Development of fetal rat intestine in organ and monolayer culture

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.     

Influences of dexamethasone on the maturation of fetal mouse intestinal mucosa in organ culture

The effects of glucocorticoids on the maturation of the fetal small intestinal mucosa have been studied using duodenal explants resected at 17 days of gestation and cultured in a serum-free medium in the presence or absence of dexamethasone (30-300 ng/ml). Dexamethasone (a) increases specifically alkaline phosphatase, maltase, trehalase and sucrase activities and (b) allows an accumulation of goblet cells along the villi at a faster rate than that occurring in utero. These results indicate that glucocorticoids influence directly the differentiation of absorptive cells and goblet cells in the small intestine during the fetal period.     

Permissive effect of glutamine on the differentiation of fetal mouse small intestine in organ culture

The proximal third of the small intestine of 15-day-old mouse embryo can be cultured for 72 h at 37 degrees C. When Trowell-T8 medium is used, the integrity of the explants is maintained, but villi do not form and absorptive cells are poorly differentiated. However, when Leibovitz L-15 or RPMI-1640 medium is used, one can observe the formation of medium-sized villi, and absorptive cells in the explants are more differentiated. Since the chemical composition of T8 medium is quite different from that of the other two media, we decided to test the importance of two major differences, i.e., three amino acids and five vitamins, in order to find out which element(s) is necessary to permit the formation of intestinal villi. Subsequent testing demonstrated that the three amino acids are responsible for the effect on differentiation, and that glutamine is the only critical difference between T8 and the two other media. The results show that the addition of L-glutamine to T8 medium permits the formation of villi, the initiation of absorptive cell differentiation, an increase in DNA synthesis, and finally, an increase in the number of epithelial cells. These findings indicate that undifferentiated fetal mouse small intestine is able to express its phenotype in organ culture, even without any extrinsic regulatory influences, provided that L-glutamine is present at a sufficient level in the culture medium. The use of inhibitors indicated that L-glutamine may be essential as an energetic substrate and/or a precursor for glucosamine.     

Role of thyroxine and insulin on the development of the fetal mouse duodenum in organ culture

The role of thyroxine and insulin in the regulation of proliferation and differentiation of the immature duodenal epithelium of the fetal mouse was investigated using an organ culture method with a serum-free medium. Thyroxine (10 nM) stimulates specifically the activity of maltase. Insulin (125 mU/mL) remains without effect on the maturation of all hydrolytic functions studied. Each hormone significantly increases the percentages of brush border enzyme activities liberated in the medium and reduces the amount of glucose released in the medium. In the presence of dexamethasone (76 nM) the effect of thyroxine on maltase activity is still observed. Finally, thyroxine and insulin do not modify the labelling index in the duodenal crypts of the explants in the presence or absence of dexamethasone. These findings indicate that thyroxine and insulin can act directly on the development of the fetal mouse duodenum at the end of gestation. Nevertheless, their implication in prenatal development of the gut functions appears to be of minor importance.     

Development of fetal thymocytes in organ culture: effect of corticosteroids.

Corticosteroids affect the development of fetal foregut-derived organs in which epithelial-mesenchymal interactions are associated with the developmental process. The thymus is one such organ and is profoundly sensitive to corticosteroids when mature. In this study corticosterone (CS) effects on fetal thymocyte development were investigated using a fetal thymus organ culture system which allows the growth, differentiation, and function of developing thymocytes to be monitored in vitro. CS inhibited, but did not block growth of fetal thymocytes, although the appearance of mature thymocytes was inhibited, similar to previously reported effects of interleukin 2 (IL2). CS enhanced the proportion of Mac1+, Ia+ and FcR+ cells and maintained high levels of IL2 receptor (IL2R) positive immature cells. Functional cytotoxic cells were detected in CS-treated organ cultures which expressed a Thy 1-, CD8- phenotype, atypical for thymus derived killer cells. While this cytotoxicity may be stimulated by CS, it could simply be due to a relative depletion of the main pool of thymocytes. These cytotoxic cells may have a role in directing apoptotic mechanisms occurring during thymocyte development.     

Development of fibroblast-type-II cell communications in fetal rabbit lung organ culture.

The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.     

Development of human fetal lung in organ culture compared with in utero ontogeny

Summary In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes. Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial immunoreactivity to cytokeratins CK 7,8, and 18 decreases with differentiation both in utero and in organ culture, whereas reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0±7.3 wk. In organ culture, capillary blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost synchronous in human lung in utero but clearly dissociate in organ culture.     

Culture of fetal mouse liver in plastic chambers: A new technique for organ culture

Summary A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO₂-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate the potential of this culture method for the study of physiological and pathological processes. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan and Science Technology Agency, Japan.     

Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Development of fetal rat ovaries responsiveness to LH during organ culture.

The responsiveness of fetal neonatal rat ovaries to LH was investigated in vitro using three complementary approaches. First, steroid production was assessed after culture. In control media, detectable levels of estrogens (estradiol and estrone) and progesterone were only observed from day 6 postpartum and during the second week of life respectively. In the presence of LH (100 ng/ml) ovaries produced both estrogens and progesterone from day 4 postpartum and the response to LH was enhanced with IBMX supplementation in the medium. Second, 3 beta-HSD activity was measured with either LH or (Bu)2 cAMP (1mM). Irrespective to the time-period studies (Bu)2 cAMP stimulated this enzyme whereas the stimulation with LH occurred only from day 5 postpartum Third, specific hCG binding was assessed and we found that it occurred only on days 7 and 10. However, when fetal ovaries were pretreated for 48 h with (Bu)2 cAMP, a specific hCG binding could be detected and progesterone production was enhanced in response to LH. An effect of the nucleotide via a stimulation of the neuraminidase activity did not seem to be involved. Lastly treatment of 18-day-old fetal ovaries with cholera toxin (10nM) or forskolin (1 microM) was found to stimulate progesterone production and VIP (0.1 to 1 microM) stimulated both the 3-HSD activity and the estradiol production. These data suggest that the absence of steroidogenic response to LH before day 4 postpartum could be explained by the absence of receptors, though the LH transmembrane signal-transduction system is functional in fetal ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Culture of fetal mouse liver in plastic chambers: a new technique for organ culture.

A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO2-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate that potential of this culture method for the study of physiological and pathological processes.     

Changes in the properties of fetal rat liver during organ culture

Summary Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine, aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro. This investigation was supported by grants from the National Institute of Child Health and Human Development (RO 1 HD09715), National Cancer Institute (CA 14194), and United States Public Health Service General Research Support Grant RR 5589.     

Long-term organ culture of mouse mammary gland

Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin, aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues.     

Effect of cortisol on human fetal lung in organ culture

Human fetal lung tissue obtained during the second trimester was cultured as organ culture with or without cortisol. The effect of cortisol on the phospholipid metabolism, as related to the appearance of osmiophilic lamellar bodies and the localisation of newly incorporated choline, was studied. In cortisol-treated explants, the concentration of saturated lecithins and the incorporation of (Me-3H)-choline into saturated lecithins increases significantly concomitantly with an increased number of osmiophilic lamellar bodies. The labelled choline is predominantly associated with these bodies. The obtained results indicate that cortisol accelerates the synthesis of pulmonary surfactant in the human fetal lung as early as in the second trimester.     

Optimization of fetal lung organ culture for surfractant biosynthesis

Summary Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical diffeentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studies byelectron microscopy tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O₂-CO₂ instead of air-CO₂ for gassing the explants slighlty decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle’s minimum essential and its Dulbeccco’s modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same peiod. Ham’s F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.     

Development of porcine embryos from the one-cell stage to blastocyst in mouse oviducts maintained in organ culture

Successful development of porcine embryos from the one-cell stage to the blastocyst stage has been accomplished using mouse oviducts in organ culture. One-cell embryos were transferred to mouse oviducts maintained in organ culture and were cultured for 6 days. Control embryos from each donor pig were cultured in a modified Krebs-Ringer bicarbonate medium. Thus control and experimental embryos obtained from the same individual pig could be directly compared. At the end of the culture period, all embryos were scored for the stage of development attained and stained to allow the cell number of each embryo to be counted. In medium alone, only 35.7% of the one-cell embryos reached the morula or blastocyst stage, whereas 78.1% of the one-cell embryos transferred to mouse oviducts reached the morula or blastocyst stage. Of those embryos reaching the morula or blastocyst stage, cell numbers were similar for the two treatments (medium alone vs. oviduct culture). The procedure described for mouse oviduct organ culture provides a simple method for culturing early-stage pig embryos to the morula or blastocyst stage prior to embryo transfer.     

Toxicity of gentamicin in organ culture of fetal rat intestine.

The effect of gentamicin in the culture of fetal rat intestine was studied. Fetal rat intestine was cultured with gentamicin or kanamycin at the concentration between 4 to 200 micrograms/ml. Kanamycin did not have influence on lactase, maltase and ALP activities. On the other hand, gentamicin caused decrease of lactase and ALP activities at the concentration of 40 and 200 micrograms/ml compared with the activities at 4 micrograms/ml. Maltase activities did not decrease with gentamicin. Our data suggest that gentamicin could affect lactase and ALP activities and lower concentration should be used in the culture.