Dissociation of Epithelial Cells from Rabbit Trachea and Small Intestine with Demonstration of APUD Endocrine Cells K . S. SONSTEGARD,' E. CUTZ AND V. WONG Department of Pathology and t h e Research Institute, T h e Hospital f o r Sick Children, and t h e University of Toronto, Toronto, Ontario, Canada
ABSTRACT In this study the entire epithelial lining of tracheas and a 15-cm segments of small intestine were dissociated into individual cell components after 45-minute incubation with 1% pronase. Light and electron microscopy of isolated cells confirmed good morphologic preservation of various epithelial cell types dissociated from the trachea and small intestinal mucosa. Of particular interest was the recovery and preservation of APUD endocrine cells, which are known to be widely dispersed amongst various non-endocrine epithelial cells in both the trachea and small intestine. The APUD cells were demonstrated in dissociated cell preparations by a formaldehyde-induced fluorescence method, Grimelius' silver nitrate stain, and electron microscopy. The isolated APUD cells retained their characteristic features, e.g., amine-handling properties, argyrophilia and cytoplasmic dense-core vesicles. The cell dissociation method described in this report provides high yields of viable epithelial cells in single cell suspensions which are suitable for further cell separation into homogeneous populations of single kinds of cells, including the APUD endocrine cells. Availability of methods for isolation of tracheal and intestinal APUD cells will facilitate further studies, in vitro, on secretory, metabolic and functional aspects of these cells.
Amine and polypeptide hormone-producing APUD cells (Amine Precursor Uptake and Decarboxylase Activity) constitute a system of dispersed endocrine cells distributed predominantly in organs derived from the primitive gut (Pearse, '69). The unifying concept of the APUD endocrine system is based on common histochemical and ultrastructural characteristics and a possible common derivation for these cells from the neural crest (Pearse, '69, ' 7 3 ) . In spite of recent advances in the morphologic and cytochemical characterization of APUD cells, information is still fragmentary regarding the secretory products and physiologic function of these cells. One of the major problems is the dispersed distribution and the relatively small number of APUD cells. The APUD cells in the tracheobronchial epithelium, referred to as Kultschitzky cells (K cells), have been only recently identified (Bensch et al., '65; Lauweryns and Peuskens, '69; Hage, '72; Cutz and AM. J . ANAT., 1 4 7 ; 357-374
Conen, '72; Cutz et al., '74, '75). It had been previously shown that K cells of the lung give rise to carcinoid and oat cell carcinomas of the lung (Bensch et al., '68; Hattori et al., '72). Ectopic hormone production and various endocrine syndromes associated with these lung tumors have been recognized (Omen, '70; Rees and Ratcliffe, '74). Although these data suggest that K cells in normal lungs may have a n endocrine function, hormone production by these cells has yet to be demonstrated. New approaches and techniques for the study of these APUD cells are desirable. This report describes a method for the enzymatic dissociation of epithelial cells from rabbit trachea wth recovery of intact K cells in single cell suspensions. Since the K cells of the tracheal epithelium seem identical to those in the intra-pulmonary Accepted July 30, '76. 1 Post-doctoral fellow of the National Research Council of Canada.
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bronchi (Ericson et al., ’72; Cutz et al., ’75), and because of its accessibility, this epithelium serves as a convenient source of “lung” K cells. In a parallel study, epithelial cells were dissociated from rabbit small intestine with the same enzymatic procedure. MATERIALS A N D METHODS
Tracheas and proximal segments of small intestine were used from ten adult New Zealand white rabbits (6-7 lbs). The animals were killed by Nembutal injection into a n ear vein, and lungs and trachea were immediately removed e n bloc. The trachea was sectioned below the cricoid cartilage and cut free of the lungs a t the bifurcation. This segment of trachea was usually 5-6 cm long, with a n internal diameter of approximately 0.4 cm. For the dissociation of cells from the small intestine, a 15 cm proximal segment was used. Cell dissociation procedures The following procedures consistently provided the most satisfactory results. After thorough washing with cold Jokliks medium (Modified Minimal Essential Medium, GIBCO), each end of the trachea was securely fastened to a piece of tygon tubing, one end of which was connected to a perfusion pump (Braun, Quigley - Rochester, New York) fitted with a 50-ml syringe containing a solution of 1% pronase (Pronase E, EM Laboratories, Elmsford, New York) in Jokliks medium. The lumen of the trachea was filled to capacity with enzyme solution and the opposite end of tubing clamped. The trachea, with the attached tubings clamped, was then placed in an Erlenmeyer flash containing warmed (37°C) Jokliks medium and a n atmosphere of 5% COz in air. The flask was agitated gently in a shaker water bath at 37°C for 30 to 45 minutes. One end of the tubing was then reconnected to the perfusion pump and the tracheal lumen perfused with warmed Jokliks medium. The perfusates, containing dissociated cells, were collected at room temperature in 200 ml of Jokliks medium supplemented with 20% fetal calf serum. The trachea was then refilled with enzyme and incubated as before for a n additional 15 minutes, after which cells were collected by perfusion. The volume of the cells in suspension
was adjusted according to the number of dissociated cells, to avoid reaggregation and to dilute the pronase. The procedure for dissociation of cells from the small intestine was modified as follows. The lumen of the intestinal segment was washed with cold Jokliks medium and everted onto a metal rod covered by dialysis tubing. The serosal surface of the everted intestine was then inflated by air in order to expose the epithelial surface maximally to the enzyme solution. The metal rod was connected to an electric motor and the preparation with the intestinal “lumen” on the outside was inserted vertically into a glass cylinder containing 80 ml of 1 % pronase in Jokliks medium and rotated 150 rpm at 37°C for 30 minutes. The cell suspensions were collected in 200 ml of 40% fetal calf serum in Jokliks medium, adjusted to a volume of 400 ml. Methods for evaluation of dissociated cells The numbers of cells and their viability were assessed in a hemocytometer chamber by the Trypan Blue Exclusion method. The qualitative assessment of dispersed cells was made on slides prepared by a cytocentrifuge (Shandon Scientific Co. Inc., Sewickley, Pennsylvania). The sedimented cells were fixed in Bouin’s soIution and stained with H & E, PAS, WHO-keratinmucin stain or Grinielius’ silver nitrate (Cutz et al., ’75). For the forrrialdeh yde-induced fluorescence (FIF) method the cell suspensions were examined with or without prior incubation with IL-DOPA as previously reported (Cutz et al., ’74). The cells were sedimented onto glass slides with the use of a cytocentriftige. The samples were then freeze-dried in a VirTis automatic freeze drier at minus 40°C overnight, and exposed to paraformaldehyde gas (relative humidity 50-60% ) at 80°C for one to two hours. Samples not exposed to paraformaldehyde gas were used as controls. The samples were examined with a Leitz Orthoplan fluorescence microscope (HB-200 bulb, BG12, UG-1 activating filters and 53, 46, 65, 47 barrier filters). For electron microscopy, cell pellets were fixed in 2% glutaraldehyde in phosphate buffer (pH 7. 4), post-fixed in 1% osmic acid and embedded in Epon. One-micron
DISSOCIATION OF APUD ENDOCRINE CELLS
Epon sections were stained by toluidine blue and PAS methods. Ultrathin sections were stained with uranyl acetate and lead ci tra te . RESULTS
Light microscopy of dissociated cells from trachea and intestine After 45 minutes’ incubation of trachea with 1% pronase a n average yield was 20 x lo6 cells. As observed by phase contrast microscopy, the dissociated tracheal epithelial cells were predominantly in a single-cell suspension and the majority of cells appeared rounded (fig. 1). Their viability ranged from 80 to 9 8 % . Isolated ciliated cells retained their ciliary beat. Following the completion of enzyme treatment the trachea showed complete denudation of the epithelial lining with a n intact basement membrane left behind. The higher yield of cells from the small intestinal segments treated with 1% pronase for 30 minutes (130 X lo6) was due to a larger epithelial surface, rather than greater effectiveness of the dissociation procedure. The dissociated intestinal cells were also i n a single-cell suspension and spherical in shape. The viability of these cells ranged between 70 and 90%. The segments of intestine after the enzyme treatment showed complete removal of epithelial cells from the villi, but small patches of cells remained in the depths of the crypts. In H & E-stained, cytocentrifuged cell preparations from both the trachea and small intestine, the majority of cells had the cytologic features of epithelial cells. Among the differentiated cell types, ciliated cells in the samples of trachea and enterocytes in the samples of intestine were the most frequent cell types present. In samples stained for mucin by PAS or WHO, goblet cells were easily identified. No contamination with connective tissue elements was observed and only occasional red cells, lymphoid cells and macrophages were present. Although the cytocentrifuged cell preparations provided a fast and simple method for general morphological assessment of dissociated cells, the cytological detail was superior i n 1-, Epon sections of cell pellets. The single cell distribution of dissociated epithelial cells and their good morphologi-
359
cal preservation were confirmed in cell pellets obtained from both the trachea (fig. 2 ) and small intestine (fig. 3 ) . At higher magnification, the various epithelial cells showed excellent preservation of the cytoplasm and nuclei (fig. 4 ) . I n Epon sections stained with the PAS method, mucous granules in goblet cells appeared well preserved (fig. 5 ) . I n cytocentrifuged samples stained with the method of Grimelius, argyrophilic (AG) cells were identified in both the trachea (fig. 6 ) and small intestine samples (fig. 7). The shape of dissociated AG cells was cuboidal or spherical, and the cytoplasm contained numerous fine, dark brown or black granules (insets, figs. 6, 7). The appearance of silver-positive cytoplasmic granules was comparable to those found in AG cells in tissue sections. In cell preparations from trachea examined by the FIF method, cells with specific yellow-green cytoplasmic fluorescence were identified only in samples previously incubated with L-DOPA (fig. 8 ) . The intensity of fluorescence i n dissociated AG cells was similar to that of AG cells in tissue sections. In non-incubated samples and in control samples not exposed to paraformaldehyde vapor no positive cells were found. In the samples of intestine, cells with yellow cytoplasmic fluorescence were present even in samples without L-DOPA incubation (fig. 9 ) . However, no fluorescent cells were seen in samples not exposed to paraformaldehyde vapor, confirming the specificity of the FIF reaction. Electron microscopy of cells dissociated from trachea and small intestine The overall ultrastructural preservation of tracheal epithelial cells after pronase treatment was excellent as judged by the cytoplasmic and nuclear morphology of various cell types. In ultrathin sections, cells appeared in various planes of section, but the differentiated cell types could usually be identified by their characteristic ultrastructural features (fig. lo). The ciliated and goblet cells were the most frequent cell types present. The cilia of the ciliated cells and mucous granules of goblet cells were well preserved. Mild to moderate degrees of ultrastructural alteration due to the effect of enzyme on cells consisted of cytoplasmic blebbing, concentra-
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tion of organelles in the perinuclear region, mild cytoplasmic vacuolation and increased density of mitochondria1 matrix. In several of the cell pellets examined, cells with cytoplasmic dense-core vesicles (DCV) characteristic of K cells were present (fig. 12). The size and morphology of DCV in dissociated K cells (fig. 13) were identical to those previously described in tissue sections of rabbit trachea. As compared to other epithelial cells, pronase effect on K cells was minimal. Except for mild dilation of smooth endoplasmic reticulum and a few cytoplasmic vacuoles, no other changes were found. The fine structural preservation of epithelial cells dissociated from the small intestine was satisfactory and was generally comparable to that of cells obtained from the trachea. However, the effects of pronase on intestinal epithleial cells was more pronounced (fig. 11). Among the various epithelial cell types, the most common were enterocytes. Due to their spherical shape and increased apical surface in the dissociated state, their brush borders appeared stretched and the microvilli shortened. Cytoplasmic blebbing and organelle condensation were more frequently found in intestinal cell preparations than in those from the trachea. On the other hand, the mucous granules in goblet cells (fig. 11) and secretory granules of Paneth cells appeared intact. A s expected, endocrine cells were more numerous in preparations from intestine than in those from the trachea. The intestinal endocrine cells were also remarkably well preserved and retained their characteristic ultrastructural features (fig. 14). The most frequently encountered type of endocrine cells contained dense, pleiomorphic DCV measuring up to 250 n m in diameter (fig. 15). Based on ultrastructural criteria, this type of endocrine cell corresponds to the enterochromaffin cell type (Forssmann, '70). Other types of endocrine cells usually found in the upper duodenum were also present, but in this study no attempt was made to quantitate the various types of endocrine cells. DISCUSSION
Recent advances in cell separation technology have facilitated the investigation
and characterization of specific types of viable cells isolated from a variety of tissues and organs (Pretlow et al., '75). Studies on homogeneous populations of cells provide more meaningful information in regard to a specific cell type than do tissue sections or tissue homogenates composed of a mixture of cells. This approach, using isolated cells, has recently been applied to studies i n vitro on amine and polypeptide hormone production by APUD cells dissociated from anterior pituitary (Hopkins and Farquhar, '73), endocrine pancreas (Orci et al., '73) and carotid body (Pietruschka, '74). The endocrine APUD cells in the tracheobronchial and gastrointestinal mucosae are known to be widely distributed among the various non-endocrine epithelial cells (Cutz et al., '75; Pearse, '69). Therefore it would be of great advantage to isolate and study t.hese cells in preparations composed of a homogeneous population of a single kind of cells. The various enzymatic and physical methods for cell dissociation and problems connected with these procedures have been discussed previously (Shortman, '72; Waymouth, '74; Pretlow et al., '75). These studies have emphasized that the cell dissociation method to be employed must be tailored to recovery of a particular cell type, because some types of cells may be selectively destroyed during the dissociation procedure. In addition, the isolated cell preparations must conform to the requirements of subsequent studies, e.g., techniques for further separation into individual kinds of cells, biochemistry, tissue culture or other studies. Therefore, it is important to evaluate critically the cell preparations following the dissociation procedure, particularly by monitoring their morphologic arid cytochemical preservation. In this study a proteolytic enzyme, pronase, was found to be effective in dissociating the epithelial lining from both the trachea and sm.311 intestine into individual cells. In spite of a relatively high concentration of enzyrne necessary to obtain single cell suspen!;ions in a short time, the majority of dissociated cells remained viable. In addition, the morphology of various epithelial cell types was well preserved at
DISSOCIATION OF APUD ENDOCRINE CELLS
both the light and electron microscopic levels. Proteolytic enzymes, including pronase, are known to effect the surface of cell membranes, but most of these changes are considered reversible (Poste, '71; Wallach, '72). Pronase-induced changes were also noted in this study and at the ultrastructural level were manifested as cell membrane blebbing and condensation and redistribution of organelles. These changes were more pronounced in the cells isolated from the small intestine than those from the trachea. The enterocytes showed the most marked changes, whereas secretory cells, such as goblet cells, Paneth cells and APUD endocrine cells, were generally well preserved. This finding suggests that there is a variation in susceptibility to proteolytic enzymes not only between different tissues but also between various cells in the same tissue. In order to monitor the recovery and preservation of APUD cells in dissociated cell preparations, we used histochemical methods similar to those employed for identification of these cells in tissue sections (Pearse, '69). The FIF method demonstrates the amine-handling properties of APUD cells and is one of the primary characteristics. Because of the low endogenous amine content in APUD cells of the trachea, prior incubation with amine precursor is required (Ericson et al., '72; Cutz et al., '75). The demonstration of intracellular amines i n dissociated tracheal APUD cells after incubation with L-DOPA indicates that the decarboxylating mechanism in isolated APUD cells remained intact. The APUD cells in the GI tract are known to contain large amounts of endogenous amines, particularly serotonin, and, therefore, the intracellular amine can be shown without prior incubation with amine precursors (Owman et al., '73). Although argyrophilia, as shown by the method of Grimelius, is considered a secondary feature of APUD cells, it is a useful and relatively simple method for demonstration of these cells by light microscopy. The presence of argyrophilic granules in dissociated APUD cells confirmed the good preservation of these cells and showed that the enzyme treatment did not interfere with this staining reaction. At the ultrastructural level the cyto-
36 1
plasm of APUD cells contains characteristic cytoplasmic granules referred to as dense-core vesicles (DCV). These granules are considered to be the storage site of amines and polypeptide hormones produced by the APUD cells (Owman et al., '73). It is of great importance to evaluate the preservation of DCV in dissociated APUD cells, because ultrastructural changes following pronase treatment have been recently reported in cytoplasmic granules of cells from anterior pituitary and endocrine pancreas (Schofield and Orci, '75; Orci et al., '73). However, no such changes were found in the present study. The ultrastructural appearance of DCV in pronase-dissociated APUD cells was comparable to that described in tissue sections (Cutz et al., '75; Forssmann, '70). The APUD cells in trachea and bronchi of human and various other animal species have been identified only recently and the function of these cells remains unknown (Lauweryns and Peuskens, '69; Ericson et al., '72; Cutz et al., '74, '75). Although it has been suggested that these cells may have a n endocrine function and, in addition to amines, may produce polypeptide hormones, so far no direct evidence has been obtained. The endocrine function of the G I tract has been studied more extensively and a variety of hormonal substances have been isolated and characterized from GI tissues (Pearse, '74). However, many questions still remain to be answered, particularly in regard to the physiologic function of GI hormones and their cellular origin. Availability of methods for dissociation and monitoring of APUD cells isolated from the epithelial lining of trachea and intestine will provide an excellent tool for studies on metabolism and secretory activity of these cells in vitro. Because single cell suspensions can be obtained, these preparations are usable for further cell separation into specific cell types, including the APUD endocrine cells. In our laboratory, studies are in progress using velocity sedimentation techniques for the further separation and characterization of APUD cells from trachea and small intestine. These methods may provide concentrated or homogeneous cell suspensions of APUD cells for direct biochemical studies,
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as well as for isolation and characterization of their secretory products. LITERATURE CITED Bensch, K. G., C. B. Gordon and L. R. Miller 1965 Studies on the bronchial counterpart of the Kultschitzky (argentaffin) cell and innervation of bronchial glands. J. Ultrastruct. Res., 12: 668-686. Bensch, K. G., B. Corrin, R. Pariente and H. Spencer 1968 Oat cell carcinoma of the lung. Its origin and relationship to bronchial carcinoid. Cancer (Piladelphia), 22: 1163-1172. Cutz, E., and P. E. Conen 1973 Endocrine-like cells in human fetal lungs. Anat. Rec., 173: 115-122. Cutz, E., W. Chan, V. Wong and P. E. Conen 1974 Endocrine cells i n rat fetal lungs. Ultrastructural and histochemical study. Lab. Invest., 30: 458-464. 1975 Ultrastructure and fluorescence histochemistry of endocrine ( APUD-type) cells in trachea of human and various animal species. Cell Tiss. Res., 158: 425-437. Ericson, L. E., R. HBkanson, B. Larson, Ch. OWman and F. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124: 532-545. Forssmann, W. G. 1970 Ultrastructure of hormone-producing cells of the upper gastrointestinal tract. In: Origin, Chemistry, Physiology and Pathophysiology of the Gastrointestinal Hormones. W. Creutzfeldt, ed. F. K. Schattauer, Stuttgard-New York, pp. 32-95. Hage, E. 1972 Endocrine cells in the bronchial mucosa of human foetuses. Acta path. microbiol. scand., Sec. A, 80: 225-234. Hattori, S . , M. Matsuda, R. Tateishi, M. Nishihara and T. Horai 1972 Oat-cell carcinoma of the lung. Clinical and morphological studies in relation to its histogenesis. Cancer (Philadelphia), 30: 1014-1024. Hopkins, C., and M. Farquhar 1973 Hormone secretion by cells dissociated from rat anterior pituitaries. J. Cell Biol., 59: 276-303. Lauweryns, J. M., and J. C. Peuskens 1969 Argyrophil (kinin and amine producing? ) cells in human infant airway epithelium. Life Sci., 8: 577-585. Omenn, G. S . 1970 Ectopic polypeptide hormone production by tumours. Ann. Int. Med., 72: 136-138.
Orci, L., A. A. Like, M. Amherdt, B. Blondel, Y. Kanazawa, E. B. "arliss, A. E. Lambert, C. B. Wollheim and A. E:. Renold 1973 Monolayer cell culture of neonatal rat pancreas: An ultrastructural and biochemical study of functioning endocrine cells. J. Ultrastruct. Res., 43: 270-297. Orci, L., M. Amherdt, J. C. Henquin, A. E. Lambert, R. M. Unger and A. IS. Renold 1973 Pronase effect on pancreatic beta cell secretion and morphology. Science, 180: 647-649. Owman, Ch., R. HPkanson and F. Sundler 1973 Occurrence and function of amines in endocrine cells produ'zing polypeptide hormones. Fed. Proc., 32: 1785-1791. Pearse, A. G. E. 1969 The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J. Histochem. Cytochem., 17: 303-3 13. 1973 Cell migration and the alimentary system: Endocrine contributions of the neural crest to the gut and its derivatives. Digestion, 8: 372-385. 1974 The gut as an endocrine organ. Brit. J. Hosp. Medicine (May), pp. 697-704. Pietruschka, F. 19'74 Cytochemical demonstration of catecholamines in cells of the carotid body in primary tissue culture. Cell Tiss. Res., 151: 317-321. Poste, G. 1971 Tissue dissociation with proteolytic enzymes. Exp. Cell Res., 65: 359-367. Pretlow, T. G., E. I;. Weir and J. G. Zettergren 1975 Problems Connected with the Separation of different Kinds of Cells. In: Int. Rev. Exp. Path. G. W. Richter and M. A. Epstein, eds. Academic Press, New York-London, 14: 91-204. Rees, H. L., and J. G. Ratcliffe 1974 Ectopic hormone production by non-endocrine tumours. Clin. Endocrin., 3: 263-299. Schofield, J. G., and L. Orci 1975 Release of growth hormone from ox pituitary slices after pronase treatment. J. Cell Biol., 65: 223-227. Shortman, K. 1979: Physical procedures for the separation of aniinal cells. Annu. Rev. Biophys. and Bio. Eng., 1: 93-130. Wallach, D. F. H. 1972 The disposition of proteins in the plasma membranes of animal cells; analytical approaches using controlled peptidolysis and protein. Biochim. Biophys. Acta, 265: 61-68. Waymouth, C. 19'74 To disaggregate or not to disaggregate. Injury and cell disaggregation. transient or permanent? In Vitro, 10: 97-111:
PLATES
PLATE 3 EXPLANATION O F FIGURES
364
1
Phase contrast micrograph of a cell suspension from rabbit trachea obtained after 30-minute incubation in 1% Pronase. The dissociated epithelial cells are spherical and in single-cell suspension. x 450.
2
Low-magnification light micrograph (LM) of cell pellet from trachea. The large epithelial cells represent ciliated and goblet cells. The dissociated cells are well preserved and are distributed as single cells. One-micron Epon section, Toluidine blue stain. x 400.
3
LM of a cell pellet from dissociated intestinal cells. The shape and distribution of isolated cells are similar to those in figure 2. Onemicron Epon section, Toluidine blue stain. x 400.
DISSOCIATION O F APUD ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 1
365
PLATE 2 EXPLANATION OF FIGURES
4 Higher-magnification LM of cells dissociated from trachea with good cytological preservation of ciliated ( c i ) and goblet (Go) cells. The smaller cells represent basal cells. One-micron Epon section, Toluidine blue stain. x 1,000.
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5
Well preserved mucus granules i n dissociated intestinal goblet cells. One-micron Epon section, PAS stain. x 1,000.
6
Two argyrophilic (AG) cells (arrowheads) among dissociated epithelial cells from the trachea. Cytocentrifuge preparation, Grimelius’ stain. :< 320. Inset: Fine black, argyrophilic granules i n the cytoplasm of isolated AG cell from trachea. Cytocentrifuge preparation, Grimelius’ stain. x 1,000.
7
Isolated AG cells (arrowheads) i n intestinal cell suspensicn. Cytocentrifuge p r e p aration, Grimelius’ stain. x 320. Inset: T h e argyrophilic granules i n intestinal AG cells appear larger than from trachea. Cytocentrifuge preparation, Grimelius’ stain. x 1,000.
8
Intense cytoplasmic fluorescence (white arrowhead) i n a cell dissociated from tracheal epithelium. Incubation i n vitro with L-DOPA, cytocentrifuge preparation, FIF. x 800.
9
Two intensely fluorescent cells (white arrowheads) among dispersed intestinal epithelial cells. Not incubated with L-DOPA, cytocentrifuge preparation, FIF. x 800.
DISSOCIATION O F APUD ENDOCRINE CELLS K. S . Sonstegard, E. Cutz and V. Wong
PLATE 2
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PLATE 3 EXPLANATION O F FIGURES
10
Low-magnification electron micrograph of cell pellet from trachea. The ciliated (Ci) and goblet ( G o ) cells appear in various planes of section. There is good ultrastructural preservation of nuclei and cytoplasm of the various cell types. x 2,000.
11 Low-magnification electron micrograph of dissociated intestinal epithelial cells. There is good preservation of nuclei, but the cytoplasm of enterocytes (En) shows some vacuolation, and the cell membrane of goblet cells ( G o ) shows cytoplasmic blebs (arrow). x 2,000.
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DISSOCIATION OF APUD ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 3
369
PLATE 4 EXPLANATION OF FIGURES
3 70
12
Electron micrograph of an isolated K cell from trachea shows excellent ultrastructural preservation, including the cytoplasmic granules. Nu, nucleus; Va, vacuoles. x 18,000.
13
Higher magnification of cytoplasmic DCV from the K cell shown in figure 12. The central core and the limiting membrane (arrows) are well preserved. Mi, mitochondria. x 30,000.
DISSOCIATION OF APUD ENDOCRINE CELLS K . S. Sonstegard, E. Cutz and V. Wong
PLATE 4
PLATE 5 EXPLANATION O F FIGURES
14
Electron micrograph of endocrine cell isolated from small intestine. Peripherally located dense granules represent DCV. Nu, nucleus; Va, vacuoles. x 11,000.
15 Pleiomorphic DCV (arrows) from cell shown i n figure 14. This type of cytoplasmic granule is characteristic of enterochromaffin cells. Mi, mitochondria; Nu, nucleus. x 23,000.
3 72
DISSOCIATION OF A P U D ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 5
ABSTRACT In this study the entire epithelial lining of tracheas and a 15-cm segments of small intestine were dissociated into individual cell components after 45-minute incubation with 1% pronase. Light and electron microscopy of isolated cells confirmed good morphologic preservation of various epithelial cell types dissociated from the trachea and small intestinal mucosa. Of particular interest was the recovery and preservation of APUD endocrine cells, which are known to be widely dispersed amongst various non-endocrine epithelial cells in both the trachea and small intestine. The APUD cells were demonstrated in dissociated cell preparations by a formaldehyde-induced fluorescence method, Grimelius' silver nitrate stain, and electron microscopy. The isolated APUD cells retained their characteristic features, e.g., amine-handling properties, argyrophilia and cytoplasmic dense-core vesicles. The cell dissociation method described in this report provides high yields of viable epithelial cells in single cell suspensions which are suitable for further cell separation into homogeneous populations of single kinds of cells, including the APUD endocrine cells. Availability of methods for isolation of tracheal and intestinal APUD cells will facilitate further studies, in vitro, on secretory, metabolic and functional aspects of these cells.
Amine and polypeptide hormone-producing APUD cells (Amine Precursor Uptake and Decarboxylase Activity) constitute a system of dispersed endocrine cells distributed predominantly in organs derived from the primitive gut (Pearse, '69). The unifying concept of the APUD endocrine system is based on common histochemical and ultrastructural characteristics and a possible common derivation for these cells from the neural crest (Pearse, '69, ' 7 3 ) . In spite of recent advances in the morphologic and cytochemical characterization of APUD cells, information is still fragmentary regarding the secretory products and physiologic function of these cells. One of the major problems is the dispersed distribution and the relatively small number of APUD cells. The APUD cells in the tracheobronchial epithelium, referred to as Kultschitzky cells (K cells), have been only recently identified (Bensch et al., '65; Lauweryns and Peuskens, '69; Hage, '72; Cutz and AM. J . ANAT., 1 4 7 ; 357-374
Conen, '72; Cutz et al., '74, '75). It had been previously shown that K cells of the lung give rise to carcinoid and oat cell carcinomas of the lung (Bensch et al., '68; Hattori et al., '72). Ectopic hormone production and various endocrine syndromes associated with these lung tumors have been recognized (Omen, '70; Rees and Ratcliffe, '74). Although these data suggest that K cells in normal lungs may have a n endocrine function, hormone production by these cells has yet to be demonstrated. New approaches and techniques for the study of these APUD cells are desirable. This report describes a method for the enzymatic dissociation of epithelial cells from rabbit trachea wth recovery of intact K cells in single cell suspensions. Since the K cells of the tracheal epithelium seem identical to those in the intra-pulmonary Accepted July 30, '76. 1 Post-doctoral fellow of the National Research Council of Canada.
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K. S. SONSTEGARD, E. CUTZ AND V. WONG
bronchi (Ericson et al., ’72; Cutz et al., ’75), and because of its accessibility, this epithelium serves as a convenient source of “lung” K cells. In a parallel study, epithelial cells were dissociated from rabbit small intestine with the same enzymatic procedure. MATERIALS A N D METHODS
Tracheas and proximal segments of small intestine were used from ten adult New Zealand white rabbits (6-7 lbs). The animals were killed by Nembutal injection into a n ear vein, and lungs and trachea were immediately removed e n bloc. The trachea was sectioned below the cricoid cartilage and cut free of the lungs a t the bifurcation. This segment of trachea was usually 5-6 cm long, with a n internal diameter of approximately 0.4 cm. For the dissociation of cells from the small intestine, a 15 cm proximal segment was used. Cell dissociation procedures The following procedures consistently provided the most satisfactory results. After thorough washing with cold Jokliks medium (Modified Minimal Essential Medium, GIBCO), each end of the trachea was securely fastened to a piece of tygon tubing, one end of which was connected to a perfusion pump (Braun, Quigley - Rochester, New York) fitted with a 50-ml syringe containing a solution of 1% pronase (Pronase E, EM Laboratories, Elmsford, New York) in Jokliks medium. The lumen of the trachea was filled to capacity with enzyme solution and the opposite end of tubing clamped. The trachea, with the attached tubings clamped, was then placed in an Erlenmeyer flash containing warmed (37°C) Jokliks medium and a n atmosphere of 5% COz in air. The flask was agitated gently in a shaker water bath at 37°C for 30 to 45 minutes. One end of the tubing was then reconnected to the perfusion pump and the tracheal lumen perfused with warmed Jokliks medium. The perfusates, containing dissociated cells, were collected at room temperature in 200 ml of Jokliks medium supplemented with 20% fetal calf serum. The trachea was then refilled with enzyme and incubated as before for a n additional 15 minutes, after which cells were collected by perfusion. The volume of the cells in suspension
was adjusted according to the number of dissociated cells, to avoid reaggregation and to dilute the pronase. The procedure for dissociation of cells from the small intestine was modified as follows. The lumen of the intestinal segment was washed with cold Jokliks medium and everted onto a metal rod covered by dialysis tubing. The serosal surface of the everted intestine was then inflated by air in order to expose the epithelial surface maximally to the enzyme solution. The metal rod was connected to an electric motor and the preparation with the intestinal “lumen” on the outside was inserted vertically into a glass cylinder containing 80 ml of 1 % pronase in Jokliks medium and rotated 150 rpm at 37°C for 30 minutes. The cell suspensions were collected in 200 ml of 40% fetal calf serum in Jokliks medium, adjusted to a volume of 400 ml. Methods for evaluation of dissociated cells The numbers of cells and their viability were assessed in a hemocytometer chamber by the Trypan Blue Exclusion method. The qualitative assessment of dispersed cells was made on slides prepared by a cytocentrifuge (Shandon Scientific Co. Inc., Sewickley, Pennsylvania). The sedimented cells were fixed in Bouin’s soIution and stained with H & E, PAS, WHO-keratinmucin stain or Grinielius’ silver nitrate (Cutz et al., ’75). For the forrrialdeh yde-induced fluorescence (FIF) method the cell suspensions were examined with or without prior incubation with IL-DOPA as previously reported (Cutz et al., ’74). The cells were sedimented onto glass slides with the use of a cytocentriftige. The samples were then freeze-dried in a VirTis automatic freeze drier at minus 40°C overnight, and exposed to paraformaldehyde gas (relative humidity 50-60% ) at 80°C for one to two hours. Samples not exposed to paraformaldehyde gas were used as controls. The samples were examined with a Leitz Orthoplan fluorescence microscope (HB-200 bulb, BG12, UG-1 activating filters and 53, 46, 65, 47 barrier filters). For electron microscopy, cell pellets were fixed in 2% glutaraldehyde in phosphate buffer (pH 7. 4), post-fixed in 1% osmic acid and embedded in Epon. One-micron
DISSOCIATION OF APUD ENDOCRINE CELLS
Epon sections were stained by toluidine blue and PAS methods. Ultrathin sections were stained with uranyl acetate and lead ci tra te . RESULTS
Light microscopy of dissociated cells from trachea and intestine After 45 minutes’ incubation of trachea with 1% pronase a n average yield was 20 x lo6 cells. As observed by phase contrast microscopy, the dissociated tracheal epithelial cells were predominantly in a single-cell suspension and the majority of cells appeared rounded (fig. 1). Their viability ranged from 80 to 9 8 % . Isolated ciliated cells retained their ciliary beat. Following the completion of enzyme treatment the trachea showed complete denudation of the epithelial lining with a n intact basement membrane left behind. The higher yield of cells from the small intestinal segments treated with 1% pronase for 30 minutes (130 X lo6) was due to a larger epithelial surface, rather than greater effectiveness of the dissociation procedure. The dissociated intestinal cells were also i n a single-cell suspension and spherical in shape. The viability of these cells ranged between 70 and 90%. The segments of intestine after the enzyme treatment showed complete removal of epithelial cells from the villi, but small patches of cells remained in the depths of the crypts. In H & E-stained, cytocentrifuged cell preparations from both the trachea and small intestine, the majority of cells had the cytologic features of epithelial cells. Among the differentiated cell types, ciliated cells in the samples of trachea and enterocytes in the samples of intestine were the most frequent cell types present. In samples stained for mucin by PAS or WHO, goblet cells were easily identified. No contamination with connective tissue elements was observed and only occasional red cells, lymphoid cells and macrophages were present. Although the cytocentrifuged cell preparations provided a fast and simple method for general morphological assessment of dissociated cells, the cytological detail was superior i n 1-, Epon sections of cell pellets. The single cell distribution of dissociated epithelial cells and their good morphologi-
359
cal preservation were confirmed in cell pellets obtained from both the trachea (fig. 2 ) and small intestine (fig. 3 ) . At higher magnification, the various epithelial cells showed excellent preservation of the cytoplasm and nuclei (fig. 4 ) . I n Epon sections stained with the PAS method, mucous granules in goblet cells appeared well preserved (fig. 5 ) . I n cytocentrifuged samples stained with the method of Grimelius, argyrophilic (AG) cells were identified in both the trachea (fig. 6 ) and small intestine samples (fig. 7). The shape of dissociated AG cells was cuboidal or spherical, and the cytoplasm contained numerous fine, dark brown or black granules (insets, figs. 6, 7). The appearance of silver-positive cytoplasmic granules was comparable to those found in AG cells in tissue sections. In cell preparations from trachea examined by the FIF method, cells with specific yellow-green cytoplasmic fluorescence were identified only in samples previously incubated with L-DOPA (fig. 8 ) . The intensity of fluorescence i n dissociated AG cells was similar to that of AG cells in tissue sections. In non-incubated samples and in control samples not exposed to paraformaldehyde vapor no positive cells were found. In the samples of intestine, cells with yellow cytoplasmic fluorescence were present even in samples without L-DOPA incubation (fig. 9 ) . However, no fluorescent cells were seen in samples not exposed to paraformaldehyde vapor, confirming the specificity of the FIF reaction. Electron microscopy of cells dissociated from trachea and small intestine The overall ultrastructural preservation of tracheal epithelial cells after pronase treatment was excellent as judged by the cytoplasmic and nuclear morphology of various cell types. In ultrathin sections, cells appeared in various planes of section, but the differentiated cell types could usually be identified by their characteristic ultrastructural features (fig. lo). The ciliated and goblet cells were the most frequent cell types present. The cilia of the ciliated cells and mucous granules of goblet cells were well preserved. Mild to moderate degrees of ultrastructural alteration due to the effect of enzyme on cells consisted of cytoplasmic blebbing, concentra-
360
K. S. SONSTEGARD, E. CUTZ AND V. WONG
tion of organelles in the perinuclear region, mild cytoplasmic vacuolation and increased density of mitochondria1 matrix. In several of the cell pellets examined, cells with cytoplasmic dense-core vesicles (DCV) characteristic of K cells were present (fig. 12). The size and morphology of DCV in dissociated K cells (fig. 13) were identical to those previously described in tissue sections of rabbit trachea. As compared to other epithelial cells, pronase effect on K cells was minimal. Except for mild dilation of smooth endoplasmic reticulum and a few cytoplasmic vacuoles, no other changes were found. The fine structural preservation of epithelial cells dissociated from the small intestine was satisfactory and was generally comparable to that of cells obtained from the trachea. However, the effects of pronase on intestinal epithleial cells was more pronounced (fig. 11). Among the various epithelial cell types, the most common were enterocytes. Due to their spherical shape and increased apical surface in the dissociated state, their brush borders appeared stretched and the microvilli shortened. Cytoplasmic blebbing and organelle condensation were more frequently found in intestinal cell preparations than in those from the trachea. On the other hand, the mucous granules in goblet cells (fig. 11) and secretory granules of Paneth cells appeared intact. A s expected, endocrine cells were more numerous in preparations from intestine than in those from the trachea. The intestinal endocrine cells were also remarkably well preserved and retained their characteristic ultrastructural features (fig. 14). The most frequently encountered type of endocrine cells contained dense, pleiomorphic DCV measuring up to 250 n m in diameter (fig. 15). Based on ultrastructural criteria, this type of endocrine cell corresponds to the enterochromaffin cell type (Forssmann, '70). Other types of endocrine cells usually found in the upper duodenum were also present, but in this study no attempt was made to quantitate the various types of endocrine cells. DISCUSSION
Recent advances in cell separation technology have facilitated the investigation
and characterization of specific types of viable cells isolated from a variety of tissues and organs (Pretlow et al., '75). Studies on homogeneous populations of cells provide more meaningful information in regard to a specific cell type than do tissue sections or tissue homogenates composed of a mixture of cells. This approach, using isolated cells, has recently been applied to studies i n vitro on amine and polypeptide hormone production by APUD cells dissociated from anterior pituitary (Hopkins and Farquhar, '73), endocrine pancreas (Orci et al., '73) and carotid body (Pietruschka, '74). The endocrine APUD cells in the tracheobronchial and gastrointestinal mucosae are known to be widely distributed among the various non-endocrine epithelial cells (Cutz et al., '75; Pearse, '69). Therefore it would be of great advantage to isolate and study t.hese cells in preparations composed of a homogeneous population of a single kind of cells. The various enzymatic and physical methods for cell dissociation and problems connected with these procedures have been discussed previously (Shortman, '72; Waymouth, '74; Pretlow et al., '75). These studies have emphasized that the cell dissociation method to be employed must be tailored to recovery of a particular cell type, because some types of cells may be selectively destroyed during the dissociation procedure. In addition, the isolated cell preparations must conform to the requirements of subsequent studies, e.g., techniques for further separation into individual kinds of cells, biochemistry, tissue culture or other studies. Therefore, it is important to evaluate critically the cell preparations following the dissociation procedure, particularly by monitoring their morphologic arid cytochemical preservation. In this study a proteolytic enzyme, pronase, was found to be effective in dissociating the epithelial lining from both the trachea and sm.311 intestine into individual cells. In spite of a relatively high concentration of enzyrne necessary to obtain single cell suspen!;ions in a short time, the majority of dissociated cells remained viable. In addition, the morphology of various epithelial cell types was well preserved at
DISSOCIATION OF APUD ENDOCRINE CELLS
both the light and electron microscopic levels. Proteolytic enzymes, including pronase, are known to effect the surface of cell membranes, but most of these changes are considered reversible (Poste, '71; Wallach, '72). Pronase-induced changes were also noted in this study and at the ultrastructural level were manifested as cell membrane blebbing and condensation and redistribution of organelles. These changes were more pronounced in the cells isolated from the small intestine than those from the trachea. The enterocytes showed the most marked changes, whereas secretory cells, such as goblet cells, Paneth cells and APUD endocrine cells, were generally well preserved. This finding suggests that there is a variation in susceptibility to proteolytic enzymes not only between different tissues but also between various cells in the same tissue. In order to monitor the recovery and preservation of APUD cells in dissociated cell preparations, we used histochemical methods similar to those employed for identification of these cells in tissue sections (Pearse, '69). The FIF method demonstrates the amine-handling properties of APUD cells and is one of the primary characteristics. Because of the low endogenous amine content in APUD cells of the trachea, prior incubation with amine precursor is required (Ericson et al., '72; Cutz et al., '75). The demonstration of intracellular amines i n dissociated tracheal APUD cells after incubation with L-DOPA indicates that the decarboxylating mechanism in isolated APUD cells remained intact. The APUD cells in the GI tract are known to contain large amounts of endogenous amines, particularly serotonin, and, therefore, the intracellular amine can be shown without prior incubation with amine precursors (Owman et al., '73). Although argyrophilia, as shown by the method of Grimelius, is considered a secondary feature of APUD cells, it is a useful and relatively simple method for demonstration of these cells by light microscopy. The presence of argyrophilic granules in dissociated APUD cells confirmed the good preservation of these cells and showed that the enzyme treatment did not interfere with this staining reaction. At the ultrastructural level the cyto-
36 1
plasm of APUD cells contains characteristic cytoplasmic granules referred to as dense-core vesicles (DCV). These granules are considered to be the storage site of amines and polypeptide hormones produced by the APUD cells (Owman et al., '73). It is of great importance to evaluate the preservation of DCV in dissociated APUD cells, because ultrastructural changes following pronase treatment have been recently reported in cytoplasmic granules of cells from anterior pituitary and endocrine pancreas (Schofield and Orci, '75; Orci et al., '73). However, no such changes were found in the present study. The ultrastructural appearance of DCV in pronase-dissociated APUD cells was comparable to that described in tissue sections (Cutz et al., '75; Forssmann, '70). The APUD cells in trachea and bronchi of human and various other animal species have been identified only recently and the function of these cells remains unknown (Lauweryns and Peuskens, '69; Ericson et al., '72; Cutz et al., '74, '75). Although it has been suggested that these cells may have a n endocrine function and, in addition to amines, may produce polypeptide hormones, so far no direct evidence has been obtained. The endocrine function of the G I tract has been studied more extensively and a variety of hormonal substances have been isolated and characterized from GI tissues (Pearse, '74). However, many questions still remain to be answered, particularly in regard to the physiologic function of GI hormones and their cellular origin. Availability of methods for dissociation and monitoring of APUD cells isolated from the epithelial lining of trachea and intestine will provide an excellent tool for studies on metabolism and secretory activity of these cells in vitro. Because single cell suspensions can be obtained, these preparations are usable for further cell separation into specific cell types, including the APUD endocrine cells. In our laboratory, studies are in progress using velocity sedimentation techniques for the further separation and characterization of APUD cells from trachea and small intestine. These methods may provide concentrated or homogeneous cell suspensions of APUD cells for direct biochemical studies,
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K. S . SONSTEGARD, E. CUTZ AND V. WONG
as well as for isolation and characterization of their secretory products. LITERATURE CITED Bensch, K. G., C. B. Gordon and L. R. Miller 1965 Studies on the bronchial counterpart of the Kultschitzky (argentaffin) cell and innervation of bronchial glands. J. Ultrastruct. Res., 12: 668-686. Bensch, K. G., B. Corrin, R. Pariente and H. Spencer 1968 Oat cell carcinoma of the lung. Its origin and relationship to bronchial carcinoid. Cancer (Piladelphia), 22: 1163-1172. Cutz, E., and P. E. Conen 1973 Endocrine-like cells in human fetal lungs. Anat. Rec., 173: 115-122. Cutz, E., W. Chan, V. Wong and P. E. Conen 1974 Endocrine cells i n rat fetal lungs. Ultrastructural and histochemical study. Lab. Invest., 30: 458-464. 1975 Ultrastructure and fluorescence histochemistry of endocrine ( APUD-type) cells in trachea of human and various animal species. Cell Tiss. Res., 158: 425-437. Ericson, L. E., R. HBkanson, B. Larson, Ch. OWman and F. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124: 532-545. Forssmann, W. G. 1970 Ultrastructure of hormone-producing cells of the upper gastrointestinal tract. In: Origin, Chemistry, Physiology and Pathophysiology of the Gastrointestinal Hormones. W. Creutzfeldt, ed. F. K. Schattauer, Stuttgard-New York, pp. 32-95. Hage, E. 1972 Endocrine cells in the bronchial mucosa of human foetuses. Acta path. microbiol. scand., Sec. A, 80: 225-234. Hattori, S . , M. Matsuda, R. Tateishi, M. Nishihara and T. Horai 1972 Oat-cell carcinoma of the lung. Clinical and morphological studies in relation to its histogenesis. Cancer (Philadelphia), 30: 1014-1024. Hopkins, C., and M. Farquhar 1973 Hormone secretion by cells dissociated from rat anterior pituitaries. J. Cell Biol., 59: 276-303. Lauweryns, J. M., and J. C. Peuskens 1969 Argyrophil (kinin and amine producing? ) cells in human infant airway epithelium. Life Sci., 8: 577-585. Omenn, G. S . 1970 Ectopic polypeptide hormone production by tumours. Ann. Int. Med., 72: 136-138.
Orci, L., A. A. Like, M. Amherdt, B. Blondel, Y. Kanazawa, E. B. "arliss, A. E. Lambert, C. B. Wollheim and A. E:. Renold 1973 Monolayer cell culture of neonatal rat pancreas: An ultrastructural and biochemical study of functioning endocrine cells. J. Ultrastruct. Res., 43: 270-297. Orci, L., M. Amherdt, J. C. Henquin, A. E. Lambert, R. M. Unger and A. IS. Renold 1973 Pronase effect on pancreatic beta cell secretion and morphology. Science, 180: 647-649. Owman, Ch., R. HPkanson and F. Sundler 1973 Occurrence and function of amines in endocrine cells produ'zing polypeptide hormones. Fed. Proc., 32: 1785-1791. Pearse, A. G. E. 1969 The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J. Histochem. Cytochem., 17: 303-3 13. 1973 Cell migration and the alimentary system: Endocrine contributions of the neural crest to the gut and its derivatives. Digestion, 8: 372-385. 1974 The gut as an endocrine organ. Brit. J. Hosp. Medicine (May), pp. 697-704. Pietruschka, F. 19'74 Cytochemical demonstration of catecholamines in cells of the carotid body in primary tissue culture. Cell Tiss. Res., 151: 317-321. Poste, G. 1971 Tissue dissociation with proteolytic enzymes. Exp. Cell Res., 65: 359-367. Pretlow, T. G., E. I;. Weir and J. G. Zettergren 1975 Problems Connected with the Separation of different Kinds of Cells. In: Int. Rev. Exp. Path. G. W. Richter and M. A. Epstein, eds. Academic Press, New York-London, 14: 91-204. Rees, H. L., and J. G. Ratcliffe 1974 Ectopic hormone production by non-endocrine tumours. Clin. Endocrin., 3: 263-299. Schofield, J. G., and L. Orci 1975 Release of growth hormone from ox pituitary slices after pronase treatment. J. Cell Biol., 65: 223-227. Shortman, K. 1979: Physical procedures for the separation of aniinal cells. Annu. Rev. Biophys. and Bio. Eng., 1: 93-130. Wallach, D. F. H. 1972 The disposition of proteins in the plasma membranes of animal cells; analytical approaches using controlled peptidolysis and protein. Biochim. Biophys. Acta, 265: 61-68. Waymouth, C. 19'74 To disaggregate or not to disaggregate. Injury and cell disaggregation. transient or permanent? In Vitro, 10: 97-111:
PLATES
PLATE 3 EXPLANATION O F FIGURES
364
1
Phase contrast micrograph of a cell suspension from rabbit trachea obtained after 30-minute incubation in 1% Pronase. The dissociated epithelial cells are spherical and in single-cell suspension. x 450.
2
Low-magnification light micrograph (LM) of cell pellet from trachea. The large epithelial cells represent ciliated and goblet cells. The dissociated cells are well preserved and are distributed as single cells. One-micron Epon section, Toluidine blue stain. x 400.
3
LM of a cell pellet from dissociated intestinal cells. The shape and distribution of isolated cells are similar to those in figure 2. Onemicron Epon section, Toluidine blue stain. x 400.
DISSOCIATION O F APUD ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 1
365
PLATE 2 EXPLANATION OF FIGURES
4 Higher-magnification LM of cells dissociated from trachea with good cytological preservation of ciliated ( c i ) and goblet (Go) cells. The smaller cells represent basal cells. One-micron Epon section, Toluidine blue stain. x 1,000.
366
5
Well preserved mucus granules i n dissociated intestinal goblet cells. One-micron Epon section, PAS stain. x 1,000.
6
Two argyrophilic (AG) cells (arrowheads) among dissociated epithelial cells from the trachea. Cytocentrifuge preparation, Grimelius’ stain. :< 320. Inset: Fine black, argyrophilic granules i n the cytoplasm of isolated AG cell from trachea. Cytocentrifuge preparation, Grimelius’ stain. x 1,000.
7
Isolated AG cells (arrowheads) i n intestinal cell suspensicn. Cytocentrifuge p r e p aration, Grimelius’ stain. x 320. Inset: T h e argyrophilic granules i n intestinal AG cells appear larger than from trachea. Cytocentrifuge preparation, Grimelius’ stain. x 1,000.
8
Intense cytoplasmic fluorescence (white arrowhead) i n a cell dissociated from tracheal epithelium. Incubation i n vitro with L-DOPA, cytocentrifuge preparation, FIF. x 800.
9
Two intensely fluorescent cells (white arrowheads) among dispersed intestinal epithelial cells. Not incubated with L-DOPA, cytocentrifuge preparation, FIF. x 800.
DISSOCIATION O F APUD ENDOCRINE CELLS K. S . Sonstegard, E. Cutz and V. Wong
PLATE 2
367
PLATE 3 EXPLANATION O F FIGURES
10
Low-magnification electron micrograph of cell pellet from trachea. The ciliated (Ci) and goblet ( G o ) cells appear in various planes of section. There is good ultrastructural preservation of nuclei and cytoplasm of the various cell types. x 2,000.
11 Low-magnification electron micrograph of dissociated intestinal epithelial cells. There is good preservation of nuclei, but the cytoplasm of enterocytes (En) shows some vacuolation, and the cell membrane of goblet cells ( G o ) shows cytoplasmic blebs (arrow). x 2,000.
368
DISSOCIATION OF APUD ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 3
369
PLATE 4 EXPLANATION OF FIGURES
3 70
12
Electron micrograph of an isolated K cell from trachea shows excellent ultrastructural preservation, including the cytoplasmic granules. Nu, nucleus; Va, vacuoles. x 18,000.
13
Higher magnification of cytoplasmic DCV from the K cell shown in figure 12. The central core and the limiting membrane (arrows) are well preserved. Mi, mitochondria. x 30,000.
DISSOCIATION OF APUD ENDOCRINE CELLS K . S. Sonstegard, E. Cutz and V. Wong
PLATE 4
PLATE 5 EXPLANATION O F FIGURES
14
Electron micrograph of endocrine cell isolated from small intestine. Peripherally located dense granules represent DCV. Nu, nucleus; Va, vacuoles. x 11,000.
15 Pleiomorphic DCV (arrows) from cell shown i n figure 14. This type of cytoplasmic granule is characteristic of enterochromaffin cells. Mi, mitochondria; Nu, nucleus. x 23,000.
3 72
DISSOCIATION OF A P U D ENDOCRINE CELLS K. S. Sonstegard, E. Cutz and V. Wong
PLATE 5
