In Vitro Cell. Dev. Biol. 28A:128-135, February 1992 © 1992 Tissue Culture Association 0883-8364/92 $01.50+0.00
GROWTH OF HUMAN RENAL CORTICAL TISSUE ON COLLAGEN GEL SUNG-GOO CHANGt, KAROLY TOTH, JENNIFER D. BLACK, HARRY K. SLOCUM, SCOTT D. PERRAPATO, ROBERT P. HUBEN, ~D YOUCEF M. RUSTUM2 Grace Cancer Drug Center and Experimental Therapeutics (S-G. C., K. T. , J. D. B., H. K. S., Y. M. R.) and Department of Urologic Oncology (S-G. C., S. D. P., R. P. H.), Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263 (Received 18 March 1991; accepted 10 September 1991)
SUMMARY
A model system for 3-dimensional "native-state" culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 8 6 : 2 0 1 3 - 2 0 1 7 ; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemica] analysis for pankeratin (epithelia] cell origin), vimentin (mesenehymal cell origin), and p-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (aHTdR). In the first 10 days, explants showed SHTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these ceils showed labeling by SHTdR and immunostaining positive for pankeratin and p-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules, aHTdR-labeled cells in tubule structures were observed even 2 too. after Passage I, 6 too. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology. Key words: three-dimensional collagen gel culture; human kidney; tubule growth; histology; immunohistochemistry; electron microscopy; (SH)thymidine; p-g]ycoprotein. I~rrRODUC'nON
studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), and p-glycoprotein [associated with toxic drug ettlux capability and proximal tubules (8,21,23,25)], transmission electron microscopy (TEM), and incorporation of tritiated thymidine. A summary of these studies has appeared previously in abstract form (5).
Various well-established in vitro models (mostly animal and a few human) are used in renal physiology, biochemistry, and toxicology. Model systems include perfused whole and sliced kidneys, isolated tubules, isolated cells in primary and established culture, and isolated organelles (1,7,10,12,16,19,20,22,24,27). An in vitro model in which normal human renal cell growth in three-dimensional tissue form would be a valuable addition to these models. The origins and applications of various three-dimensional culture systems have been reviewed recently (13). Hoffman et al. (11,14,26) have developed a native-state, threedimensional, collagen gel-supported primary culture system that allows human cancers and normal tissues to grow in vitro with maintenance of tissue structure and differentiated markers functions for 2 wk. Perrapato et al. (17) also demonstrated that this gel culture system can support human genitourinary tumors in three-dimensional growth. In the present study we have applied this system to grow human kidney cortical tissue as a long-term culture. The cultures were
MATERIALS~ D METHODS Tissue procurement. Normal renal cortical tissue from 11 patients, identified by frozen section at the time of radical nephrectomy, was transported in a sterile container to laboratory laminar flow hoods. ColLagengel culture (histoculture). Tissues were explnnted as has been described by Hoffman et al. (14). Briefly, the kidney samples were divided into 1 to 2-ram-diameter pieces, and five pieces were placed on top of previously hydrated Spongostan gels (I)< 1 )< 1 era) (Health Design Indust., Rochester, NY). Spongostan is an absorbent, water-insoluble sponge prepared from highly purified gelatin. Each gel occupied one well of a six-well plate (Falcon, Lincoln Park, NJ). Two and a half milliliters of Eagle's minimal essential medium (MEM) (GIBCO,Grand Island, NY) supplemented with 10% fetal bovine serum {GIBCO), 50 gg/ml gentamicin final concentration (GIBCO), and cefotaxime (Hoechst, Somerville, NJ) final concentration 1 mg/ml were added. C,entamicin, routinely used in this culture system for primary explant of tissues (14), was used at a concentration 100X less than the IDaoreported for 1-h exposure in a renal tubule cell line (27). The final volume of medium was sufficient to reach to the upper gel surface, withoutimmersing it. Covered culture plates were maintained in
l Present address: Department of Urology, Kyung Hee University Medical Center, 1 Hoegi-dong Dongdaemun-Ku, Seoul 130-702, Korea. 2 To whom correspondence should be addressed. 128
HUMAN RENAL CORTEX ON 3-DIMENSIONAL COLLAGEN GEL a humidified 5% CO2 incubator at 37 ° C. Cultures underwent sterile media changes every 72 h. Growth of explant specimens. Cultured explants of five 2-mm cubes from each cultured well resulted in growth within 24 wk to the point where the small pieces coalesced into a single small tissue mass. Twelve of the 90 primary cultured explants were lost due to fungal contamination leaving 78 individual cultures available for evaluation. We have observed recently that these cultures include fibrohlast and endothelial-like cells growing as a monolayer on the bottom of some wells. Passage. Cultured explants were divided into four sections and reimplanted individually onto fresh collagen gel when they occupied most of the gel surface. Histology. Individual specimens were fixed in 10% phosphate buffered formalin (pH 7.4) for 24 h and embedded in paraffin. Sections 5-#m thick from each culture specimen were all stained with Harris' hematoxylin and eosin (H&E). The periodic acid-Schiff (PAS) reaction was performed in selected cases (four cultures) for the identification of basement membrane in the explanted tissue and around the newly formed tubules in collagen gel (18). The primary specimen and individually cultured specimens harvested at various times ( 10,13,15,18,20,25,32,37,153 days) were examined by a pathologist (K. T.), and histologic evaluations conducted. Sections from the paraffin-embedded tissue blocks (four cultures) were used for the immunohistochemical detection of cytokeratin, vimentin, and P-glycoprotein (Pgp). Those cultures in which epithelial-like cell growth was most active were selected. Immunohistochemical techniques. Cytokeratins and vimentin are cytoskeletal antigens of the intermediate filament classes. To determine the epithelial or mesenchymal origin of growing cells in the gels, monoclonal anti-pankeratin (basic marker for epithelial cells) and monoclonal anti-vimentin (marker for cells of mesenchymal origin) reactivity was studied by
129
the avidin-biotin affinity immunoperoxidase system (Lipshaw's immunotag kits, Detroit, MI) according to the manufacturer's instructions. P-glycoprotein is a unique membrane transport protein, a product of the multidrug resistance gene (mdrl), highly expressed in some tumors and in certain normal tissues (8,21,23,25). Pgp expression seems to be a new specific marker for epithelial cells of proximal tubules in kidney (8,21,23). In this study, Pgp expression was studied for this reason in the newly formed tubules in the gel using C219, a monoclonal antibody against Pgp (Centocor, Malveru, PA), by a recently developed immunoperoxidase "sandwich" method (4). The sensitivity of this method allows use of formalin-fixed, paraffin-embedded materials. As a negative and positive control for Pgp detection a previously well characterized human epidermoid carcinoma cell line KB31 (drug sensitive parent line with no detectable Pgp) and KB24C1 (a multidrug resistant subline with overexpressing Pgp) were utilized. Electron microscopy. Renal tissue specimens cultured in collagen gel (two specimens 48 days postprimary explant date and two specimens 40 days post-first passage) were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 ° C. The fixed tissues were then washed extensively in phosphate buffer, postfixed in 1% phosphate buffered osmium tetroxide, dehydrated in a graded alcohol series, and embedded in Spurr's resin. Ultrathin sections were prepared using a Porter-Blum MT-1 uhramicrotome and stained with uranyl acetate and lead citrate before examination with a Siemens Elmiskop 101. DNA precursor uptake. Thirty-seven selected cultures were exposed to medium containing 4 #Ci/ml [methyl-all] thymidine (83 Ci/mmol specific activity) for 72 h. Fixation, embedding, sectioning, and deparaffinization were completed as previously described (17). Specimen slides were exposed to Kodak NTB liquid emulsion (Kodak, Rochester, NY) for 10 days at 4 ° C, followed by standard development in D19 Kodak developer and
Fro. 1. Histology of cultured human renal cortical tissue. A, 10 days culture. Note tubule formation within the collagen gel at this early growth stage at the rim of explant, H&E X480; B, 20 days culture. The epithelial-like cells arc forming tubulelike structures resembling the kidney tubules in vivo. The ceils are organized and grown in tissue form. Degenerative cells are seen within the tubule lumen, H&E )
GROWTH OF HUMAN RENAL CORTICAL TISSUE ON COLLAGEN GEL SUNG-GOO CHANGt, KAROLY TOTH, JENNIFER D. BLACK, HARRY K. SLOCUM, SCOTT D. PERRAPATO, ROBERT P. HUBEN, ~D YOUCEF M. RUSTUM2 Grace Cancer Drug Center and Experimental Therapeutics (S-G. C., K. T. , J. D. B., H. K. S., Y. M. R.) and Department of Urologic Oncology (S-G. C., S. D. P., R. P. H.), Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263 (Received 18 March 1991; accepted 10 September 1991)
SUMMARY
A model system for 3-dimensional "native-state" culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 8 6 : 2 0 1 3 - 2 0 1 7 ; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemica] analysis for pankeratin (epithelia] cell origin), vimentin (mesenehymal cell origin), and p-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (aHTdR). In the first 10 days, explants showed SHTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these ceils showed labeling by SHTdR and immunostaining positive for pankeratin and p-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules, aHTdR-labeled cells in tubule structures were observed even 2 too. after Passage I, 6 too. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology. Key words: three-dimensional collagen gel culture; human kidney; tubule growth; histology; immunohistochemistry; electron microscopy; (SH)thymidine; p-g]ycoprotein. I~rrRODUC'nON
studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), and p-glycoprotein [associated with toxic drug ettlux capability and proximal tubules (8,21,23,25)], transmission electron microscopy (TEM), and incorporation of tritiated thymidine. A summary of these studies has appeared previously in abstract form (5).
Various well-established in vitro models (mostly animal and a few human) are used in renal physiology, biochemistry, and toxicology. Model systems include perfused whole and sliced kidneys, isolated tubules, isolated cells in primary and established culture, and isolated organelles (1,7,10,12,16,19,20,22,24,27). An in vitro model in which normal human renal cell growth in three-dimensional tissue form would be a valuable addition to these models. The origins and applications of various three-dimensional culture systems have been reviewed recently (13). Hoffman et al. (11,14,26) have developed a native-state, threedimensional, collagen gel-supported primary culture system that allows human cancers and normal tissues to grow in vitro with maintenance of tissue structure and differentiated markers functions for 2 wk. Perrapato et al. (17) also demonstrated that this gel culture system can support human genitourinary tumors in three-dimensional growth. In the present study we have applied this system to grow human kidney cortical tissue as a long-term culture. The cultures were
MATERIALS~ D METHODS Tissue procurement. Normal renal cortical tissue from 11 patients, identified by frozen section at the time of radical nephrectomy, was transported in a sterile container to laboratory laminar flow hoods. ColLagengel culture (histoculture). Tissues were explnnted as has been described by Hoffman et al. (14). Briefly, the kidney samples were divided into 1 to 2-ram-diameter pieces, and five pieces were placed on top of previously hydrated Spongostan gels (I)< 1 )< 1 era) (Health Design Indust., Rochester, NY). Spongostan is an absorbent, water-insoluble sponge prepared from highly purified gelatin. Each gel occupied one well of a six-well plate (Falcon, Lincoln Park, NJ). Two and a half milliliters of Eagle's minimal essential medium (MEM) (GIBCO,Grand Island, NY) supplemented with 10% fetal bovine serum {GIBCO), 50 gg/ml gentamicin final concentration (GIBCO), and cefotaxime (Hoechst, Somerville, NJ) final concentration 1 mg/ml were added. C,entamicin, routinely used in this culture system for primary explant of tissues (14), was used at a concentration 100X less than the IDaoreported for 1-h exposure in a renal tubule cell line (27). The final volume of medium was sufficient to reach to the upper gel surface, withoutimmersing it. Covered culture plates were maintained in
l Present address: Department of Urology, Kyung Hee University Medical Center, 1 Hoegi-dong Dongdaemun-Ku, Seoul 130-702, Korea. 2 To whom correspondence should be addressed. 128
HUMAN RENAL CORTEX ON 3-DIMENSIONAL COLLAGEN GEL a humidified 5% CO2 incubator at 37 ° C. Cultures underwent sterile media changes every 72 h. Growth of explant specimens. Cultured explants of five 2-mm cubes from each cultured well resulted in growth within 24 wk to the point where the small pieces coalesced into a single small tissue mass. Twelve of the 90 primary cultured explants were lost due to fungal contamination leaving 78 individual cultures available for evaluation. We have observed recently that these cultures include fibrohlast and endothelial-like cells growing as a monolayer on the bottom of some wells. Passage. Cultured explants were divided into four sections and reimplanted individually onto fresh collagen gel when they occupied most of the gel surface. Histology. Individual specimens were fixed in 10% phosphate buffered formalin (pH 7.4) for 24 h and embedded in paraffin. Sections 5-#m thick from each culture specimen were all stained with Harris' hematoxylin and eosin (H&E). The periodic acid-Schiff (PAS) reaction was performed in selected cases (four cultures) for the identification of basement membrane in the explanted tissue and around the newly formed tubules in collagen gel (18). The primary specimen and individually cultured specimens harvested at various times ( 10,13,15,18,20,25,32,37,153 days) were examined by a pathologist (K. T.), and histologic evaluations conducted. Sections from the paraffin-embedded tissue blocks (four cultures) were used for the immunohistochemical detection of cytokeratin, vimentin, and P-glycoprotein (Pgp). Those cultures in which epithelial-like cell growth was most active were selected. Immunohistochemical techniques. Cytokeratins and vimentin are cytoskeletal antigens of the intermediate filament classes. To determine the epithelial or mesenchymal origin of growing cells in the gels, monoclonal anti-pankeratin (basic marker for epithelial cells) and monoclonal anti-vimentin (marker for cells of mesenchymal origin) reactivity was studied by
129
the avidin-biotin affinity immunoperoxidase system (Lipshaw's immunotag kits, Detroit, MI) according to the manufacturer's instructions. P-glycoprotein is a unique membrane transport protein, a product of the multidrug resistance gene (mdrl), highly expressed in some tumors and in certain normal tissues (8,21,23,25). Pgp expression seems to be a new specific marker for epithelial cells of proximal tubules in kidney (8,21,23). In this study, Pgp expression was studied for this reason in the newly formed tubules in the gel using C219, a monoclonal antibody against Pgp (Centocor, Malveru, PA), by a recently developed immunoperoxidase "sandwich" method (4). The sensitivity of this method allows use of formalin-fixed, paraffin-embedded materials. As a negative and positive control for Pgp detection a previously well characterized human epidermoid carcinoma cell line KB31 (drug sensitive parent line with no detectable Pgp) and KB24C1 (a multidrug resistant subline with overexpressing Pgp) were utilized. Electron microscopy. Renal tissue specimens cultured in collagen gel (two specimens 48 days postprimary explant date and two specimens 40 days post-first passage) were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 ° C. The fixed tissues were then washed extensively in phosphate buffer, postfixed in 1% phosphate buffered osmium tetroxide, dehydrated in a graded alcohol series, and embedded in Spurr's resin. Ultrathin sections were prepared using a Porter-Blum MT-1 uhramicrotome and stained with uranyl acetate and lead citrate before examination with a Siemens Elmiskop 101. DNA precursor uptake. Thirty-seven selected cultures were exposed to medium containing 4 #Ci/ml [methyl-all] thymidine (83 Ci/mmol specific activity) for 72 h. Fixation, embedding, sectioning, and deparaffinization were completed as previously described (17). Specimen slides were exposed to Kodak NTB liquid emulsion (Kodak, Rochester, NY) for 10 days at 4 ° C, followed by standard development in D19 Kodak developer and
Fro. 1. Histology of cultured human renal cortical tissue. A, 10 days culture. Note tubule formation within the collagen gel at this early growth stage at the rim of explant, H&E X480; B, 20 days culture. The epithelial-like cells arc forming tubulelike structures resembling the kidney tubules in vivo. The ceils are organized and grown in tissue form. Degenerative cells are seen within the tubule lumen, H&E )
