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Cyto-Dynamic Properties of Urinary Neoplasms: III. Cultivation in Vitro of Carcinoma of the Kidney
THE JOURNAL OF UROLOGY
Vol. 69, No. 1, January 1953 Printed in U.S.A.
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS: III. CULTIVATION IN VITRO OF CARCINOMA OF THE KIDNEY R.G.BUNGE From the State University of Iowa, College of Medicine, Iowa City, Iowa
In previous reports the cultivation in vitro of transitional cell carcinoma of the ureter1 and maintenance of ''hypernephroma"2 have been reported. The opportunity to view growing carcinoma of the kidney has been intriguing since there are many curious histological observations about the neoplasm itself. Within a
Fm. 1. Original explant, carcinoma of kidney, growth pattern resembles that of cultures (H & E stain).
single specimen the morphology varies greatly and the exact meaning of these variations has not been understood. (Caspersson A & B cells?) The question of origin of the neoplasm has not been thoroughly established, whether one portion of the nephron contributes to the original prototype or whether different portions of the renal unit can give rise to different types of renal neoplasm or to the variations within a simple neoplasm. The detection of hypernephroma cells in urinary sediments has been difficult. The opportunity to test various anticarcinogenic agents against this type of tumor has been presented by cultivation in vitro. METHODS
Tissue was obtained aseptically from a fresh surgical specimen, histology of which is presented in figure 1. The tissue was cut up into small fragments approx1 2
Bunge, R. G. and Stein, R. J.: J. Urol., 64: 646, 1950. Stein, R. J. and Bunge, R. G.: J. Urol., 66: 103, 1951. 18
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
19
imately 1 mm. square, washed several times with Simm's solution to which 100 units of penicillin per ml. and 5 mg. of streptomycin had been added. The fragments were then transplanted into square roller tubes whose interior surfaces had
"'": FIG. 2. A, 7-day culture in White's medium. Some fibrocytic proliferation and epithelial "sprouts." No active growth of epithelium. B, 8-day culture in equal parts of autologous serum and White's medium. Epithelial outgrowth tending to form bridge. C, other growths projecting outward and then proliferating parallel to explant.
been prepared in advance with a thin layer of rooster plasma. Media employed were equal parts of autologous serum and White's synthetic medium and White's medium alone.3 Tubes were placed in a roller drum and incubated at a tempera' White, P.R., Growth, 10: 231, 1946.
20
R. G. BUNGE
ture of 37°C. The medium was changed whenever indicated by the· pH of the neutral red indicator. This was on an average of every 3 days. OBSERVATIONS
In White's medium, very little proliferation occurred, although the edges of the explant remained clear and appeared to be viable (fig. 2, A). The addition of autologous serum to the medium resulted in some slight proliferation, although the cultures never became stabilized. During the first 24 hours there was a minimal amount of sloughing and rounding of the edges of the explants. In the cultures containing equal parts of autologous serum and White's medium, the proliferation began after this 24 hour period. It was noted that multiple stalk-like sheets of epithelium appeared which, within the next 24 to 48 hours, proliferated to a point where they curved toward one another, forming a bridge effect around the periphery of the culture (fig. 2, B). A minimal amount of fibrocytic proliferation was noted. The cells very often presented huge nuclei and pseudopodia. No definite tubular formation was noted. In a previous report we had observed the presence of large multinucleated cells in the urines collected from kidneys containing carcinoma. At that time we were uncertain as to whether these were neoplastic cells or were associated with hypernephroma. It has been suggested that they represented proliferating tubular cells. In stained preparations of the cultures, many multinucleated giant cells similar to those seen in the urinary sediment were observed. It is not contended that the multinucleated cells in the urinary sediment are malignant, but the similarity is made as a matter of record. In previous attempts to grow this neoplasm in tissue cultures, using balanced salt solution, chick embryo juice and human placental cord serum, almost pure fibrocytic growth has resulted. It was not until autologous serum was used that an apparent suppression of fibrocytic growth occurred and epithelium grew out. CONCLUSIONS
Carcinoma of the kidney has been cultivated in equal parts of autologous serum and White's medium, and its growth pattern described. White's medium alone (with rooster plasma coagulum) maintains carcinoma of the kidney, but does not support active growth.
Vol. 69, No. 1, January 1953 Printed in U.S.A.
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS: III. CULTIVATION IN VITRO OF CARCINOMA OF THE KIDNEY R.G.BUNGE From the State University of Iowa, College of Medicine, Iowa City, Iowa
In previous reports the cultivation in vitro of transitional cell carcinoma of the ureter1 and maintenance of ''hypernephroma"2 have been reported. The opportunity to view growing carcinoma of the kidney has been intriguing since there are many curious histological observations about the neoplasm itself. Within a
Fm. 1. Original explant, carcinoma of kidney, growth pattern resembles that of cultures (H & E stain).
single specimen the morphology varies greatly and the exact meaning of these variations has not been understood. (Caspersson A & B cells?) The question of origin of the neoplasm has not been thoroughly established, whether one portion of the nephron contributes to the original prototype or whether different portions of the renal unit can give rise to different types of renal neoplasm or to the variations within a simple neoplasm. The detection of hypernephroma cells in urinary sediments has been difficult. The opportunity to test various anticarcinogenic agents against this type of tumor has been presented by cultivation in vitro. METHODS
Tissue was obtained aseptically from a fresh surgical specimen, histology of which is presented in figure 1. The tissue was cut up into small fragments approx1 2
Bunge, R. G. and Stein, R. J.: J. Urol., 64: 646, 1950. Stein, R. J. and Bunge, R. G.: J. Urol., 66: 103, 1951. 18
CYTO-DYNAMIC PROPERTIES OF URINARY NEOPLASMS
19
imately 1 mm. square, washed several times with Simm's solution to which 100 units of penicillin per ml. and 5 mg. of streptomycin had been added. The fragments were then transplanted into square roller tubes whose interior surfaces had
"'": FIG. 2. A, 7-day culture in White's medium. Some fibrocytic proliferation and epithelial "sprouts." No active growth of epithelium. B, 8-day culture in equal parts of autologous serum and White's medium. Epithelial outgrowth tending to form bridge. C, other growths projecting outward and then proliferating parallel to explant.
been prepared in advance with a thin layer of rooster plasma. Media employed were equal parts of autologous serum and White's synthetic medium and White's medium alone.3 Tubes were placed in a roller drum and incubated at a tempera' White, P.R., Growth, 10: 231, 1946.
20
R. G. BUNGE
ture of 37°C. The medium was changed whenever indicated by the· pH of the neutral red indicator. This was on an average of every 3 days. OBSERVATIONS
In White's medium, very little proliferation occurred, although the edges of the explant remained clear and appeared to be viable (fig. 2, A). The addition of autologous serum to the medium resulted in some slight proliferation, although the cultures never became stabilized. During the first 24 hours there was a minimal amount of sloughing and rounding of the edges of the explants. In the cultures containing equal parts of autologous serum and White's medium, the proliferation began after this 24 hour period. It was noted that multiple stalk-like sheets of epithelium appeared which, within the next 24 to 48 hours, proliferated to a point where they curved toward one another, forming a bridge effect around the periphery of the culture (fig. 2, B). A minimal amount of fibrocytic proliferation was noted. The cells very often presented huge nuclei and pseudopodia. No definite tubular formation was noted. In a previous report we had observed the presence of large multinucleated cells in the urines collected from kidneys containing carcinoma. At that time we were uncertain as to whether these were neoplastic cells or were associated with hypernephroma. It has been suggested that they represented proliferating tubular cells. In stained preparations of the cultures, many multinucleated giant cells similar to those seen in the urinary sediment were observed. It is not contended that the multinucleated cells in the urinary sediment are malignant, but the similarity is made as a matter of record. In previous attempts to grow this neoplasm in tissue cultures, using balanced salt solution, chick embryo juice and human placental cord serum, almost pure fibrocytic growth has resulted. It was not until autologous serum was used that an apparent suppression of fibrocytic growth occurred and epithelium grew out. CONCLUSIONS
Carcinoma of the kidney has been cultivated in equal parts of autologous serum and White's medium, and its growth pattern described. White's medium alone (with rooster plasma coagulum) maintains carcinoma of the kidney, but does not support active growth.