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.     

Metanephric development in serum-free organ culture

Summary A new mouse metanephric organ culture system has been developed to study mammalian renal development. The system permits in vitro organotypic differentiation in a serum-free, hormone supplemented medium consisting of Dulbecco’s minimal essential medium (MEM) and Ham’s F12 medium supplemented with insulin, 5 μg/ml; PGE₁, 25 ng/ml; T₃, 3.2 pg/ml; hydrocortisone, 5 μg/ml; and transferrin, 5 μg/ml. In this system, metanephric development continues morphologically beyond the S-shaped tubule stage. A well differentiated proximal tubule forms with a well defined brush border, specialized intercellular connections, and an apical endocytic network. In addition, a unique devascularized glomerulus, with highly differentiated podocytes surrounding areas of basement membrane, forms entirely from epithelial elements. The present organ culture model goes beyond the limitations of previously described systems in that it does not require separation of nephrogenic blastema from ureteric bud, nor require animal serum or nonspecific tissue extracts for metanephric development. The model is thus suited for morphological, biochemical, and endocrinological study of normal and abnormal renal organogenesis.     

Growth factor requirements of organogenesis in serum-free metanephric organ culture

Summary In order to define humoral growth factors which may regulate mammalian renal development, the growth requirements of fetal metanephric organogenesis were studied in serum-free murine organ culture. Metanephric growth, determined by cell proliferation and protein content, and metanephric differentiation, determined morphometrically as epithelial glomerular formation, were compared and contrasted following 144 hours of organ culture incubation in basal medium, basal medium supplemented with 10% fetal bovine serum, and basal medium supplemented with various combinations of growth factors. The basal medium was composed of equal volumes of Dulbecco's modified Eagle's medium and Hams' F-12 medium. Five humoral growth factors were studied in the following concentrations: selenium, 6.8×10⁻⁹ M; insulin, 8.3×10⁻⁷ M; triiodothyronine, 2×10⁻⁹ M; transferrin, 6.2×10⁻⁸ M; and prostaglandin E₁, 7.1×10⁻⁸ M. Results showed that transferrin and prostaglandin E₁ were necessary for optimal growth in the system and that prostaglandin E₁ was necessary for maximal metanephric differentiation. Such data provide guidelines for the creation of serum-free medium for future fetal renal cell and tissue culture systems, and provide insight into the factors which may regulate normal and abnormal renal embryogenesis and the reparative processes of renal hyperplasia and hypertrophy which follow renal injury. A preliminary report of this work was presented at the Ninth International Congress of Nephrology, Los Angeles, June 1984. These studies were supported in part by Basil O'Connor Starter Research Grant 5-349 from the March of Dimes Birth Defects Foundation and New Investigator Research Award I-R23-AM34891-01 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases of the National Institutes of Health (Both to Dr. Avner). Editor's Statement The determination of effects of extracellular components on the introduction and maintenance of differentiation is an area for which serum-free culture techniques are particularly suited. The approaches described in this report utilize morphometric techniques to quantitate differentiation in serum-free fetal organ culture in addition to standard methodologies for assessing growth. The purely epithelial nature of the cultures used in these studies also provides some interesting advantages in the design of experiments aimed, at determining the importance of cell-cell interactions at various stages in the differentiative process. David W. Barnes     

Cell injury and regeneration of human epithelium in organ culture

Human esophageal, tracheal, and pancreatic ductal fragments were collected at autopsy after a postmortem interval of 12 hours or less and maintained in explant organ culture for 30 days. The viability and growth of the explants was assessed by morphology, LDH enzyme release, and cellular outgrowth. The viability and growth of the bronchial explant epithelium was directly related to the postmortem interval. Esophageal epithelial regeneration followed the desquamation of the superficial cell layers. Pancreatic epithelia appeared to grow more slowly and with less outgrowth than the other tissues. Epithelial cell growth along the explant surface and onto the culture dish appeared to proceed through the well-characterized process that follows cell injury, i.e., flattening, migration, replication, and differentiation. Thus, sufficient numbers of viable epithelial cells capable of regeneration were present in routine autopsy epithelium, but there was considerable variation from tissue to tissue and case to case. The most effective and accurate approach to follow when evaluating and predicting the growth and viability of these explants is by using a combination of morphologic, enzymatic and biologic assays. Errors in the interpretation of viability are possible when only one assay method is utilized. These tissues grown in explant organ culture are suitable for studies on the mechanism and response of epithelia to cell injury, recovery and wound healing.Abbreviations 4F-1G 4% formaldehyde, 1% glutaraldehyde - HIFBS heat inactivated fetal bovine serum - IA immediate autopsy - LDH lactate dehydrogenase - OsO₄ osmium tetroxide - RA routine autopsy     

Effects of culture conditions on the synthesis of human chorionic gonadotropin by placental organ cultures

Summary Culture conditions for maintaining first trimester human placenta in organ culture, which enhance the secretion of human chorionic gonadotropin (hCG), are described. Nutrient medium, oxygen tension and Gelfoam support matrix infuence the synthesis of hCG by these cultures. Placental tissue remained viable for the duration of experiments (12 days) as judged by the incorporation of tritiated thymidine into DNA and the lack of release of incorporated [¹²⁵Iiododeoxyuridine. Optimal conditions for hCG synthesis in placental organ culture included an atmosphere of 95% air and 5% CO₂ (approximately 20% O₂), CMRL 1066 medium containing fetal human or bovine serum, insulin, hydrocortisone and retinal acetate. Multiple pieces of placenta could be cultured in the same dish with an additive effect on hCG secretion. The functional responsiveness of these placental cultures was demonstrated by modulation of hCG synthesis with theophylline and 3′5′ dibutyryl cyclic AMP. Presented in part at the meeting of the American Association for Cancer Research, April 1978. This work is being submitted in partial fulfillment of the Ph.D. requirements in the Department of Biology, Catholic University of America.     

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Karyology of primary human fetal cell cultures

Summary Cell cultures were established from the biopsies of lung, skin and kidney from each of nine human fetuses, and chromosome analyses were performed on material through the fifth subculture. Kidney cell cultures generally showed a higher level of polyploidy than lung or skin. The frequencies of hyperdiploid cells and those with structural abnormalities were consistent with the low levels found in cultures of human lymphocytes. The data provide a normal cytogenetic baseline for human fetal material which may be useful in a variety of studies. Supported by Food and Drug Administration contracts FDA 74-51 and 74-52. Authors are listed alphabetically.     

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.     

Evaluation of metanephric maturation in a human fetal kidney explant model

Summary We have developed a unique human fetal kidney explant model to study the role of the insulinlike growth factor (IGF) system in metanephric development. Kidneys from 10–18 wk gestation human abortuses were maintained in serum-free conditions and defined medium, which was shown to support the induction and differentiation of the viable metanephric blastema. Histologically the tissue remained viable to 192 h of serum-free culture, while metanephric differentiation, reflected by a shrinking nephrogenic zone and the formation of maturing S-shape and glomerular forms, was accelerated and occurred between 48 and 96 h. In the nephrogenic zone, a significant decrease in IGF-II gene expression occurred, which reflected the differentiation of the metanephric blastema cell mass. IGF-II expression persisted, however, in the expanded interstitial mesenchyme. With differentiation over 48 h an increase in IGFBP-2 and WT1 gene expression by Northern blot analysis occurred, and was localized by in situ hybridization to the differentiating glomerular epithelial cell mass. Analysis of the explant-conditioned media by Western ligand blot demonstrated an increase in the rate of IGF binding protein (IGFBP)-2 peptide production by the differentiating explant, consistent with an increase in IGFBP-2 gene expression and with metanephric differentiation. This pattern of temporal and spatial gene expression closely approximates that of normal in vivo fetal renal development and of glomerular epithelial cell differentiation.     

Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney

Summary Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture. The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM),d-valine-containing modified alpha-MEM (mALPHA) andl-valine mALPHA. The mean number of cumulative population doublings (CPDL) was significantly (P<0.001) enhanced with thel-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) ord-valine mALPHA (18.3 CPDL) media. In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures. Surface-associated fibronectin was absent in these cell cultures. AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in thel-valine mALPHA. The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells. The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation. Research was supported by a NIEHS (ES 3101) grant to S. M. D’Ambrosio and a NCI grant (CA21104) to J. E. Trosko.     

Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13-18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 degrees C in a mixture of 95% air - 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished gamma-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained 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.     

Anin vitro,serum-free organ culture technique for the study of development and growth of the dermal skeleton in fish

Summary To develop a serum-free, chemically definedin vitro organ culture system enabling the study of epithelial-mesenchymal interactions in development and growth of fish dermal skeleton, we investigatedin vitro continuation of scale regeneration in the cichlid fishHemichromis bimaculatus. The culture medium in our system is based on Leibovitz medium (L-15) supplemented with vitamin C, additional amino acids and HEPES. With this basis medium, we examined the effects of all trans-retinoic acid, dexamethasone, and prostaglandin-E2 (PG-E2), factors known to exert an effect on development and growth of teeth and bone in mammalian culture systems, on thein vitro regeneration of scales. These effects were compared with those obtained by supplementation of the basis medium with newborn and fetal calf serum. To evaluate our culture system, the medium that allowed to mimick in the best possible way thein vivo regeneration of scales (i.e., the basis medium plus dexamethasone and PG-E2) was also tested on thein vitro development of teeth in the same fish species. Our serum-free, chemically defined organ culture system enablesin vitro development and growth of both scales and teeth. With this model culture system, it is possible to evaluate thein vitro effects of hormones, growth factors, and other substances on growth and development of dermal skeleton in fish.     

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.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Medium-term organ culture for teleost fish retinae

Whole retinae from Midas cichlids Cichlasoma citrinellum were maintained successfully in superfusion culture for 21 days post-removal and continued to exhibit normal light- and circadian-driven cone movements.     

Effects of peptides and steroid hormones on cell kinetic parameters of normal human breast tissue in organ culture

Summary Several studies have shown the importance of different hormones in the regulation of mammary tsssue growth. The use of organ culture techniques has shown tremendous value for the knowledge of cell proliferation in human breast tissue. Therefore, the purpose of these studies was to analyze the length of the cell cycle, DNA-labeling index, mitotic index, and growth fraction under the effects of insulin, hydrocortisone, and 17-β estradiol in 5-d organ culture. Normal tissues obtained from patients who underwent breast surgery for benign lesions were individually cultured at 37°C (95% air:5% CO₂ in Medium 199). Autoradiographic studies indicated that the hormones shortened the length of cell cycle of normal breast tissue in 5-d organ cultures. From the growth fraction studies we concluded that the hormones may have stimulated the cells to reenter the cell cycle from G₀ because these values were increased by the hormones used. Estrogen can alter the S phase duration with a consequent increase in the rate of DNA synthesis which may explain the high DNA-labeling index observed in the present studies. Supported by Public Health Service grant CA38921 from the National Cancer Institute, Bethesda, MD, and by an Institutional grant from the United Foundation of Greater Detroit.     

Outward-rectifying chloride channels in cultured adult and fetal human nasal epithelial cells

Summary The patch-clamp technique was used to characterize ion channels in the apical membranes of cultured human nasal epithelial cells, dissociated from fetal nasal mucosa and from adult nasal polyps. Outward-rectifying chloride channels were found in 4.3% of the cell-attached patches from fetal cells (n=258) and in 3.1% of the patches from adult cells (n=320). After exeision the number of patches containing active chloride channels increased threefold to 13% of the patches from the fetal cells and 10% from adult cells. The single-channel conductance at 0 mV in symmetrical 150mm NaCl solutions was 24.3 ±0.9 pS (n=28) and 26.0 ± 1.2 pS (n=30), respectively, in adult and fetal cells and showed outward rectification in the potential range from –80 to +80 mV. In fetal cells as well as in adult cells the channels were anion selective, and were almost impermeable for larger anions and monovalent cations. In cell-free patches the channels were Ca²⁺ independent. In most of the channels the open probability was voltage independent and high (±0.86); in 20% of the channels, however, the open probability increased with depolarization. In conclusion, fetal nasal epithelial cells contain chloride channels in their apical membranes with singlechannel properties and regulatory mechanisms similar to those found in cells from adults.     

A continuous-flow method of organ culture

Summary A method of perfusion organ culture is described in which explants cultured at the airmedium interface are bathed by a continuous flow of nutrient medium. Morphological studies on the fetal rat lung indicate that explant development in this system is comparable to that obtained using standard organ-culture dishes. Medium supply is easily manipulated and continuous sampling of the effluent stream is possible without disturbing the immediate explant environment. The basic design facilitates secretory-response studies on cultured organ explants as demonstrated by a study of glucose-stimulated insulin release by the neonatal rat pancreas. This work was supported by U. S. Public Health Service Training Grant No. GM 00114.