DEVELOPMENTAL
45, 304-317 (1975)
BIOLOGY
Differentiation GIORCIO Department
of Medicine,
and Maturation DERITIS, Harvard
of Cultured
Z. MYRON
Medical
FALCHUK,
Fetal Rat Jejunum
AND JERRY
S. TRIER
School and Peter Bent Brigham Hospital, 02115 Accepted
March
Boston, Massachusetts
24, 1975
To determine whether differentiation and maturation of mammalian intestinal mucosa require influences available only in vivo or whether they can occur in vitro, fetal rat jejunum was cultured in chemically defined medium using organ culture methodology. Segments of jejunum from B-day fetal rats were cultured in modified Liebowitz L-15 medium in room air at 37°C. Segments harvested after 24, 48, and 72 hr of culture were examined by light and electron microscopy. Uncultured jejunum from B-day fetuses had either no or very few rudimentary villi and was lined largely by undifferentiated stratified epithelium. Goblet cells were not seen. In contrast, villi were present in the majority of 24-hr cultures, and simple columnar rather than stratified epithelium predominated. After 48 and 72 hr, villi were present in over 90% of cultured jejunal segments and stratified epithelium had disappeared. Goblet cells were seen in jejunal segments cultured 48 hr or longer in 47 of 74 fetuses. Electron microscopy further documented progressive differentiation of the epithelium during culture. Microvilli increased in number and height, a terminal web developed in the apical cytoplasm and the number of apical vesicles, mitochondria and formed elements of endoplasmic reticulum increased in absorptive cells. Jejunal lactase and alkaline phosphatase activity increased nine- and sevenfold, respectively, during 72 hr of culture, while the activity of the mitochondrial enzyme, ornithine carbamoyl transferase, increased fourfold. These observations indicate that jejunum from 18-day fetal rats can be cultured in vitro for at least 72 hr in chemically defined medium and that, during culture, maturation of the jejunal mucosa takes place with the appearance of villi, conversion of stratified to columnar epithelium and differentiation of individual epithelial cells. INTRODUCTION
During the last 4 days of the 22-day gestation period of fetal rats, growth and differentiation of the small intestine are rapid (12, 17, 23). The mucosal surface of the jejunum of 18-day fetuses is lined with a layer of stratified, relatively undifferentiated epithelial cells and villi are absent. During the next 4 days of gestation, villi form and greatly increase the mucosal surface area. As the villi develop, the stratified epithelial lining is replaced by a single layer of more differentiated columnar epithelial cells. There is evidence that this morphological differentiation is accompanied by functional maturation. The small intestine of fetal rats can absorb and transport protein molecules such as horseradish peroxidase as early as 2-3 days before birth (26). Additionally, the activity of digestive enzymes in the brush border of 304 Copyright All rights
0
1975 by of reproduction
Academic Press, Inc. in any
form
reserved.
the intestinal absorptive cells of mice and rats, such a leucylnaphthylamidase and lactase, increases markedly during the last few days of gestation (18,24). We have recently documented that mucosa of the small intestine from adult mammals can be maintained in vitro for up to 48 hr by using organ culture methology (3, 16). To’ determine whether the rapid maturation and differentiation of rat small intestine, which occur during the few days prior to birth, require influences available only in uivo in the uterine environment or whether such maturation can occur in uitro, we prepared organ cultures of isolated segments of 18-day fetal rat jejunum which we then examined after l-3 days of in vitro culture. MATERIALS
Timed-pregnant were anesthetized
AND
METHODS
Sprague-Dawley rats with ether on the 18th
DF&TIS,
FALCHUK
AND TRIER
day of gestation. Using sterile technique, the 18-day fetuses were removed at laparotomy, the fetal jejunum was exposed, cut into seven to ten segments approximately 2-3 mm in length and placed into a petri dish containing Leibovitz L-15 medium (19). One segment was fixed immediately for baseline morphological studies and the others were cultured using the method of Hoorn (14) as modified by Dolin et al. (8). Briefly, two or three segments of jejunum were placed into sterile plastic culture plates, 5.5 cm in diameter. The plates had previously been scored with a scalpel blade. Leibovitz L-15 medium (19), 1.25 ml, supplemented with a final concentration of 0.2% bovine albumin, 2.0 mM glutamine, 100 U/ml of crystalline penicillin G and 100 pg/ml of streptomycin was added to each culture dish. In addition 0.5 pg/ml of pentagastrin was added because of its recently demonstrated trophic effect on intestinal epithelium (15, 20). The dishes were cultured at a temperature of 36.5”C in room air for 24, 48, and 72 hr on a rocker platform that completed one full cycle per minute. Whenever sufficient tissue was available, tissue from the same fetus cultured for all three time periods was examined. In some instances, tissue cultured for only two of the three time periods was available for comparison to baseline segments. Baseline and cultured jejunal segments were fixed by immersion for 90 min in chilled 2.5% glutaraldehyde buffered to pH 7.4 in 0.1 M cacodylate buffer containing 0.05% calcium chloride. After repeated rinsing in 0.1 M cacodylate buffer for 90 min, tissues were placed in chilled 1% osmium tetroxide buffered to pH 7.4 in 0.1 M cacodylate buffer. Tissues were then rapidly dehydrated in graded concentrations of ethanol and embedded in epoxy resin using Luft’s (22) method. The embedded tissues were then carefully oriented and mounted on aluminum rods for sectioning. Sections of the entire block were then cut 1 pm thick with a Sorvall
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305
MT-2 microtome, mounted on glass slides and stained with Richardson’s stain (28). For electron microscopy, appropriate areas were selected and the blocks were trimmed. Sections were cut with diamond knives, mounted on uncoated copwith uranyl per mesh grids, stained acetate (30) and lead citrate (27) and examined with a Philips EM-300 electron microscope. To determine the influence of culture of fetal intestine on jejunal alkaline phosphatase, lactase, sucrase, and ornithine carbamoyl transferase (OCT) activities, the uncultured jejunum of 18-day fetuses and jejunal segments cultured for 24,48, and 72 hr were homogenized in 1 ml of 0.9% sodium chloride. An aliquot of each homogenate was used for determination of tissue protein (21); the remainder was stored at -85°C until the enzyme assays were performed. Lactase and sucrase activities were determined by a modification of the method of Dahlqvist (6). Aliquots of homogenates were added to tubes containing 0.056 M lactose or 0.056 M sucrose in 0.1 M maleate buffer at pH 6.0 and incubated at 37°C in a shaking water bath for l-3 hr. The reaction was stopped by placing the tubes into a boiling water bath. A unit of disaccharidase activity represented one micromole of glucose liberated per minute per gram of tissue protein. Alkaline phosphatase was determined by the method of Bessey et at. (1). Homogenate was added to tubes containing 200 ~1 of 0.15 mM p-nitrophenyl phosphate in 0.0625 M glycine buffer at pH 10.3 to which 0.625 mM MgCl, and 0.125 mM Zn acetate had been added. Following incubation at 37°C for lo-30 min, the reaction was stopped by addition of 1 ml of 0.02 N NaOH. A unit of phosphatase activity represented one micromole of p-nitrophenol liberated per minute per gram of tissue protein. OCT was measured according to the method of Snodgrass (29). Homogenates were added to tubes containing 7.5 mM
306
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DEVELOPMENTAL BIOL~CY
ornithine-HCl in 0.27 M triethanolamine (TEA), pH 7.7, and the reaction was started by addition of 0.0171 M carbamoyl phosphate in 0.27 M TEA. After 15-30 min of incubation at 37”C, the reaction was stopped by adding 5% trichloracetic acid-0.5% phosphotungstic acid. Following centrifugation at 5000g for 30 min, the citrulline liberated in the supernatant fluid was measured using the method of Ceriotti (5). A unit of OCT activity represents one micromole of citrulline formed per hour per gram of tissue protein.
cytoplasm (Fig. 1A). Secondary lumina (up to 30 pm in diameter) were often observed in the deeper layers of the epithelium (17). In the remaining fetuses, including those that had developed rudimentary villi, islands of simple cuboidal and columnar epithelium were seen interspersed among the more abundant stratified epithelium. No recognizable goblet or endocrine cells were seen in the jejunal epithelium of any of the 18-day old fetuses. In most jejunal segments harvested after 24 hr of culture, there was morphological evidence of maturation of the mucosal RESULTS architecture (Table 1). Small villi were present in segments from 15 of 25 fetuses Light Microscopy (Fig. 1B). The epithelial lining consisted The results of the light microscopic ob- exclusively of simple columnar epithelium servations are summarized in Table 1. in segments cultured for 24 hr from 21 Villi were not seen in jejunal segments fetuses. In cultured jejunum from 4 fetuses, from 39 of 45 l&day fetuses prior to cul- islands of stratified epithelium persisted ture. In the jejunum of the other 6 l&day and were interspersed among more abunfetuses, a few very small rudimentary villi dant areas of columnar epithelium. Goblet were observed. Usually only one and never cells could be identified in jejunal segmore than two such villi were seen per ments from 4 of 25 fetuses. jejunal cross-section. These villi consisted There was morphological evidence of of aggregates of mesenchyme which pro- further maturation of the jejunal mucosa in jected into the small jejunal lumen and segments harvested after 48 and 72 hr of which were lined by a mixture of stratified culture. Villi were present in over 90% of and immature-appearing simple columnar the fetal segments cultured for these longer epithelial cells. In 18 of 45 fetuses, only periods (Table 1). These villi were consiststratified epithelium, 3-12 cells thick, was ently taller and more developed (Fig. 1C) observed lining the jejunum (Fig. 1A). The than those seen after 24 hr of culture. Their individual cells of the stratified epithelium epithelial lining was composed of fairly showed little differentiation. Cell width tall, columnar cells, Mitotic figures were often approximated cell height and nuclei regularly observed and were most abunwere large in relation to the amount of dant along the lower half of the villi. TABLE
1
MORPHOLOGICAL FEATURES OF MATURATION Sample Present l&Day fetuses (45)” 24-Hr cultures (25Y 46.Hr cultures (39)’ 7%Hr cultures (35F’ a Number * Number
Epithelium
Villi
6 15 36 32
of fetuses examined. of fetal jejuna cultured.
Absent 39 10 3 3
Stratified
Columnar
18 0 0 0
0 21 39 35
Goblet cells Mixed 27 4 0 0
Present 0 4 22 25
Absent 45 21 17 10
DERITIS, FALCHUK AND TRIER
Organ. Culture
of Fetal
Intestine
FIG. 1. (A), Jejunal mucosa from an l&day rat fetus consisting of stratified, relatively undifferentiatedappearing epithelial cells. (B), Mucosa from a jejunal segment which had been cultured for 24 hr from the same fetus as A composed of small but definite villi and lined primarily by columnar epithelium. (C), Mucosa from a jejunal segment which had been cultured for 72 hr from the same fetus as A with well-formed villi lined by a single layer of relatively differentiated-appearing columnar epithelial cells. Richardson’s stain; x 440.
Stratification of the epithelium was not seen in any of the segments cultured for 48 or more hr. Goblet cells, sufficiently well differentiated so that they could be identified with the light microscope, were present in segments cultured 48 hr or longer from 47 of 74 fetuses. Occasionally, epithelial endocrine cells could be recognized with the light microscope. In many jejunal segments cultured for 24 hr and in most segments cultured 48 or 72
hr, a layer of simple columnar epithelial cells extended along the serosal surface, especially near the cut edge of the segments. Presumably, epithelization of the serosal surface was initiated by proliferation of the epithelial cells at the extreme lateral edge of the cultured segments. The light microscopic appearance of most of these cells was similar to that of the epithelial cells which lined the lumen of the cultured segment. However, at the
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DEVELOPMENTAL BIOLOGY
leading edge of this sheet of ceils, immature-appearing sequamoid and cuboidal cells, often in mitosis, were common. Villi were not seen along the epithelialized portion of the serosal side of the segments. Some degenerative changes involving both epithelium and mesenchyme were evident in approximately one-third of the 48- and 72-hr cultures. These were patchy in distribution and usually involved only a small portion of the cultured jejunal segment. These degenerative changes were characterized by decreased cytoplasmic staining, nuclear pycnosis and the presence of large, lysosome-like inclusions. Electron
VOLUME 45, 1975
lumnar epithelium were taller. In most epithelial cells the nuclei were basally located. The microvilli on the luminal surface were much more numerous (Fig. 4). The relative amount of granular endoplasmic reticulum was increased. Moreover,
Microscopy
The fine structure of the stratified jejunal epithelial cells from uncultured l&day fetal rats revealed only limited evidence of differentiation. Microvilli, approximately 0.1 pm in width and up to 0.6 pm in length were distributed sparsely along the luminal border (Figs. 2 and 3A) of the surface cells. There was no terminal web in the subapical cytoplasm. Nuclei were located more or less in the center of the cells. A small Golgi complex was often seen in the perinuclear cytoplasm. In some sections, this Golgi complex was located beneath the nucleus (Fig. 2); in other sections it was located above and along the side of the nucleus. A few mitochondria and occasional strands of granular endoplasmic reticulum were scattered throughout the cytoplasm (Figs. 2 and 3A). The remainder of the cytoplasm was filled with numerous unattached ribosomes. Cells not in contact with the gut lumen had similar structural features; however, most had no microvilli except for those cells that bordered secondary lumina. Sizable intercellular spaces were common between adjacent epithelial cells (Fig. 2). The fine structure of the epithelium of jejunal segments cultured for 24 hr appeared more mature than uncultured baseline tissue from 18-day fetuses. The individual cells which formed the simple co-
FIG. 2. Surface epithelial cells from uncultured jejunal mucosafrom an 18-day rat fetus. The cells are relatively undifferentiated with only a few microvilli (V) on the apical surface, a few strands of granular endoplasmic reticulum (R), a few mitochondria (M) and a small, infranuclear Golgi complex (G). The remaining cytoplasm contains numerous unattached rihosomes. x 9800.
DFBITIS,
FALCHUK
AND TRIER
Organ
Culture
of Fetal
Intestine
FIG. 3. (A), Luminal surface of uncultured jejunal mucosa from an 18-day fetal rat. Relatively few microvilli are present and there is no terminal web in the apical cytoplasm. (B), Jejunal epithelium which had been cultured for 48 hr from the same fetus as A. The microvilli are increased in number and there is now evidence of a terminal web (T) in the apical cytoplasm. (C). Jejunal epithelium which had been cultured for 72 hr from the same fetus as A. There is further development of the microvilli and the number of apical vesicles (arrows) is greater than in A. x 17,000.
the mitochondria in many of the cells were much larger (Fig. 4) than those in epithelial cells of uncultured jejunum from 1% day fetuses. A similar transient increase in mitochondrial size also occurs in uiuo in fetal rat jejunal epithelium during the 19th and 20th days of gestation (Mathan, M., Moxey, P.,, and Trier, J., unpublished
observations). Golgi cisternae were seen in the supranuclear and paranuclear cytoplasm but, in contrast to uncultured jejunum from 18-day fetuses, not in the infranuclear cytoplasm. Epithelial cells generally were closely opposed to one another with some interdigitation of lateral cytoplasmic processes of adjacent cells (Fig. 4).
310
DEVELOPMENTAL
BIOLOGY
FIG. 4. Apical two-thirds of an absorptive cell from a jejunal segment that had been cultured for 24 hr from the same fetus as in Fig. 2. The striking features are an increase in the number of microvilli (V) and in the size of the mitochondria (M). x 16,000.
Thus, the intercellular spaces between epithelial cells were consistently smaller than those of uncultured jejunum from M-day fetuses. The fine structural features of the epithelium of jejunal segments cultured for 48 and for 72 hr were very similar and will, therefore, be described jointly. There was
VOLUME 45, 1975
FIG. 5. Villous absorptive cells from a jejunal segment that had been cultured for 72 hr from the same fetus as Fig. 2. There is morphological evidence of significant maturation. The cells are now tall, columnar cells, the microvilli (V) are more abundant, the terminal web (T) is partially developed, the number of mitochondria and amount of granular endoplasmic reticulum are increased and there is a well-developed supranuclear Golgi complex (G). x 8200.
morphological evidence of further differentiation at both time intervals when compared to epithelial cell structure in 24-hr cultures. Absorptive cells were tall and
DERITIS,
FALCHUK
AND TRIER
FIG. 6. Higher magnification of the supranuclear region of an absorptive cell from a jejunal segment cultured for 72 hr illustrating the well-developed supranuclear Golgi complex (G). x 23,000.
columnar with basally located nuclei. Their apical surface was lined by more closely packed microvilli (Figs. 3B, C, and 5). In many cells, an incompletely formed but recognizable terminal web was apparent in the apical cytoplasm just beneath the microvilli. The number of small apical vesicles in the apical cytoplasm was generally greater than the number seen in the luminal epithelial cells of 18-day uncultured jejunum (compare Figs. 3A and C). However, these apical vesicles did not show evidence of the particles applied to the outer leaflet of the unit membrane which characterize the apical endocytic complex of near-term fetal rats (12) and of suckling rats (11, 31). Moreover, apical tubular profiles were uncommon. A well-developed Golgi complex was evident in the supranuclear and paranuclear cytoplasm (Figs. 5 and 6). The remaining cytoplasm contained mitochondria that were much more numerous and considerably smaller in size than those seen in the epithelium of 24-hr cultures, some lysosome-like structures
Organ Culture of Fetal Intestine
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primarily in the apical and supranuclear cytoplasm and many unattached ribosomes. Adjacent epithelial cells were generally very closely applied to one another and there was extensive interdigitation of cytoplasmic processes along their lateral cytoplasmic surfaces (Fig. 5). Striking elongation of some of the microvilli of a few absorptive cells was evident in many of the 48- and 72-hr cultures and in a few of the 24-hr cultures. Microvillous lengths as great as 4 pm were not uncommon (Fig. 7). Branching of the microvilli was often seen in association with the elongation (Fig. 7). Goblet cells were seen in segments from most of the fetuses cultured for 48 and 72 hr. They included immature forms that contained relatively few apical mucous granules as well as many mature-appearing cells characterized by many apical mucous granules (Fig. B), an abundant network of granular endoplasmic reticulum and a well-developed supranuclear Golgi complex. Endocrine epithelial cells were seen in many of the 48- and 72-hr cultures (Fig. 9) but not in 24-hr cultures. However, there appeared to be fewer identifiable endocrine epithelial cells in the 48- and 72-hr cultures than in uncultured jejunum from 20- and 21-day fetuses. Enzyme Studies The results of lactase, alkaline phosphatase and OCT determinations in cultured and in uncultured jujunal mucosa are summarized in Table 2. Mean lactase and alkaline phosphatase activities in the jejunum of baseline uncultured 18-day fetuses were 2.4 and 8 units, respectively. Following 24 hr of culture, mean lactase activity had increased to 4.1 units and mean alkaline phosphatase activity had increased to 25.9 units. After 72 hr of culture, there was a more than ninefold increase over baseline activities of lactase to 21.8 units and a more than sevenfold increase of alkaline
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DEVELOPMENTAL BIOLOGY
VOLUME 45, 1975
FIG. 7. Apical portion of villous absorptive cells from a jejunal segment cultured for 72 hr showing the striking elongation and branching (arrows) of microvilli seen in a few cells in many of the cultures. x 13,000.
phosphatase activity to 58.6 units (Table 2). The activities of lactase and alkaline phosphatase in jejunal segments cultured for 72 hr were 37 and 21%, respectively, of that found in uncultured jejunum from 21-day fetuses. No appreciable sucrase activity was found in either cultured or uncultured jejunum from any of the fetuses. Ornithine carbamoyl transferase activity in uncultured jejunum from 18-day fetuses averaged 49.8 units. During culture, specific activity of this enzyme increased progressively reaching 201 units after 72 hr of culture. The activity of OCT in segments cultured for 72 hr was 110% of that found in uncultured jejunum from 21-day fetuses.
DISCUSSION
Our observations indicate that jejunum from 18-day fetal rats can be cultured in uitro in a chemically defined medium for at least 72 hr. In general, tissue preservation was satisfactory. Mitoses were regularly seen, suggesting continued epithelial cell proliferation throughout the culture period. In some regions of some 48- and 72-hr cultures, the presence of autophagic vacuoles in epithelial and mesenchymal cells suggested that some cellular degeneration was taking place. These changes were spotty and unpredictable; at times they were evident in only one of three segments cultured in a single plate. There was evidence of significant differ-
D&ITS,
FALCHUK
AND TRIER
Organ Culture of Fetal Intestine
FIG. 8. Goblet cell cytoplasm from a jejunal segment cultured for 72 hr. The cell contains typical granules (X) and abundant elements of endoplasmic reticulum (R). x 24,000.
313
mucous
314
DEVELOPMENTAL BIOLOGY
FIG. 9. Endocrine epithelial cell from a jejunal (arrows) are seen in the cytoplasm. x 13,000.
VOLUME 45, 1975
segment cultured
TABLE
for 72 hr. Typical
secretory
granules
2
ENZYME ACTIVITY IN CULTURED AND UNCULTURED FETAL RAT JWUNUM Sample 18-Day fetuses 24-Hr cultures 48-Hr cultures 72-Hr cultures 19-Day fetuses 20-Day fetuses 21-Day fetuses a One unit * 1 unit = c 1 unit = d Mean +
(uncultured)
(uncultured) (uncultured) (uncultured)
Lactase unitsa 2.4 4.1 6.9 21.8 7.3 43.7 58.4
zt 0.P i 0.8 zt 0.6 zt 6.0 * 0.1 + 2.6 + 8
Alkaline
phosphatase units*
8.0 25.9 37.1 58.6 65.6 174.2 280
+ 0.7 * 7.4 * 6.6 + 3.6 * 0.5 + 32 zt 64
Ornithine carbamoyl transferase units’ 49.8 74.6 137 201 137 175 182
+ 8 * 17 * 40 * 33 zt 48 * 33 + 37
= 1 rmol of glucose liberated per gram of protein per minute. 1 rmol p-nitrophenol liberated per gram of protein per minute. 1 pmol citrulline formed per gram of protein per hour. SE of four to eight determinations
entiation of the isolated jejunal segments during the culture period. Whereas the jejunum of M-day rats is usually without villi and lined largely by undifferentiated stratified epithelium, in the majority of segments cultured for 24-72 hr, villi had appeared and the stratified epithelium had matured to a simple columnar epithelial lining. In addition to architectural matura-
tion of the jejunal mucosa, there was evidence of significant cytological differentiation of the individual epithelial cells during culture. The amount of cytoplasm occupied by unattached ribosomes decreased substantially. The number of microvilli along the luminal surface, the amount of Golgi material and formed elements of endoplasmic reticulum and the number of
DERITIS,
FALCHUK
AND TRIER
mitochondria increased strikingly. In many segments readily identifiable, well-differentiated goblet cells and endocrine cells were obvious. Finally, the specific activities of the brush border enzymes, alkaline phosphatase and lactase, increased more than seven- and ninefold, respectively, while the specific activity of the intracellular mitochondrial enzyme, ornithine carbamoyl transferase, increased more than fourfold during 72 hr of culture. These increased enzyme activities correlate well with the observed development of the brush border and proliferation of mitochondria in epithelial cells during culture. These findings indicate that fetal mammalian small intestine can undergo differentiation in vitro when cultured in a chemically defined medium in the absence of many of the hormonal and metabolic influences to which it is exposed both from maternal and extra-intestinal fetal tissues during normal in utero gestation. Such maturation and differentiation of fetal tissue cultured in defined medium is not without precedent. It is well established that other fetal organs such as pancreas and salivary glands show evidence of significant differentiation when cultured in vitro (2, 9, 10). It has also been documented that embryonic chick duodenum remains viable for several days when maintained in vitro in organ culture (7, 13, 25). In these studies, cultures were initiated at a time when villi had formed and goblet cells already were present in the chick embryos. However, Moog and Kirsch (25) and Dobbins and co-workers (7) noted that further elongation of duodenal villi and morphological evidence of maturation of absorptive cells occurred during 2-4 days of culture of intestine obtained from 16-day chick embryos. Although the degree of differentiation and maturation of isolated cultured jejunal segments in our studies was substantial, it was not identical to the differentiation which occurs in vivo in rat fetuses between the 18th and 21st days of gestation. First,
“f-gun Culture of Fetal Intestine
315
villi were shorter in cultured segments than in comparably aged fetuses. Second, the apical endocytic complex of tubules and pinocytotic vesicles, which is at least partially developed in the intestinal epithelium of 20- and 21-day fetal rats (12, 26), was not obvious in segments of jejunum from 18-day fetuses which had been cultured for 48 and 72 hr, although the number of apical vesicles increased modestly during culture. It is possible that the paucity of macromolecules such as protein and glycoprotein in the chemically defined medium interfered with the development of this structural feature. Moreover, it is possible that the endocytic complex may have developed in culture had fetal ileum rather that jejunum been selected for culture. Third, endocrine epithelial cells, though present, seemed less numerous in cultures than in the jejunum of 20- and 21-day fetuses. Fourth, the specific activities of the brush border enzymes, lactase and alkaline phosphatase, increased less in culture than during a comparable time period of fetal development in utero. It was of interest that strikingly elongated and branched microvilli were seen on the apical surface of some of the epithelial cells of cultured fetal rat jejunum (Fig. 7). Recently, Burgess and Grey (4) reported similarily elongated and branched microvilli in the epithelium of ll-day chick duodenum cultured on chorioallantoic membrane of host chick embryos in the presence of cytochalasin B but not in its absence. The mechanism responsible for the elongation of microvilli and the reason for their patchy distribution in our cultures is not clear. However, since our culture system did not contain cytochalaain B, our observations indicate that elongation and branching of microvilli in cultured fetal small intestine is not a specific response to cytochalasin B. In conclusion, it is hoped that the organ culture system described in this report may provide a useful model for the study of factors that may regulate and influence
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DEVELOPMENTAL BIOLOGY
organogenesis and differentiation of the mucosa of mammalian small intestine. We are grateful for the valuable technical assistance provided by Elaine Markezin, Pamela C. Moxey, and Johanna Rogers. This study was supported by National Institutes of Health Grants No. AMDD 17537 and AMDD 17684 and a grant from the John A. Hartford Foundation, Inc. Dr. Falchuk was supported in part by a Nelson E. Weeks-Medical Foundation of Boston fellowship. REFERENCES 1. BESSEY, 0. A., LOWRY, 0. H., and BROCK, M. J. (1946). A method for the rapid determination of alkaline phosphatase with five cubic milliletens of serum. J. Biol. Chem. 164, 321-329. 2. BORGHESE, E. (1950). Explantation experiments on the influence of the connective tissue capsule on the development of the epithelial part of the submandibular gland of Mus musculus. J. Anat. 84, 303-318. 3. BROWNING, T. H., and TRIER, J. S. (1969). Organ culture of mucosal biopsies of human small intestine. J. Clin. Invest. 48, 1423-1432. 4. BURGESS, D. R., and GREY, R. D. (1974). Alterations in morphology of developing microvilli elicited by cytochalasin B. J. Cell Biol. 62, 566-574. 5. CERIOTTI, G., and GAZZANIGA, A. (1966). A sensitive method for serum ornithine carbamoyl transferase determination. Clin. Chim. Acta 14, 57-62. 6. DAHLQVIST, A. (1968). Assay of intestinal disaccharidases. Anal. Biachem. 22, 99-107. 7. DOBBINS, W. O., III, HUMANS, J. C., and MCCARTHY, K. S. (1967). A light and electron microscopic study of duodenal epithelium of chick embryos cultured in the presence and absence of hydrocortisone. Gastroenterology 53, 557-574. 8. DOLIN, R., BLACKLOW, N. R., MALMGREN, R. A., and CHANOCK, R. M. (19701. Establishment of human fetal intestinal organ cultures for growth of viruses. J. Infect. Dis. 122, 227-231. 9. GOLOSOW, N., and GROBSTEIN, C. (1962). Epithelio-mesenchymal interaction in pancreatic morphogenesis. Develop. Biol. 4, 242-255. 10. GROBSTEIN, C. (1953). Analysis in vitro of the early organization of the rudiment of the mouse submandibular gland. J. Morphol. 93, 19-34. 11. Hayward, A. F. 1967. Changes in fine structure of developing intestinal epithelium associated with pinocytosis. J. Anat. 102, 57-70. 12. HERMOS, J. A., MATHAN, M., and TRIER, J. S. (1971). DNA synthesis and proliferation by villous epithelial cells in fetal rats. J. Cell Biol. 50, 255-258.
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13. HIJMANS, J. C. (19721. Effect of colchicine and hydrocortisone on invertase activity in the duodenum of the chick embryo. Gastroenterology 63, 279-287. 14. HOORN, B. (1966). Organ cultures of ciliated epithelium for the study of respiratory viruses. Acta Path. Microbial. &and. 66, Suppl. 183, l-37. 15. JOHNSON, L. R., and GUTHRIE, P. D. (1974). Mucosal DNA synthesis: A short term index of the trophic action of gastrin. Gastroenterology 67, 453-459. 16. KAGNOFF, M. F., DONALDSON,R. M., WINTRIER, J. S. (1972). Organ culture of rabbit small intestine: Prolonged in vitro steady state protein synthesis and secretion and secretory IgA secretion. Gastroenterology 63, 541-551. 17. KAMMARAAD, A. (1942). The development of the gastro-intestinal tract of the rat. J. Morphol. 70, 323-342. 18. KOLDOVSK?, 0. (19691. Digestion and absorption of carbohydrates. In “Development of the Functions of the Small Intestine in Mammals and Man,” pp. 25-95. Karger, Basel. 19. LEIBOVITZ, A. (1963). The growth and maintenance of tissue cell cultures in free gas exchange with the atmosphere. Amer. J. Hyg. 78,173-180. 20. LICHTENBERGER, L. M., MILLER, L. R., ERWIN, D. N., and JOHNSON, L. R. (1973). Effect of pentagastrin on adult rat duodenal cells in culture. Gastroenterology 65, 242-251. 21. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 22. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytel. 9, 409-414. 23. MATHAN, M., HERMOS, J. A., and TRIER, J. S. (1972). Structural features of the epitheliomesenchymal interface of rat duodenal mucosa during development. J. Cell Biol. 52, 577-588. 24. Moot, F., BIRKENMEIER, E. H., and GLAZIER, H. S. (1971). Leucylnaphthylamidase in the small intestine of the mouse: Normal development and influence of cortisone and antibiotics. Develop. Biol. 25, 398-419. 25. Moot, F., and KIRSCH, M. H. (19551. Quantitative determination of phosphatase activity in chick embryo duodenum cultured in fluid media with and without hydrocortisone. Nature (London) 175, 722-723. 26. ORLIC, D., and LEV, R. (1973). Fetal rat intestinal absorption of horseradish peroxidase from swallowed amniotic fluid. J. Cell Biol. 56, 106-119. 27. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. 28. RICHARDSON, K. C., JARETT, L., and FINKE, E. H.
D&ITIS, FALCHUKAND TRIER (1960). Embedding in epoxy resin for ultrathin sectioning in electron microscopy. Stain Technol. 35, 313-323. P. J. (1968). The effects of pH on the 29. SNODGRASS,
kinetics of human liver ornithine-carbamoyl phosphate transferase. Biochemistry 7, 30473051.
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30. WATSON,M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. Biophys.
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31. WISSIG,S. F., and GRANEY,D. 0. (1968). Membrane modifications in the apical endocytic complex of ileal epithelial cells. J. Cell Biol. 39, 564-579.
45, 304-317 (1975)
BIOLOGY
Differentiation GIORCIO Department
of Medicine,
and Maturation DERITIS, Harvard
of Cultured
Z. MYRON
Medical
FALCHUK,
Fetal Rat Jejunum
AND JERRY
S. TRIER
School and Peter Bent Brigham Hospital, 02115 Accepted
March
Boston, Massachusetts
24, 1975
To determine whether differentiation and maturation of mammalian intestinal mucosa require influences available only in vivo or whether they can occur in vitro, fetal rat jejunum was cultured in chemically defined medium using organ culture methodology. Segments of jejunum from B-day fetal rats were cultured in modified Liebowitz L-15 medium in room air at 37°C. Segments harvested after 24, 48, and 72 hr of culture were examined by light and electron microscopy. Uncultured jejunum from B-day fetuses had either no or very few rudimentary villi and was lined largely by undifferentiated stratified epithelium. Goblet cells were not seen. In contrast, villi were present in the majority of 24-hr cultures, and simple columnar rather than stratified epithelium predominated. After 48 and 72 hr, villi were present in over 90% of cultured jejunal segments and stratified epithelium had disappeared. Goblet cells were seen in jejunal segments cultured 48 hr or longer in 47 of 74 fetuses. Electron microscopy further documented progressive differentiation of the epithelium during culture. Microvilli increased in number and height, a terminal web developed in the apical cytoplasm and the number of apical vesicles, mitochondria and formed elements of endoplasmic reticulum increased in absorptive cells. Jejunal lactase and alkaline phosphatase activity increased nine- and sevenfold, respectively, during 72 hr of culture, while the activity of the mitochondrial enzyme, ornithine carbamoyl transferase, increased fourfold. These observations indicate that jejunum from 18-day fetal rats can be cultured in vitro for at least 72 hr in chemically defined medium and that, during culture, maturation of the jejunal mucosa takes place with the appearance of villi, conversion of stratified to columnar epithelium and differentiation of individual epithelial cells. INTRODUCTION
During the last 4 days of the 22-day gestation period of fetal rats, growth and differentiation of the small intestine are rapid (12, 17, 23). The mucosal surface of the jejunum of 18-day fetuses is lined with a layer of stratified, relatively undifferentiated epithelial cells and villi are absent. During the next 4 days of gestation, villi form and greatly increase the mucosal surface area. As the villi develop, the stratified epithelial lining is replaced by a single layer of more differentiated columnar epithelial cells. There is evidence that this morphological differentiation is accompanied by functional maturation. The small intestine of fetal rats can absorb and transport protein molecules such as horseradish peroxidase as early as 2-3 days before birth (26). Additionally, the activity of digestive enzymes in the brush border of 304 Copyright All rights
0
1975 by of reproduction
Academic Press, Inc. in any
form
reserved.
the intestinal absorptive cells of mice and rats, such a leucylnaphthylamidase and lactase, increases markedly during the last few days of gestation (18,24). We have recently documented that mucosa of the small intestine from adult mammals can be maintained in vitro for up to 48 hr by using organ culture methology (3, 16). To’ determine whether the rapid maturation and differentiation of rat small intestine, which occur during the few days prior to birth, require influences available only in uivo in the uterine environment or whether such maturation can occur in uitro, we prepared organ cultures of isolated segments of 18-day fetal rat jejunum which we then examined after l-3 days of in vitro culture. MATERIALS
Timed-pregnant were anesthetized
AND
METHODS
Sprague-Dawley rats with ether on the 18th
DF&TIS,
FALCHUK
AND TRIER
day of gestation. Using sterile technique, the 18-day fetuses were removed at laparotomy, the fetal jejunum was exposed, cut into seven to ten segments approximately 2-3 mm in length and placed into a petri dish containing Leibovitz L-15 medium (19). One segment was fixed immediately for baseline morphological studies and the others were cultured using the method of Hoorn (14) as modified by Dolin et al. (8). Briefly, two or three segments of jejunum were placed into sterile plastic culture plates, 5.5 cm in diameter. The plates had previously been scored with a scalpel blade. Leibovitz L-15 medium (19), 1.25 ml, supplemented with a final concentration of 0.2% bovine albumin, 2.0 mM glutamine, 100 U/ml of crystalline penicillin G and 100 pg/ml of streptomycin was added to each culture dish. In addition 0.5 pg/ml of pentagastrin was added because of its recently demonstrated trophic effect on intestinal epithelium (15, 20). The dishes were cultured at a temperature of 36.5”C in room air for 24, 48, and 72 hr on a rocker platform that completed one full cycle per minute. Whenever sufficient tissue was available, tissue from the same fetus cultured for all three time periods was examined. In some instances, tissue cultured for only two of the three time periods was available for comparison to baseline segments. Baseline and cultured jejunal segments were fixed by immersion for 90 min in chilled 2.5% glutaraldehyde buffered to pH 7.4 in 0.1 M cacodylate buffer containing 0.05% calcium chloride. After repeated rinsing in 0.1 M cacodylate buffer for 90 min, tissues were placed in chilled 1% osmium tetroxide buffered to pH 7.4 in 0.1 M cacodylate buffer. Tissues were then rapidly dehydrated in graded concentrations of ethanol and embedded in epoxy resin using Luft’s (22) method. The embedded tissues were then carefully oriented and mounted on aluminum rods for sectioning. Sections of the entire block were then cut 1 pm thick with a Sorvall
Organ Culture of Fetal Intestine
305
MT-2 microtome, mounted on glass slides and stained with Richardson’s stain (28). For electron microscopy, appropriate areas were selected and the blocks were trimmed. Sections were cut with diamond knives, mounted on uncoated copwith uranyl per mesh grids, stained acetate (30) and lead citrate (27) and examined with a Philips EM-300 electron microscope. To determine the influence of culture of fetal intestine on jejunal alkaline phosphatase, lactase, sucrase, and ornithine carbamoyl transferase (OCT) activities, the uncultured jejunum of 18-day fetuses and jejunal segments cultured for 24,48, and 72 hr were homogenized in 1 ml of 0.9% sodium chloride. An aliquot of each homogenate was used for determination of tissue protein (21); the remainder was stored at -85°C until the enzyme assays were performed. Lactase and sucrase activities were determined by a modification of the method of Dahlqvist (6). Aliquots of homogenates were added to tubes containing 0.056 M lactose or 0.056 M sucrose in 0.1 M maleate buffer at pH 6.0 and incubated at 37°C in a shaking water bath for l-3 hr. The reaction was stopped by placing the tubes into a boiling water bath. A unit of disaccharidase activity represented one micromole of glucose liberated per minute per gram of tissue protein. Alkaline phosphatase was determined by the method of Bessey et at. (1). Homogenate was added to tubes containing 200 ~1 of 0.15 mM p-nitrophenyl phosphate in 0.0625 M glycine buffer at pH 10.3 to which 0.625 mM MgCl, and 0.125 mM Zn acetate had been added. Following incubation at 37°C for lo-30 min, the reaction was stopped by addition of 1 ml of 0.02 N NaOH. A unit of phosphatase activity represented one micromole of p-nitrophenol liberated per minute per gram of tissue protein. OCT was measured according to the method of Snodgrass (29). Homogenates were added to tubes containing 7.5 mM
306
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DEVELOPMENTAL BIOL~CY
ornithine-HCl in 0.27 M triethanolamine (TEA), pH 7.7, and the reaction was started by addition of 0.0171 M carbamoyl phosphate in 0.27 M TEA. After 15-30 min of incubation at 37”C, the reaction was stopped by adding 5% trichloracetic acid-0.5% phosphotungstic acid. Following centrifugation at 5000g for 30 min, the citrulline liberated in the supernatant fluid was measured using the method of Ceriotti (5). A unit of OCT activity represents one micromole of citrulline formed per hour per gram of tissue protein.
cytoplasm (Fig. 1A). Secondary lumina (up to 30 pm in diameter) were often observed in the deeper layers of the epithelium (17). In the remaining fetuses, including those that had developed rudimentary villi, islands of simple cuboidal and columnar epithelium were seen interspersed among the more abundant stratified epithelium. No recognizable goblet or endocrine cells were seen in the jejunal epithelium of any of the 18-day old fetuses. In most jejunal segments harvested after 24 hr of culture, there was morphological evidence of maturation of the mucosal RESULTS architecture (Table 1). Small villi were present in segments from 15 of 25 fetuses Light Microscopy (Fig. 1B). The epithelial lining consisted The results of the light microscopic ob- exclusively of simple columnar epithelium servations are summarized in Table 1. in segments cultured for 24 hr from 21 Villi were not seen in jejunal segments fetuses. In cultured jejunum from 4 fetuses, from 39 of 45 l&day fetuses prior to cul- islands of stratified epithelium persisted ture. In the jejunum of the other 6 l&day and were interspersed among more abunfetuses, a few very small rudimentary villi dant areas of columnar epithelium. Goblet were observed. Usually only one and never cells could be identified in jejunal segmore than two such villi were seen per ments from 4 of 25 fetuses. jejunal cross-section. These villi consisted There was morphological evidence of of aggregates of mesenchyme which pro- further maturation of the jejunal mucosa in jected into the small jejunal lumen and segments harvested after 48 and 72 hr of which were lined by a mixture of stratified culture. Villi were present in over 90% of and immature-appearing simple columnar the fetal segments cultured for these longer epithelial cells. In 18 of 45 fetuses, only periods (Table 1). These villi were consiststratified epithelium, 3-12 cells thick, was ently taller and more developed (Fig. 1C) observed lining the jejunum (Fig. 1A). The than those seen after 24 hr of culture. Their individual cells of the stratified epithelium epithelial lining was composed of fairly showed little differentiation. Cell width tall, columnar cells, Mitotic figures were often approximated cell height and nuclei regularly observed and were most abunwere large in relation to the amount of dant along the lower half of the villi. TABLE
1
MORPHOLOGICAL FEATURES OF MATURATION Sample Present l&Day fetuses (45)” 24-Hr cultures (25Y 46.Hr cultures (39)’ 7%Hr cultures (35F’ a Number * Number
Epithelium
Villi
6 15 36 32
of fetuses examined. of fetal jejuna cultured.
Absent 39 10 3 3
Stratified
Columnar
18 0 0 0
0 21 39 35
Goblet cells Mixed 27 4 0 0
Present 0 4 22 25
Absent 45 21 17 10
DERITIS, FALCHUK AND TRIER
Organ. Culture
of Fetal
Intestine
FIG. 1. (A), Jejunal mucosa from an l&day rat fetus consisting of stratified, relatively undifferentiatedappearing epithelial cells. (B), Mucosa from a jejunal segment which had been cultured for 24 hr from the same fetus as A composed of small but definite villi and lined primarily by columnar epithelium. (C), Mucosa from a jejunal segment which had been cultured for 72 hr from the same fetus as A with well-formed villi lined by a single layer of relatively differentiated-appearing columnar epithelial cells. Richardson’s stain; x 440.
Stratification of the epithelium was not seen in any of the segments cultured for 48 or more hr. Goblet cells, sufficiently well differentiated so that they could be identified with the light microscope, were present in segments cultured 48 hr or longer from 47 of 74 fetuses. Occasionally, epithelial endocrine cells could be recognized with the light microscope. In many jejunal segments cultured for 24 hr and in most segments cultured 48 or 72
hr, a layer of simple columnar epithelial cells extended along the serosal surface, especially near the cut edge of the segments. Presumably, epithelization of the serosal surface was initiated by proliferation of the epithelial cells at the extreme lateral edge of the cultured segments. The light microscopic appearance of most of these cells was similar to that of the epithelial cells which lined the lumen of the cultured segment. However, at the
308
DEVELOPMENTAL BIOLOGY
leading edge of this sheet of ceils, immature-appearing sequamoid and cuboidal cells, often in mitosis, were common. Villi were not seen along the epithelialized portion of the serosal side of the segments. Some degenerative changes involving both epithelium and mesenchyme were evident in approximately one-third of the 48- and 72-hr cultures. These were patchy in distribution and usually involved only a small portion of the cultured jejunal segment. These degenerative changes were characterized by decreased cytoplasmic staining, nuclear pycnosis and the presence of large, lysosome-like inclusions. Electron
VOLUME 45, 1975
lumnar epithelium were taller. In most epithelial cells the nuclei were basally located. The microvilli on the luminal surface were much more numerous (Fig. 4). The relative amount of granular endoplasmic reticulum was increased. Moreover,
Microscopy
The fine structure of the stratified jejunal epithelial cells from uncultured l&day fetal rats revealed only limited evidence of differentiation. Microvilli, approximately 0.1 pm in width and up to 0.6 pm in length were distributed sparsely along the luminal border (Figs. 2 and 3A) of the surface cells. There was no terminal web in the subapical cytoplasm. Nuclei were located more or less in the center of the cells. A small Golgi complex was often seen in the perinuclear cytoplasm. In some sections, this Golgi complex was located beneath the nucleus (Fig. 2); in other sections it was located above and along the side of the nucleus. A few mitochondria and occasional strands of granular endoplasmic reticulum were scattered throughout the cytoplasm (Figs. 2 and 3A). The remainder of the cytoplasm was filled with numerous unattached ribosomes. Cells not in contact with the gut lumen had similar structural features; however, most had no microvilli except for those cells that bordered secondary lumina. Sizable intercellular spaces were common between adjacent epithelial cells (Fig. 2). The fine structure of the epithelium of jejunal segments cultured for 24 hr appeared more mature than uncultured baseline tissue from 18-day fetuses. The individual cells which formed the simple co-
FIG. 2. Surface epithelial cells from uncultured jejunal mucosafrom an 18-day rat fetus. The cells are relatively undifferentiated with only a few microvilli (V) on the apical surface, a few strands of granular endoplasmic reticulum (R), a few mitochondria (M) and a small, infranuclear Golgi complex (G). The remaining cytoplasm contains numerous unattached rihosomes. x 9800.
DFBITIS,
FALCHUK
AND TRIER
Organ
Culture
of Fetal
Intestine
FIG. 3. (A), Luminal surface of uncultured jejunal mucosa from an 18-day fetal rat. Relatively few microvilli are present and there is no terminal web in the apical cytoplasm. (B), Jejunal epithelium which had been cultured for 48 hr from the same fetus as A. The microvilli are increased in number and there is now evidence of a terminal web (T) in the apical cytoplasm. (C). Jejunal epithelium which had been cultured for 72 hr from the same fetus as A. There is further development of the microvilli and the number of apical vesicles (arrows) is greater than in A. x 17,000.
the mitochondria in many of the cells were much larger (Fig. 4) than those in epithelial cells of uncultured jejunum from 1% day fetuses. A similar transient increase in mitochondrial size also occurs in uiuo in fetal rat jejunal epithelium during the 19th and 20th days of gestation (Mathan, M., Moxey, P.,, and Trier, J., unpublished
observations). Golgi cisternae were seen in the supranuclear and paranuclear cytoplasm but, in contrast to uncultured jejunum from 18-day fetuses, not in the infranuclear cytoplasm. Epithelial cells generally were closely opposed to one another with some interdigitation of lateral cytoplasmic processes of adjacent cells (Fig. 4).
310
DEVELOPMENTAL
BIOLOGY
FIG. 4. Apical two-thirds of an absorptive cell from a jejunal segment that had been cultured for 24 hr from the same fetus as in Fig. 2. The striking features are an increase in the number of microvilli (V) and in the size of the mitochondria (M). x 16,000.
Thus, the intercellular spaces between epithelial cells were consistently smaller than those of uncultured jejunum from M-day fetuses. The fine structural features of the epithelium of jejunal segments cultured for 48 and for 72 hr were very similar and will, therefore, be described jointly. There was
VOLUME 45, 1975
FIG. 5. Villous absorptive cells from a jejunal segment that had been cultured for 72 hr from the same fetus as Fig. 2. There is morphological evidence of significant maturation. The cells are now tall, columnar cells, the microvilli (V) are more abundant, the terminal web (T) is partially developed, the number of mitochondria and amount of granular endoplasmic reticulum are increased and there is a well-developed supranuclear Golgi complex (G). x 8200.
morphological evidence of further differentiation at both time intervals when compared to epithelial cell structure in 24-hr cultures. Absorptive cells were tall and
DERITIS,
FALCHUK
AND TRIER
FIG. 6. Higher magnification of the supranuclear region of an absorptive cell from a jejunal segment cultured for 72 hr illustrating the well-developed supranuclear Golgi complex (G). x 23,000.
columnar with basally located nuclei. Their apical surface was lined by more closely packed microvilli (Figs. 3B, C, and 5). In many cells, an incompletely formed but recognizable terminal web was apparent in the apical cytoplasm just beneath the microvilli. The number of small apical vesicles in the apical cytoplasm was generally greater than the number seen in the luminal epithelial cells of 18-day uncultured jejunum (compare Figs. 3A and C). However, these apical vesicles did not show evidence of the particles applied to the outer leaflet of the unit membrane which characterize the apical endocytic complex of near-term fetal rats (12) and of suckling rats (11, 31). Moreover, apical tubular profiles were uncommon. A well-developed Golgi complex was evident in the supranuclear and paranuclear cytoplasm (Figs. 5 and 6). The remaining cytoplasm contained mitochondria that were much more numerous and considerably smaller in size than those seen in the epithelium of 24-hr cultures, some lysosome-like structures
Organ Culture of Fetal Intestine
311
primarily in the apical and supranuclear cytoplasm and many unattached ribosomes. Adjacent epithelial cells were generally very closely applied to one another and there was extensive interdigitation of cytoplasmic processes along their lateral cytoplasmic surfaces (Fig. 5). Striking elongation of some of the microvilli of a few absorptive cells was evident in many of the 48- and 72-hr cultures and in a few of the 24-hr cultures. Microvillous lengths as great as 4 pm were not uncommon (Fig. 7). Branching of the microvilli was often seen in association with the elongation (Fig. 7). Goblet cells were seen in segments from most of the fetuses cultured for 48 and 72 hr. They included immature forms that contained relatively few apical mucous granules as well as many mature-appearing cells characterized by many apical mucous granules (Fig. B), an abundant network of granular endoplasmic reticulum and a well-developed supranuclear Golgi complex. Endocrine epithelial cells were seen in many of the 48- and 72-hr cultures (Fig. 9) but not in 24-hr cultures. However, there appeared to be fewer identifiable endocrine epithelial cells in the 48- and 72-hr cultures than in uncultured jejunum from 20- and 21-day fetuses. Enzyme Studies The results of lactase, alkaline phosphatase and OCT determinations in cultured and in uncultured jujunal mucosa are summarized in Table 2. Mean lactase and alkaline phosphatase activities in the jejunum of baseline uncultured 18-day fetuses were 2.4 and 8 units, respectively. Following 24 hr of culture, mean lactase activity had increased to 4.1 units and mean alkaline phosphatase activity had increased to 25.9 units. After 72 hr of culture, there was a more than ninefold increase over baseline activities of lactase to 21.8 units and a more than sevenfold increase of alkaline
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DEVELOPMENTAL BIOLOGY
VOLUME 45, 1975
FIG. 7. Apical portion of villous absorptive cells from a jejunal segment cultured for 72 hr showing the striking elongation and branching (arrows) of microvilli seen in a few cells in many of the cultures. x 13,000.
phosphatase activity to 58.6 units (Table 2). The activities of lactase and alkaline phosphatase in jejunal segments cultured for 72 hr were 37 and 21%, respectively, of that found in uncultured jejunum from 21-day fetuses. No appreciable sucrase activity was found in either cultured or uncultured jejunum from any of the fetuses. Ornithine carbamoyl transferase activity in uncultured jejunum from 18-day fetuses averaged 49.8 units. During culture, specific activity of this enzyme increased progressively reaching 201 units after 72 hr of culture. The activity of OCT in segments cultured for 72 hr was 110% of that found in uncultured jejunum from 21-day fetuses.
DISCUSSION
Our observations indicate that jejunum from 18-day fetal rats can be cultured in uitro in a chemically defined medium for at least 72 hr. In general, tissue preservation was satisfactory. Mitoses were regularly seen, suggesting continued epithelial cell proliferation throughout the culture period. In some regions of some 48- and 72-hr cultures, the presence of autophagic vacuoles in epithelial and mesenchymal cells suggested that some cellular degeneration was taking place. These changes were spotty and unpredictable; at times they were evident in only one of three segments cultured in a single plate. There was evidence of significant differ-
D&ITS,
FALCHUK
AND TRIER
Organ Culture of Fetal Intestine
FIG. 8. Goblet cell cytoplasm from a jejunal segment cultured for 72 hr. The cell contains typical granules (X) and abundant elements of endoplasmic reticulum (R). x 24,000.
313
mucous
314
DEVELOPMENTAL BIOLOGY
FIG. 9. Endocrine epithelial cell from a jejunal (arrows) are seen in the cytoplasm. x 13,000.
VOLUME 45, 1975
segment cultured
TABLE
for 72 hr. Typical
secretory
granules
2
ENZYME ACTIVITY IN CULTURED AND UNCULTURED FETAL RAT JWUNUM Sample 18-Day fetuses 24-Hr cultures 48-Hr cultures 72-Hr cultures 19-Day fetuses 20-Day fetuses 21-Day fetuses a One unit * 1 unit = c 1 unit = d Mean +
(uncultured)
(uncultured) (uncultured) (uncultured)
Lactase unitsa 2.4 4.1 6.9 21.8 7.3 43.7 58.4
zt 0.P i 0.8 zt 0.6 zt 6.0 * 0.1 + 2.6 + 8
Alkaline
phosphatase units*
8.0 25.9 37.1 58.6 65.6 174.2 280
+ 0.7 * 7.4 * 6.6 + 3.6 * 0.5 + 32 zt 64
Ornithine carbamoyl transferase units’ 49.8 74.6 137 201 137 175 182
+ 8 * 17 * 40 * 33 zt 48 * 33 + 37
= 1 rmol of glucose liberated per gram of protein per minute. 1 rmol p-nitrophenol liberated per gram of protein per minute. 1 pmol citrulline formed per gram of protein per hour. SE of four to eight determinations
entiation of the isolated jejunal segments during the culture period. Whereas the jejunum of M-day rats is usually without villi and lined largely by undifferentiated stratified epithelium, in the majority of segments cultured for 24-72 hr, villi had appeared and the stratified epithelium had matured to a simple columnar epithelial lining. In addition to architectural matura-
tion of the jejunal mucosa, there was evidence of significant cytological differentiation of the individual epithelial cells during culture. The amount of cytoplasm occupied by unattached ribosomes decreased substantially. The number of microvilli along the luminal surface, the amount of Golgi material and formed elements of endoplasmic reticulum and the number of
DERITIS,
FALCHUK
AND TRIER
mitochondria increased strikingly. In many segments readily identifiable, well-differentiated goblet cells and endocrine cells were obvious. Finally, the specific activities of the brush border enzymes, alkaline phosphatase and lactase, increased more than seven- and ninefold, respectively, while the specific activity of the intracellular mitochondrial enzyme, ornithine carbamoyl transferase, increased more than fourfold during 72 hr of culture. These increased enzyme activities correlate well with the observed development of the brush border and proliferation of mitochondria in epithelial cells during culture. These findings indicate that fetal mammalian small intestine can undergo differentiation in vitro when cultured in a chemically defined medium in the absence of many of the hormonal and metabolic influences to which it is exposed both from maternal and extra-intestinal fetal tissues during normal in utero gestation. Such maturation and differentiation of fetal tissue cultured in defined medium is not without precedent. It is well established that other fetal organs such as pancreas and salivary glands show evidence of significant differentiation when cultured in vitro (2, 9, 10). It has also been documented that embryonic chick duodenum remains viable for several days when maintained in vitro in organ culture (7, 13, 25). In these studies, cultures were initiated at a time when villi had formed and goblet cells already were present in the chick embryos. However, Moog and Kirsch (25) and Dobbins and co-workers (7) noted that further elongation of duodenal villi and morphological evidence of maturation of absorptive cells occurred during 2-4 days of culture of intestine obtained from 16-day chick embryos. Although the degree of differentiation and maturation of isolated cultured jejunal segments in our studies was substantial, it was not identical to the differentiation which occurs in vivo in rat fetuses between the 18th and 21st days of gestation. First,
“f-gun Culture of Fetal Intestine
315
villi were shorter in cultured segments than in comparably aged fetuses. Second, the apical endocytic complex of tubules and pinocytotic vesicles, which is at least partially developed in the intestinal epithelium of 20- and 21-day fetal rats (12, 26), was not obvious in segments of jejunum from 18-day fetuses which had been cultured for 48 and 72 hr, although the number of apical vesicles increased modestly during culture. It is possible that the paucity of macromolecules such as protein and glycoprotein in the chemically defined medium interfered with the development of this structural feature. Moreover, it is possible that the endocytic complex may have developed in culture had fetal ileum rather that jejunum been selected for culture. Third, endocrine epithelial cells, though present, seemed less numerous in cultures than in the jejunum of 20- and 21-day fetuses. Fourth, the specific activities of the brush border enzymes, lactase and alkaline phosphatase, increased less in culture than during a comparable time period of fetal development in utero. It was of interest that strikingly elongated and branched microvilli were seen on the apical surface of some of the epithelial cells of cultured fetal rat jejunum (Fig. 7). Recently, Burgess and Grey (4) reported similarily elongated and branched microvilli in the epithelium of ll-day chick duodenum cultured on chorioallantoic membrane of host chick embryos in the presence of cytochalasin B but not in its absence. The mechanism responsible for the elongation of microvilli and the reason for their patchy distribution in our cultures is not clear. However, since our culture system did not contain cytochalaain B, our observations indicate that elongation and branching of microvilli in cultured fetal small intestine is not a specific response to cytochalasin B. In conclusion, it is hoped that the organ culture system described in this report may provide a useful model for the study of factors that may regulate and influence
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