ORGAN CULTURE OF HUMAN RECTAL MUCOSA

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.3 Printed in U.S.A. ORGAN CULTURE OF HUMAN RECTAL MUCOSA GREGORY L. EASTWOOD, M.D., AND JERRY S. TRIER, M.D. Departments of Medicine and Anatomy, Boston University School of Medicine, Boston, Massachusetts Mucosal biopsies from human rectum were cultured in vitro using a simple organ culture system. Light microscopy consistently revealed good preservation of rectal mucosa in biopsies cultured for up to 24 hr. Moreover, mucosal morphology was well preserved in some but not all biopsies cultured for as long as 48 hr. Electron microscopy revealed preservation of surface epithelial cell structure in biopsies cultured for 24 hr. Autoradiographs of biopsies cultured over thymidine- 3 R containing medium for 6 hr showed labeling of proliferating cells in the lower two-thirds of the rectal crypts. In biopsies subsequently cultured for an additional 18 hr over isotope-free medium, some of the labeled cells had migrated to the upper crypts and the absorptive surface. These studies show that mucosal morphology is maintained and that some epithelial cell proliferation continues for at least 24 hr in rectal mucosal biopsies cultured in vitro in this organ culture system. Application of organ culture techniques 1 to the culture of human small intestinal mucosal biopsies has been shown recently to maintain mucosal morphology, epithelial cell proliferation, and fat absorption for up to 24 hr.2 Utilization of this method for culture of mucosa from human large intestine would be valuable since it would permit in vitro studies of mucosal epithelial cell renewal and mucosal metabolic functions which would not be feasi- ble using current in vivo methods. Rowever, a culture method successful for mucosal tissue from the small intestine cannot be applied unreservedly to the large intestine since distinct differences exist in their mucosal morphology, physiology, and their bacteriological milieu. We therefore examined the applicability of the small bowel organ culture method to the culture of human rectal mucosa. Materials and Methods Received July 5, Accepted October 19, Rectal mucosal biopsies were obtained 7 to Presented in part at the Annual Meeting of the 10 cm proximal to the anus from healthy young American Gastroenterological Association, Dallas adults using a single port multipurpose suction Texas, May 24, biopsy tube. 3 Each subject had given informed, Address requests for reprints to: Dr. Jerry S. written consent. Biopsies were gently placed Trier, Department of Medicine, Boston University epithelial surface up and cut surface down (fig. School of Medicine, 80 East Concord Street, Boston, 1) on a stainless steel wire screen which rested Massachusetts over a small well in the center of a plastic culture dish (Falcon Plastics, Los Angeles, CaliO. This investigation was supported by Research Grant AM-14420, and Training Grant AM from To maintain a humid environment, a ring of the National Institute of Arthritis and Metabolic porous paper felt surrounding the central well Diseases, National Institutes of Health, Bethesda, was saturated with water. Sufficient organ culture medium (approximately 1 ml) was added Maryland. Dr. Trier is recipient of Research Career De to the well to barely float the mesh and biopsy velopment Award AM from the National In and an additional drop of medium was placed stitutes of Health, Bethesda, Maryland. over the surface of the biopsy. The organ cul- 375

2 376 EASTWOOD AND TRIER Vol. 64, No.3 Saturated Felt Medium FIG. 1. Cross sectional view of the organ culture plate. The biopsy rests upon a wire mesh screen suspended over a well containing medium. See text for details. ture medium consisted of nine parts Trowel T-8 medium without chloramphenicol (GIBCO, Inc., Grand Island, N. Y.), 0.9 part fetal calf serum (GIBCO), and 0.1 part solution of crystalline penicillin G (10,000 U per mil and streptomycin sulfate (10 mg per mi). To study epithelial cell proliferation 10 /-LC of thymidine- 3H (specific activity 6.7 c per mm, New England Nuclear Corp., Boston, Mass.) were added to each milliliter of medium. The culture dishes were placed in a McIntosh jar (Torsion Balance Co., Clifton, N. J.), gassed with a mixture of 95% O 2 and 5% CO 2, and placed in an incubator at 37 C within 30 min after excision of the biopsies. Biopsies from 8 subjects were cultured for 6 and 24 hr: the first 6 hr over thymidine- 3 H containing medium; the remaining time over isotope-free medium. In another experiment the mucosal biopsy was cultured over isotopefree medium for the first 21 hr and then exposed to thymidine- 3 H-containing medium for 3 hr. In four additional experiments, biopsies were cultured for 24, 36, or 48 hr, using isotope-free medium. Some biopsies were fixed in Bouin's solution, embedded in paraffin, serially sectioned, and stained with hematoxylin and eosin. Most biopsies were fixed in potassium dichromatebuffered osmium tetroxide' and embedded in Epon Specimens were cut out of the Epon, carefully oriented, and remounted so that sections could be cut along the full length of the rectal crypts. Sections for autoradiography were cut 1 /-L thick from Epon-embedded specimens with glass knives using a Sorval MT-2 ultramicrotome. Slides were dipped in Hford K-5 photographic emulsion diluted 1: 1 with distilled water, and exposed in light-proof boxes at 2 C for 4 to 6 weeks. The slides were then developed in Kodak D 19, fixed, and stained by immersion for 15 sec in toluidine blue at 80 C. Previous studies have shown that the epithelial proliferative zone in the large bowel is normally confined to the lower two-thirds of the crypts. 6, 7 Using an eyepiece micrometer, we divided the crypts into upper one-third and lower two-thirds and counted the total and labeled epithelial cell nuclei in the lower twothirds of the crypts. These results were expressed as percentage of epithelial cell nuclei that were labeled to provide an index of proliferative activity. Only nuclei of cells having a grain density of at least 4 times greater than background were scored as labeled. The number of labeled epithelial cell nuclei in the upper one-third of crypts was also counted and expressed as number of labeled cells per upper third of the crypt. Labeled epithelial cells on the surface between crypts were counted and expressed as number of labeled surface cells per histological section. Only crypts in approximately the lateral thirds of the biopsy sections were used for quantitation since these were most heavily labeled (see "Results"). However, the most lateral crypt on each edge of the biopsy and the surface epithelium lateral to it were excluded because epithelial cell proliferation appeared stimulated at the cut edge of the biopsies (see "Results"). For each subject, five to 27 well oriented crypts (mean 14.6) were counted in 6 to 10 histological sections. Only every 10th section was counted to eliminate the possibility of scoring a given cell more than once. Areas for electron microscopic study were selected from toluidine blue-stained 1 /-L sections. Blocks were appropriately trimmed and thin sections were cut with a diamond knife on an LKB ultramicrotome. These were stained with uranyl acetate" and lead citrate 9 and examined with a Philips EM-300 electron microscope. Results Mucosal architecture remained normal in most rectal biopsies cultured for 6 to 24 hr (fig. 2). The number and height of the rectal crypts were unchanged and the epithelial lining of the mucosa remained intact. Crypt and surface epithelial cells appeared normal (figs. 3 and 4), and mitotic figures were abundant in the lower crypts in paraffin-embedded samples (fig. 4), although the amount of mucus in goblet cells was decreased. Degenerative changes, when present, were limited to the basal half of those crypts in the center of the biopsy. In such degenerating

3 March 1973 ORGAN CULTURE OF HUMAN RECTAL MUCOSA 377 FIG. 2. Light micrographs of rectal mucosal biopsies from a normal volunteer. A, uncultured. B, cultured for 24 hr. Surface epithelium, crypts, and lamina propria are well preserved (hematoxylin and eosin, x 160).

4 378 EASTWOOD AND TRIER Vol. 64, No.3 FIG. 3. High magnification of the absorptive surface epithelium in biopsies from a normal volunteer. A, uncultured. B, cultured for 24 hr. Although the surface cells appear normal after organ culture, extruded (short arrow) and extruding (long arrow) epithelial cells accumulate (hematoxylin and eosin, x 400). FIG. 4. High magnification of the lower portions of rectal crypts in biopsies from a normal volunteer. A, uncultured. B, cultured for 24 hr. Epithelial cells appear well preserved and a mitotic figure (arrow) is seen in the crypt from the biopsy cultured for 24 hr, although the amount of mucus in goblet cells is decreased (hematoxylin and eosin, x 500).

5 March 1973 ORGAN CULTURE OF HUMAN RECTAL MUCOSA 379 crypts, mitotic figures were uniformly absent, dark-staining intracytoplasmic bodies presumably cytolysosomes, appeared within the epithelial cells, and some degenerated epithelial cells were sloughed into the crypt lumen. Such changes were present in portions of a few biopsies after 24-hr of incubation but became more prominent after 36 to 48 hr of culture. However, even after 48 hr in vitro normalappearing crypts were seen in some of the biopsies. In several biopsies cultured for 24 hr, we noted a sheet of epithelial cells extending along the cut surface underneath the biopsies (fig. 5). This lining appeared to originate from the epithelial cells at the lateral edge of the biopsy. The cells at the leading edge of this sheet were squamoid whereas those near the lateral margin were cuboidal or columnar (fig. 5). In radioautographs, the nuclei of some of the epithelial cells at the origin of the sheet were heavily labeled. Electron microscopic examination of biopsies cultured for 24 hr revealed preservation of the surface epithelial cells and their organelles when compared with uncultured biopsies (fig. 6). Nuclei, mitochondria, endoplasmic reticulum, Golgi material, junctional complexes between cells, microvilli, and surface glycocalyx all were intact. However, when compared with uncultured biopsies, intercellular spaces were narrower, intracellular vesicles were decreased in number, lysosomes were moderately increased in size and number, and there appeared to be more glycogen within the cytoplasm after 24 hr of culture. In 8 subjects, autoradiographs from biopsies cultured for 6 hr (fig. 7) showed that a mean of 4.8% (range 3.3 to 6.0%) of epithelial cells were labeled in the lower two-thirds of the crypts. Labeled cells were occasionally seen in the upper onethird of the crypts (mean of 0.1 per crypt) but were not observed on the surface epi- FIG. 5. Light micrograph of a rectal mucosal biopsy cultured for 24 hr. A new layer of cuboidal and squamoid epithelial cells (arrows) extends from the columnar epithelial cells at the biopsy edge on the left to cover the cut surface of the biopsy (hematoxylin and eosin, x 160). Inset, higher magnification showing transition from columnar to cuboidal epithelium (x 300).

6 380 EA~TWOOD AND TRIER Vol. 64, No.3 FIG. 6. Electron micrographs of the apical portions of absorptive surface epithelial cells. A, uncultured. B, cultured for 24 hr. Nuclei (N), mitochondria (M), Golgi material (G), and microvilli (V) are well preserved. Lysosomes (L) are increased in number and intracellular vesicles and intercellular spaces are diminished (x 7000). Inset, surface glycocalyx, microvilli, junctional complex, and membrane-bounded vesicles are well preserved after 24 hr of culture (x 15,000). thelium between crypts. Since the intensity of the labeling decreased toward the central core of the biopsy, only heavily labeled crypts in the lateral third of the cultured biopsies were scored. After 24 hr of culture, there were more labeled cells in the upper one-third of the crypts (mean of 0.7 per crypt), and a few labeled cells appeared on the surface (a mean of 0.1 per histological section). Such surface cells in 24-hr cultures were labeled more lightly than labeled crypt cells in 6-hr cultures. In the single experiment in which a biopsy was cultured over isotope-free medium for 21 hr and then exposed to thymidine- 3 H-containing medium for 3 hr, 7.1 % of cells were labeled in the lower two-thirds of the crypts and an occasional cell (a mean of 0.2 per crypt) was labeled in the upper third of the crypts. No labeled cells were seen on the surface.

7 March 1973 ORGAN CULTURE OF HUMAN RECTAL MUCOSA 381 Discussion These studies document that rectal mucosal biopsies can be cultured in vitro for 24 hr or longer with good preservation of mucosal architecture and histology. Similarly, the ultrastructure of surface epithelial cells from biopsies cultured 24 hr is well preserved although the number of intracellular vesicles and the size of the intracellular spaces are diminished and lysosomes are more prominent. Moreover, the normal pattern of epithelial cell renewal appears maintained throughout the culture period. Proliferative activity, as shown by thymidine-3h incorporation into newly synthesized deoxyribonucleic acid of dividing cells during the first 6 hr of culture, is virtually confined to the lower two-thirds of the crypts. Some of these labeled cells then migrate to the upper crypts and out onto the surface during the next 18 hr of culture. The demonstration that crypt nuclei still incorporate thymidine- 3 H after 21 hr of culture is more evidence that the mucosa remains viable in the organ culture system. After 24 hr in the culture system, most crypts appear normal by light microscopy. Degenerative changes, such as lysosomal enlargement, absence of mitoses, and sloughing of cells, when they develop, appear first at the base of crypts in the center of the specimen. This is not surprising since, in vivo, each cell is within but a few microns of its source of nutrition and oxygen, the capillaries. In contrast, in vitro, many cells, particularly those in the center of the biopsy, may be several hundred microns from the nutrient medium or surrounding oxygen. The capricious and reduced labeling of cells in the crypts in the center of cultured biopsies may thus reflect reduced proliferation in this portion of the biopsy as well as diminished diffusion of labeled precursor to the central core of tissue. It was of interest that the epithelial cells grew from the peripheral edges of some biopsies to cover partially the cut inferior surface. Autoradiographs demonstrated labeled cell nuclei in the columnar and cubiodal cells at the biopsy edge. Pre- FIG. 7. Light microautoradiograph showing labeling of two nuclei at the base of a crypt (arrows). The biopsy was cultured 6 hr over thymidine- 3 H-containing medium (toluidine blue, x 700). sumably, these labeled columnar cells derive from the differentiated surface cells which normally do not proliferate. A similar pattern of new epithelial growth has been described in the initial stages of healing of colonic mucosal wounds in dogs 10 and man. 11 In such wounds, mature epithelial cells adjacent to a denuded area appear first to dedifferentiate and then proliferate, eventually covering the underlying connective tissue. In previously published studies, Shorter et al. 12 incubated biopsies for 1 hr in T.e. 199 medium containing thymidine- 3 H. \Vhereas mucosal morphology was preserved, nuclear labeling was limited. The poor labeling was attributed to incomplete I diffusion of the isotope through the tissue in vitro. However, Galand et al. 13 reported adequate in vitro labeling of biopsies incubated for a total of 21/2 hr in Eagle's medium supplemented with fetal calf serum. Their biopsies were exposed to thymidine-3h for the first and last 15

8 382 EASTWOOD AND TRIER Vol. 64. No.3 min of the 21/2-hr incubation. Deschner et al. 14 incubated rectal biopsies in Eagle's basic salt solution with added human serum, antibiotics, and thymidine- 3R, and described satisfactory thymidine- 3R uptake and good tissue preservation in half the specimens after 19 hr of incubation. These earlier studies utilized tissue culture methods in which small fragments of tissue were immersed in medium and agitated by shaking or by bubbling oxygen mixtures through the medium. In contrast, the organ culture methodology used by us differs. The specimen must not be made so small that architectural relationships are lost, nor so large that its mere bulk impedes the diffusion of nutrients. Agitation of specimens is avoided. They are not immersed but maintained on the surface of the medium in a well oxygenated atmosphere. The organ culture medium which we used (Trowell T-S) contains insulin (50 mg per liter), usually lacking in tissue culture media, and much more glucose (4 g per liter) than most tissue culture media. In a brief note Johansen 15 recently described an organ culture system similar to that used by us here for rectal mucosa and earlier for culture of small intestinal mucosa. 2 She reported histological preservation of rectal mucosa in vitro for up to 90 hr and that hydrocortisone, 5 to 10 Ilg per m} of medium, "appeared to be advantageous to the cultures" whereas insulin seemed to cause "excessive epithelial cell proliferation and tissue disorder." Supporting data, however, were not presented. In pilot studies in which we omitted insulin from or added hydrocortisone to the medium, cultured tissues showed more degenerative changes than were seen with our standard method. REFERENCES 1. Trowell OA: The culture of mature organs in a synthetic medium. Exp Cell Res 16: Browning TH. Trier JS: Organ culture of mucosal biopsies of human small intestine. J Clin Invest 48: Brandborg LL. Rubin CEo Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach. small bowel and colon. Gastroenterology 37: Dalton AJ: A chrome-osmium fixative for electron microscopy. Anat Rec 121: Luft JH: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9: Lipkin M, Quastler H: Cell population kinetics in the colon of the mouse. J Clin Invest 41: , Lipkin M, Bell B, Sherlock P: Cell proliferation kinetics in the gastrointestinal tract of man. I. Cell renewal in colon and rectum. J Clin Invest 42: , Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4: , Reynolds ES: The use of lead citrate at high ph as an electron opaque stain in electron microscopy. J Cell Bioi 17: , Melnyk CS, Braucher RE, Kirsner JB: Colon mucosa response to injury. I. Morphological Etudy. Gastroenterology 51:43-49, Melnyk CS, Braucher RE, Kirsner JB: Regeneration of the human colon mucosa. Morphological and histochemical study. Gastroenterology 52: , Shorter RG, Spencer RJ, Hallenbeck GA : Kinetic studies of the epithelial cells of the rectal mucosa in normal subjects and patients with ulcerative colitis. Gut 7: , Galand P, Mainguet P, Arguello M, et al: In vitro autoradiographic studies of cell proliferation in the gastrointestinal tract of man. J Nuc\ Med 9:37-39, Deschner E, Lewis CM, Lipkin M: In vitro study of human rectal epithelial cells. I. Atypical zone of H' thymidine incorporation in mucosa of multiple polyposis. J Clin Invest 42: Johansen PG: An in vitro system for studying mucus secretion and other physiological activities in human intestinal mucosa. Experientia 26: , 1970