which histologically proved to be a transitional cell carcinoma of the ureter (fig. 1). The specimen was cut up into small fragments, approximately 1 mm. square, which were washed in Tyrode's solution (without sodium bicarbonate) to which 200 units of penicillin had been added per ml. to insure sterility. After two or three washings to remove blood and cellular debris, the fragments were transplanted into a modified roller tube designed by one of us (R. J. S.). One interior surface of the tube had been prepared in advance by the application of a thin layer of rooster plasma. The media employed were: a) four parts of human cord serum*, four parts of Tyrode's solution, two parts of 50 per cent chick-embryo extract,8 and b) White's synthetic medium. 12 The use of these two media was dictated by the fact that the serum-embryo extract medium is poorly defined chemically, whereas, a medium of known composition must be employed to study the specific nutritional and biochemical requirements of the growing cells. No literature relating to the use of synthetic medium in in vitro growth of urinary neoplasms is available. OBSERVATIONS
Proliferation occurred in both media. The first evidence of cellular activity in the cultures appeared on the third day. This lag phase has been noted in the cultivation of other adult tissues. The rate of growth and the morphologic architecture resembled very closely those seen in the human subject as suggested by duration of symptoms and histological sections. Papillomatous proliferation was noticeable (fig. 2). The cells very closely resembled those recovered from the urinary sediment. The morphology of the cells resembled those of the histologic sections (figs. 3 and 4). Typical "tadpole" cells of transitional cell carcinoma were also observed in the cultures. As compared to normal transition.al cells, the nuclei were large, nuclear cytoplasmic ratio reduced, nuclear membrane thickened and the nucleoli prominent, SUMMARY
It has been demonstrated that a human transitional cell carcinoma of the ureter can be grown in vitro both in synthetic and routine media. The opportuni' Parker, R. C.: Science, 83: 579, 1936. °Carrel, A. and Lindbergh, C. A.: The Culture of Organs. New York: Harper & Bros.,
1
1938
Werthessen, N. T.: Endocrinology, 44: 109, 1949. White, P.R.: Growth, 10: 3, 1946. * "Huma.n cord serum" is obta.ined from the umbilical cord immediately after birth.
11
12
650
RAYMOND G. BUNGE AND ROBER'r J. STEIN
ties for studying the biochemical dynamics of this particular neoplasm are evident. Chromatography, Warburg respiratory studies, the incorporation of various hormones into the medium, the assimilation of radio-active isotopes, the use of folic acid antagonists, and other techniques offer unlimited possibilities. Certain of these are now under study.
Vol. 64, No. 5, November 1950 Printed in U.S.A.
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS: I. CULTIVATION IN VITRO OF TRANSITIONAL CELL CARCINOMA OF THE URETER RAYMOND G. BUNGE
AND
ROBERT J. STEIN
Frain the Departments of Urology and of Obstetrics and Gynecology, College of Medicine, State University of Iowa, Iowa City, Iowa
The valuable contributions of histologists have, on the basis of nonviable, stained sections of tissue, provided classifications, static growth patterns, cellular details, etc. Recently, more attention has been paid to living cells, and particularly to their relationships and environment. This was Schwann's broad concept of cell study: And it has been restated by Carrel :1 "A cell depends as strictly upon its medium as the nucleus upon the cytoplasm." New avenues of investigation, new techniques, new knowledge in allied fields augur well for more complete understanding of this relationship. The investigation of disease processes has been confined, for the most part, within the boundaries of Virchow's basic formulations, and it was not until 1907 that Ross Harrison demonstrated that tissue could be grown outside of the body. 2 Since Harrison's demonstration of tissue culture, a large related and comprehensive literature has arisen, but application to human tissues, and particularly human neoplasms, has been limited, especially in urinary neoplasia.3 The technique provides an excellent opportunity to study cells in the living state and their metabolism, as well as changes which may occur due to controlled environmental alterations. It is the purpose of this and subsequent papers to describe the behavior of genito-urinary neoplasms cultivated in vitro. METHODS
There are essentially six methods for the cultivation of tissue in vitro: 1) The hanging drop method developed by Lewis and Lewis, 4 Carrel, 5 and Maximow, 6 which enables one to study cells over a short period. 2) The open watch glass method of Fell which permits the cultivation of a large quantity of tissue, but does not permit cells to be observed and gaseous exchange controlled. 7 3) The flask method of Carrel provides cultivation of tissue over a long period of time and control of the medium as well as gas tension. 5 4) The roller tube of Gey employs an ordinary test tube and retains all the features of the flask technique except that, due to the curvature of the tube, high powered microscopic observation is not possible. 8 Carrel, A.: Science, 73: 297, 1931. Harrison, R. G.: Proc. Soc. Exp. Biol. & Med., 4: 140, 1906-07. Fischer, A.: Gewebuzuchtung. Munich: Muller and Steinicke, 1930, 3rd ed. 4 Lewis, M. R. and Lewis, W. H.: Anat. Rec., 5: 277, 1911. 5 Carrel, A.: J. Exp. Med., 17: 14, 1913. 6 Matimow, A.: Carnegie Inst. Wash., Contrib. to Embryol., 16: 47, 1925. 7 Fell, H. B : Arch. Exp. Zellforsch, 7: 69, 1928. 8 Gey, G. 0.: Amer. J. Cancer, 17: 752, 1933. 646 1
2
3
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
647
Frn. 1. Paraffin section of original tumor of ureter. (H & E stain.)
Frn. 2. Papillary outgrowth of ureteral tumor cells from original explant in usual routine medium of unknown chemical composition.
648
RAYMOND G. BUNGE AND ROBERT J. STEIN
FIG. 3. Tumor cells growing in synthetic medium. Compare morphology with figure 4. Note typical "tadpole" cells as seen in urinary sediments.
FIG. 4. Cells taken directly from original tumor. Note similarity to cells growing in issue culture. (H & E stain.)
CY'.!.'0-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
649
5) The fluid cultivation method of Parker enables one to maintain tissue in a functional state without growth in a fluid medium. 9 6) The organ culture methods of Carrel and Lindbergh, 10 and Werthesson11 permit one to keep excised organs in a viable state. In this :investigation, the roller tube technique has been employed because of its simplicity and the ability to maintain the tissue in a living state for a long period. Since a fluid phase is used as the nutrient, it may be removed at any time and examined chemically. Tissue was obtained asepticaUy from a surgical specimen which histologically proved to be a transitional cell carcinoma of the ureter (fig. 1). The specimen was cut up into small fragments, approximately 1 mm. square, which were washed in Tyrode's solution (without sodium bicarbonate) to which 200 units of penicillin had been added per ml. to insure sterility. After two or three washings to remove blood and cellular debris, the fragments were transplanted into a modified roller tube designed by one of us (R. J. S.). One interior surface of the tube had been prepared in advance by the application of a thin layer of rooster plasma. The media employed were: a) four parts of human cord serum*, four parts of Tyrode's solution, two parts of 50 per cent chick-embryo extract,8 and b) White's synthetic medium. 12 The use of these two media was dictated by the fact that the serum-embryo extract medium is poorly defined chemically, whereas, a medium of known composition must be employed to study the specific nutritional and biochemical requirements of the growing cells. No literature relating to the use of synthetic medium in in vitro growth of urinary neoplasms is available. OBSERVATIONS
Proliferation occurred in both media. The first evidence of cellular activity in the cultures appeared on the third day. This lag phase has been noted in the cultivation of other adult tissues. The rate of growth and the morphologic architecture resembled very closely those seen in the human subject as suggested by duration of symptoms and histological sections. Papillomatous proliferation was noticeable (fig. 2). The cells very closely resembled those recovered from the urinary sediment. The morphology of the cells resembled those of the histologic sections (figs. 3 and 4). Typical "tadpole" cells of transitional cell carcinoma were also observed in the cultures. As compared to normal transition.al cells, the nuclei were large, nuclear cytoplasmic ratio reduced, nuclear membrane thickened and the nucleoli prominent, SUMMARY
It has been demonstrated that a human transitional cell carcinoma of the ureter can be grown in vitro both in synthetic and routine media. The opportuni' Parker, R. C.: Science, 83: 579, 1936. °Carrel, A. and Lindbergh, C. A.: The Culture of Organs. New York: Harper & Bros.,
1
1938
Werthessen, N. T.: Endocrinology, 44: 109, 1949. White, P.R.: Growth, 10: 3, 1946. * "Huma.n cord serum" is obta.ined from the umbilical cord immediately after birth.
11
12
650
RAYMOND G. BUNGE AND ROBER'r J. STEIN
ties for studying the biochemical dynamics of this particular neoplasm are evident. Chromatography, Warburg respiratory studies, the incorporation of various hormones into the medium, the assimilation of radio-active isotopes, the use of folic acid antagonists, and other techniques offer unlimited possibilities. Certain of these are now under study.