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Primary culture of chicken bursal plical epithelium
Research in Veterinary Science 1991, 50, 352-354
Primary culture of chicken bursal plical epithelium T. N U N O Y A , M. T A J I M A , Nippon Institute for Biological Science, 2221-1 Shinmachi, Ome, Tokyo 198, Japan, C. I T A K U R A , Department of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060, Japan
Plical epithelial cells were obtained by trypsin-EDTA treatment of chicken bursa of Fabricius and cultured in the presence of type IV collagen. The culture became confluent six to seven days after seeding. The grown cells showed a positive reaction for cytokeratin by immunostaining and h a d ultrastructural characteristics of the epithelial cells in vivo. The cell culture will be useful for parasitological and virological studies. SEVERAL studies have been conducted on in vitro cultures of the bursa of Fabricius aimed at investigating the interaction between B cells and bursal epithelial components. They were confined to bursal reticular epithelial cells (Boyd et al 1983a,b) or an in vitro organ culture system for alymphoid bursal epithelial cells (Eerola 1980, Eerola et al 1982). To the present authors' knowledge, no further studies have been made particularly o f the epithelium lining the inner faces of the plicae. The plical epithelial cells are divided into three definitive types of cells: the most numerous cells are tall, columnar with an oval nucleus and the two other types are goblet cells and sudanophilic cells, although the latter are few in n u m b e r (Hodges 1974). Interestingly, parasitisation in avian cryptosporidiosis occasionally occurs on the microvillus border of the plical epithelial cell layers (Fletcher et al 1975, Randall 1982), but the interaction between cryptosporidia and the epithelial cells is poorly understood. The purpose of the present study was to develop a method of culturing the bursal plical epithelium for investigation of bursal cryptosporidiosis and other studies. Specific pathogen-free White Leghorn line M chickens were obtained from the Nippon Institute for Biological Science's Laboratory Animal Research Station. The bursa of Fabricius of four-week-old chickens was used throughout the study, because the bursas at this age were considered to be big enough for the source of plical epithelial cells and to retain the proliferating capacity. After blood was withdrawn from the chicken by cardiac puncture, the bursa was asceptically excised and opened in a sterilised glass dish. Each plica was separated and cut into 1 to 2 m m 3 pieces and the pieces placed in a bottle containing equal volumes of 0.25 per cent trypsin and 0.01 per cent ethylene diaminetetraacetic acid (EDTA)in physiological saline or 0.1 per cent coUagenase (Wako Pure Chemical Industries, Japan) in phosphate buffered saline (PBS) (approximately 2 ml per bursa). The material was then stirred for either 15 or 30 minutes at room temperature, or incubated at 37°C for one hour without stirring. After tissue digestion, bottles were
allowed to stand for a minute to precipitate gross tissue fragments and the supernatants containing released cells were centrifuged at 1000 g for five minutes. The resulting pellets were resuspended in culture media (approximately 10 ml per bursa), which consisted of Eagle's m i n i m u m essential medium (Grand Island Biological), supplemented with 5 to 20 per cent fetal calf serum (Gibco), 200 iu m l - i penicillin and 200 /zg m l - l streptomycin. Ten ml of the suspension was seeded on coverslips in a 60 m m x 15 m m plastic culture dish (Becton Dickinson) and incubated at 37°C in a humidified atmosphere o f 5 per cent carbon dioxide in air. The medium was changed after overnight incubation and the cultures were maintained during the following week at 37°C. Some of the coverslips were coated with collagen type I (from rat tail) or type IV (from h u m a n placenta) (Sigma Chemical) before seeding with bursal cells. Seven milligrams of the collagen was dissolved in 20 ml of 0" 1 M acetic acid. The solution was pipetted on to coverslips in a culture dish as above and then allowed to dry at room temperature. The coverslips were sterilised by exposure to ultraviolet light as previously described (Nunoya et al 1987). The collagen-coated coverslips were washed twice with PBS to remove acetic acid before use. To examine the morphology of grown cells, some of the cultures were fixed in Bouin's fluid and stained with haematoxylin and eosin for light microscopy, and others fixed in phosphate buffered 2" 5 per cent glutaraldehyde for electron microscopy. The glutaraldehyde-fixed cells were then dehydrated in ethanol and propylene oxide and embedded in Epok 812. Silver-grey sections cut on an ultramicrotome were stained with uranyl acetate and lead citrate and examined with a JEM-100B electron microscope. The cultured cells were also processed for immunocytochemistry. The cells were washed gently in PaS and fixed in cold ethanol for 10 minutes. A monoclonal antibody to cytokeratin (CK5, Sigma Chemical) was used as a primary antibody and reacted with anti-mouse IgG peroxidase conjugate (Sigma Chemical). The cells were developed with s u b s t r a t e solutions (0.02 per cent 3-amino-9-ethyl carbazole, 0.03 per cent hydrogen peroxide, and 5 per cent N,N-dimethylformamide in 0.1 M acetate buffer, p H 5.2) and counterstained with haematoxylin. The plical epithelial cells were most efficiently isolated by incubating bursal tissue in the trypsin-EDTA mixture at 37°C for one hour. The effectiveness was confirmed by histological examination of the tissue remnants after incubation. The continuous stirring during digestion resulted in an increase of non-epithelial components such as follicular
352
Culture of bursal plical epithelium
353
FIG 1 : Confluent culture of bursal plical epithelia[ cells. Haematoxylin and eosin x 62. Inset shows a homogeneous population of large, polygonal cerls with oval nuclei x 252
FIG 3: Electron micrograph of cultured plical epithelial cells showing well-developed mitochondria, interdigitated cell membranes and desmosomes (arrows) x 6762
lymphocytes and stromal fibroblasts; the 15 minute digest became confluent three to four days after seeding but less than 10 per cent of cells spread to form small colonies of plical epithelial cells and the remaining cells were fibroblasts, and the 30 minute digest was exclusively follicular lymphocytes. Since collagenase treatment surpassed trypsin as a celldispersing agent in various experiments (Lang 1979), collagenase was substituted for the trypsin-EDTA. With this enzyme, however, no growth was observed in the released cells under the same culture conditions, although epithelial cell release from the plicae was seemingly effective. Pretreatment of coverslips with collagen was necessary for cell spread and type IV collagen gave a better result (more than 10 per cent). This collagen requirement by plical epithelial cells was considered to be a general property of epithelial cells, because their attachment and differentiation were strongly influenced by collagen, especially type IV (Wicha et al 1979). The time taken to attain confluent culture
was related to the amount of serum in the medium; between six and seven days under the optimum concentration of approximately 20 per cent. The plical epithelial cells appeared to grow in some areas from the periphery of fragmented epithelial layers and in other areas from single dispersed cells. The confluent culture stained with haematoxylin and eosin comprised a homogeneous population of large, polygonal cells with centrally positioned, pale and oval nuclei in neutrophilic cytoplasm (Fig 1). By immunostaining using anti-cytokeratin monoclonal antibody, the cytoplasm of the plical epithelial cells showed a strongly positive reaction (Fig 2). Electron microscopy revealed many mitochondria and well developed endoplasmic reticulum in the abundant cytoplasm, and highly convoluted or interdigitated cell membranes and occasional desmosomes (Fig 3). These immunocytological and electron microscopic findings in vitro were consistent with those obtained from the bursal sections (Hoshi et al 1988). The aim of the present study was to establish plical epithelial cell cultures which were largely free of other cell types present in the bursa, such as follicular lymphocytes, fibroblasts and reticuloepithelial cells. The plical epithelial cells were isolated by incubating transversely sliced bursal plicae in trypsin-EOTA solution without stirring but with occasional gentle agitation. This procedure permitted effective release of the plical epithelial cells by exposing the attachment site of the plical epithelial cell layer of the plical fragments to the enzyme and minimised contamination by other bursal components. Confluent cultures consisted of morphologically homogeneous cells which possessed characteristics similar to those of plical epithelial cells in vivo.
Acknowledgements
FIG 2: Positive immunostaining for cytokeratin is observed in cultured plical epithelial cells x 62. Inset shows distribution of positive filaments throughout the cytoplasm of the cells x 252
This work was supported in part by a Grant-in-Aid for Scientific Research number 63860039 from the Ministry of Education, Science and Culture of Japan. The authors thank Miss H. Tomioka for technical assistance.
354
T. Nunoya, M. Tajima, C. Itakura
References BOYD, R. L., WARD, H. A. &MULLER, H. K. (1983a) Bursal and thymic reticular epithelial cells in the chicken: Preparation of in vitro monolayer cultures. Journal of the Reticuloendothelial Society 34, 371-382 BOYD, R. L., WARD, H. A. & MULLER, H. K. (1983b) Bursal and thymic reticular epithelial cells in the chicken: Induction of B- and T-lymphocyte differentiation by in vitro monolayer cultures. Journal of the Reticuloendothelial Society 34, 383-393 EEROLA, E. (1980) In vitro culture of chicken bursal epithelium. Cellular Immunology 53, 162-172 EEROLA, E., LASSILA, O., GILMOUR, D. G. & TOIVANEN, A. (1982) Induction of B cell differentiation in vitro by bursal epithelium. Journal oflmmunology 128, 2652-2655 FLETCHER, O. J., MUNNELL, J. F. & PAGE, R. K. (1975) Cryptosporidiosis of the bursa of Fabricius of chickens. Avian Diseases 19, 630-639
LANG, G. (1979) Collageuase in equine cell culture preparation. Journal of Clinical Microbiology 9, 731- 733 HODGES, R. D. (1974) The Histology of the Fowl. London, Academic Press. pp 206-213 HOSHI, S., NUNOYA, T. & UEDA, S. (1988) Identification of B-L antigens on reticular epithelial ceils of the bursa of Fabricius. Microbiology and Immunology 32, 173-186 NUNOYA, T., TAJIMA, M. & YAGIHASHI, T. (1987) Decrease in catalase activity of cultured cells by Mycoplasma gallisepticum infection. Veterinary Microbiology 13, 343-351 RANDALL, C. J. (1982) Cryptosporidiosis of the bursa of Fabricius and trachea in broilers. Avian Pathology 11, 95-102 WICHA, M. S., LIOTTA, L. A. & GARBISA, S. (1979) Basement membrane collagen requirements for attachment and growth of mammary epithelium. Experimental Cell Research 124, 181 - 190
Received September 14, 1990 Accepted November 19, 1990
Primary culture of chicken bursal plical epithelium T. N U N O Y A , M. T A J I M A , Nippon Institute for Biological Science, 2221-1 Shinmachi, Ome, Tokyo 198, Japan, C. I T A K U R A , Department of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060, Japan
Plical epithelial cells were obtained by trypsin-EDTA treatment of chicken bursa of Fabricius and cultured in the presence of type IV collagen. The culture became confluent six to seven days after seeding. The grown cells showed a positive reaction for cytokeratin by immunostaining and h a d ultrastructural characteristics of the epithelial cells in vivo. The cell culture will be useful for parasitological and virological studies. SEVERAL studies have been conducted on in vitro cultures of the bursa of Fabricius aimed at investigating the interaction between B cells and bursal epithelial components. They were confined to bursal reticular epithelial cells (Boyd et al 1983a,b) or an in vitro organ culture system for alymphoid bursal epithelial cells (Eerola 1980, Eerola et al 1982). To the present authors' knowledge, no further studies have been made particularly o f the epithelium lining the inner faces of the plicae. The plical epithelial cells are divided into three definitive types of cells: the most numerous cells are tall, columnar with an oval nucleus and the two other types are goblet cells and sudanophilic cells, although the latter are few in n u m b e r (Hodges 1974). Interestingly, parasitisation in avian cryptosporidiosis occasionally occurs on the microvillus border of the plical epithelial cell layers (Fletcher et al 1975, Randall 1982), but the interaction between cryptosporidia and the epithelial cells is poorly understood. The purpose of the present study was to develop a method of culturing the bursal plical epithelium for investigation of bursal cryptosporidiosis and other studies. Specific pathogen-free White Leghorn line M chickens were obtained from the Nippon Institute for Biological Science's Laboratory Animal Research Station. The bursa of Fabricius of four-week-old chickens was used throughout the study, because the bursas at this age were considered to be big enough for the source of plical epithelial cells and to retain the proliferating capacity. After blood was withdrawn from the chicken by cardiac puncture, the bursa was asceptically excised and opened in a sterilised glass dish. Each plica was separated and cut into 1 to 2 m m 3 pieces and the pieces placed in a bottle containing equal volumes of 0.25 per cent trypsin and 0.01 per cent ethylene diaminetetraacetic acid (EDTA)in physiological saline or 0.1 per cent coUagenase (Wako Pure Chemical Industries, Japan) in phosphate buffered saline (PBS) (approximately 2 ml per bursa). The material was then stirred for either 15 or 30 minutes at room temperature, or incubated at 37°C for one hour without stirring. After tissue digestion, bottles were
allowed to stand for a minute to precipitate gross tissue fragments and the supernatants containing released cells were centrifuged at 1000 g for five minutes. The resulting pellets were resuspended in culture media (approximately 10 ml per bursa), which consisted of Eagle's m i n i m u m essential medium (Grand Island Biological), supplemented with 5 to 20 per cent fetal calf serum (Gibco), 200 iu m l - i penicillin and 200 /zg m l - l streptomycin. Ten ml of the suspension was seeded on coverslips in a 60 m m x 15 m m plastic culture dish (Becton Dickinson) and incubated at 37°C in a humidified atmosphere o f 5 per cent carbon dioxide in air. The medium was changed after overnight incubation and the cultures were maintained during the following week at 37°C. Some of the coverslips were coated with collagen type I (from rat tail) or type IV (from h u m a n placenta) (Sigma Chemical) before seeding with bursal cells. Seven milligrams of the collagen was dissolved in 20 ml of 0" 1 M acetic acid. The solution was pipetted on to coverslips in a culture dish as above and then allowed to dry at room temperature. The coverslips were sterilised by exposure to ultraviolet light as previously described (Nunoya et al 1987). The collagen-coated coverslips were washed twice with PBS to remove acetic acid before use. To examine the morphology of grown cells, some of the cultures were fixed in Bouin's fluid and stained with haematoxylin and eosin for light microscopy, and others fixed in phosphate buffered 2" 5 per cent glutaraldehyde for electron microscopy. The glutaraldehyde-fixed cells were then dehydrated in ethanol and propylene oxide and embedded in Epok 812. Silver-grey sections cut on an ultramicrotome were stained with uranyl acetate and lead citrate and examined with a JEM-100B electron microscope. The cultured cells were also processed for immunocytochemistry. The cells were washed gently in PaS and fixed in cold ethanol for 10 minutes. A monoclonal antibody to cytokeratin (CK5, Sigma Chemical) was used as a primary antibody and reacted with anti-mouse IgG peroxidase conjugate (Sigma Chemical). The cells were developed with s u b s t r a t e solutions (0.02 per cent 3-amino-9-ethyl carbazole, 0.03 per cent hydrogen peroxide, and 5 per cent N,N-dimethylformamide in 0.1 M acetate buffer, p H 5.2) and counterstained with haematoxylin. The plical epithelial cells were most efficiently isolated by incubating bursal tissue in the trypsin-EDTA mixture at 37°C for one hour. The effectiveness was confirmed by histological examination of the tissue remnants after incubation. The continuous stirring during digestion resulted in an increase of non-epithelial components such as follicular
352
Culture of bursal plical epithelium
353
FIG 1 : Confluent culture of bursal plical epithelia[ cells. Haematoxylin and eosin x 62. Inset shows a homogeneous population of large, polygonal cerls with oval nuclei x 252
FIG 3: Electron micrograph of cultured plical epithelial cells showing well-developed mitochondria, interdigitated cell membranes and desmosomes (arrows) x 6762
lymphocytes and stromal fibroblasts; the 15 minute digest became confluent three to four days after seeding but less than 10 per cent of cells spread to form small colonies of plical epithelial cells and the remaining cells were fibroblasts, and the 30 minute digest was exclusively follicular lymphocytes. Since collagenase treatment surpassed trypsin as a celldispersing agent in various experiments (Lang 1979), collagenase was substituted for the trypsin-EDTA. With this enzyme, however, no growth was observed in the released cells under the same culture conditions, although epithelial cell release from the plicae was seemingly effective. Pretreatment of coverslips with collagen was necessary for cell spread and type IV collagen gave a better result (more than 10 per cent). This collagen requirement by plical epithelial cells was considered to be a general property of epithelial cells, because their attachment and differentiation were strongly influenced by collagen, especially type IV (Wicha et al 1979). The time taken to attain confluent culture
was related to the amount of serum in the medium; between six and seven days under the optimum concentration of approximately 20 per cent. The plical epithelial cells appeared to grow in some areas from the periphery of fragmented epithelial layers and in other areas from single dispersed cells. The confluent culture stained with haematoxylin and eosin comprised a homogeneous population of large, polygonal cells with centrally positioned, pale and oval nuclei in neutrophilic cytoplasm (Fig 1). By immunostaining using anti-cytokeratin monoclonal antibody, the cytoplasm of the plical epithelial cells showed a strongly positive reaction (Fig 2). Electron microscopy revealed many mitochondria and well developed endoplasmic reticulum in the abundant cytoplasm, and highly convoluted or interdigitated cell membranes and occasional desmosomes (Fig 3). These immunocytological and electron microscopic findings in vitro were consistent with those obtained from the bursal sections (Hoshi et al 1988). The aim of the present study was to establish plical epithelial cell cultures which were largely free of other cell types present in the bursa, such as follicular lymphocytes, fibroblasts and reticuloepithelial cells. The plical epithelial cells were isolated by incubating transversely sliced bursal plicae in trypsin-EOTA solution without stirring but with occasional gentle agitation. This procedure permitted effective release of the plical epithelial cells by exposing the attachment site of the plical epithelial cell layer of the plical fragments to the enzyme and minimised contamination by other bursal components. Confluent cultures consisted of morphologically homogeneous cells which possessed characteristics similar to those of plical epithelial cells in vivo.
Acknowledgements
FIG 2: Positive immunostaining for cytokeratin is observed in cultured plical epithelial cells x 62. Inset shows distribution of positive filaments throughout the cytoplasm of the cells x 252
This work was supported in part by a Grant-in-Aid for Scientific Research number 63860039 from the Ministry of Education, Science and Culture of Japan. The authors thank Miss H. Tomioka for technical assistance.
354
T. Nunoya, M. Tajima, C. Itakura
References BOYD, R. L., WARD, H. A. &MULLER, H. K. (1983a) Bursal and thymic reticular epithelial cells in the chicken: Preparation of in vitro monolayer cultures. Journal of the Reticuloendothelial Society 34, 371-382 BOYD, R. L., WARD, H. A. & MULLER, H. K. (1983b) Bursal and thymic reticular epithelial cells in the chicken: Induction of B- and T-lymphocyte differentiation by in vitro monolayer cultures. Journal of the Reticuloendothelial Society 34, 383-393 EEROLA, E. (1980) In vitro culture of chicken bursal epithelium. Cellular Immunology 53, 162-172 EEROLA, E., LASSILA, O., GILMOUR, D. G. & TOIVANEN, A. (1982) Induction of B cell differentiation in vitro by bursal epithelium. Journal oflmmunology 128, 2652-2655 FLETCHER, O. J., MUNNELL, J. F. & PAGE, R. K. (1975) Cryptosporidiosis of the bursa of Fabricius of chickens. Avian Diseases 19, 630-639
LANG, G. (1979) Collageuase in equine cell culture preparation. Journal of Clinical Microbiology 9, 731- 733 HODGES, R. D. (1974) The Histology of the Fowl. London, Academic Press. pp 206-213 HOSHI, S., NUNOYA, T. & UEDA, S. (1988) Identification of B-L antigens on reticular epithelial ceils of the bursa of Fabricius. Microbiology and Immunology 32, 173-186 NUNOYA, T., TAJIMA, M. & YAGIHASHI, T. (1987) Decrease in catalase activity of cultured cells by Mycoplasma gallisepticum infection. Veterinary Microbiology 13, 343-351 RANDALL, C. J. (1982) Cryptosporidiosis of the bursa of Fabricius and trachea in broilers. Avian Pathology 11, 95-102 WICHA, M. S., LIOTTA, L. A. & GARBISA, S. (1979) Basement membrane collagen requirements for attachment and growth of mammary epithelium. Experimental Cell Research 124, 181 - 190
Received September 14, 1990 Accepted November 19, 1990