Differentiation-arrested rat fetal lung in primary monolayer cell culture

Summary Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO₂ of ∼142 mm Hg, cultures developed in a pO₂ of ∼30 mm Hg, close to the normal fetal arterial pO₂, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO₂ ∼30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO₂. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung. This work was supported by grants from the Medical Research Council of Canada, the Ontario Thoracic Society, and the Physicians' Services Incorporated Foundation.     

Positive influence of tetracycline on human fetal kidney in serum-free organ culture

Summary Human fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20μg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.     

An improved organ culture method for adult mammalian lung

Summary An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated with agar and 200-μm-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface. Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can be identified in the living tissue. The explants were studied with time-lapse cinematography, light microscopy, histology, and with erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased from 1 week to 1 month. This study was supported by NHLBI Grant No. HL15098-05.     

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.     

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.     

Adult rat lung in organ culture: Maintenance of histotypic structure and ability to synthesize phospholipid

Summary The maintenance of adult rat lung explants in organ culture was assessed both morphologically and biochemically. A comparison of several culture media indicated that Ham's F12K plus 0.1 μM dexamethasone, which maintained the explants for 14 days, was superior. The ability of the explants to synthesize dipalmitoylphosphatidylcholine increased with the length of cultivation to values greater than the noncultivated controls. The DNA content remained constants for 7 days, and a relatively normal structural relationship between type I and type II pneumocytes was maintained. Explants cultivated in Ham's F12K without dexamethasone did not maintain a histotypic morphology; the type II pneumocytes appeared to proliferate and the ability to synthesize phosphatidylcholine decresed. Support was given by NIH Grant HL19669 and from the Veterans Administration.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

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.     

Optimization of culture conditions for the formation of sperm cells in pollen tubes of tradescantia

A culture medium and culture conditions are described that enable generative cell division and sperm formation to occur in a large proportion (greater than 70%) of the pollen tubes of Tradescantia paludosa within six to eight hours of culture of pollen. The nature of the nitrogen source, speed of shaking, and ratio of pollen to medium are important parameters in determining the extent of sperm formation. Addition of the plant hormones indole acetic acid, gibberellic acid, and kinetin to the growth medium does not influence generative cell division.     

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.     

Hormonal regulation of beta-adrenergic receptors in fetal rabbit lung in organ culture

Explants of fetal rabbit lung were established on the 25th day of gestation. These were maintained in serum-free medium for periods up to 10 days. During this time, the cultures exhibited morphological changes typical of terminal lung differentiation. Morphological evidence was also obtained for synthesis and secretion of pulmonary surfactant in these explants. beta-Adrenergic receptors were identified in these lung explants. Exposure of the explants to 10(-7)M dexamethasone on the third day of culture resulted in a significant increase in the number of beta-adrenergic receptors in the tissue without a change in receptor affinity. The effect of dexamethasone in organ culture was dose-dependent, a maximum increase in receptor number being observed within 48 hours of incubation with a hormone concentration of 1 x 10(-7)M. Exposure of the explant tissue to 1 x 10(-7)M triiodothyronine resulted in no significant increase in the concentration of beta-adrenergic receptors and no change in receptor affinity. These results suggest that glucocorticoids may potentiate the effects of beta-adrenergic agents in the fetal lung by increasing the numbers of their receptors. The effects of triiodothyronine upon the fetal lung do not appear to be mediated by this mechanism.     

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.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

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.     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres

The relevance to the in vivo situation of in vitro toxicity studies of complex atmospheres has frequently been limited by the procedures used for the exposure of the biological samples. We have evaluated from on-road measurements the size distribution pattern and the subsequent respiratory tract deposition rates of particulate matter from urban atmospheric aerosols, which are in the range of 110 and 3 pg/cm² per min for tracheobronchial and alveolar areas, respectively. Continuous flow-through rotating chambers and a specific design for exhaust sampling and dilution with controlled adjustment of pO₂ and pCO₂ to 20% and 5%, respectively, have been developed to expose biphasic air/liquid organotypic cultures of rat lung slices to continuous flows of diluted exhausts from diesel engines with preservation of the physicochemical properties of the exhaust. The size distribution of the particulate matter and the bioavailability of pollutants were preserved, thus allowing us to closely mimic in vitro the in vivo atmosphere/tissue interactions that occur mainly through diffusion mechanisms. The toxicity response profile has been assessed in terms of tissue viability, oxidative stress, DNA injury, and the early phase of inflammatory reaction. Exhaust filtration, addition to fuel of rapeseed methyl ester, and preincubation of lung tissue with soy isoflavones modulated the toxicity response profile of exhausts. The importance of preserving both particulate matter size distribution and adsorbed pollutant bioavailability, which could not be ascertained using more classical in vitro approaches, is discussed and should be considered a prerequisite for further developments of in vitro studies to modelize in vivo inhalation of complex atmospheres. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Preparation of human hepatocytes for primary culture

Summary A method based on the use of a neutral bacterial protease was employed for the preparation of isolated human hepatocytes from surgical biopsy specimens. Cells maintained in primary culture developed ultrastructural features characteristic of human hepatocytes in vivo. They retained the ability to bind insulin; binding kinetics were similar to those of other species. The hepatocytes responded to physiological concentrations of insulin by an increase in glycogen synthesis.