Long term organ culture of rat liver rudiment in a synthetic medium: morphological and biochemical development.

Embryonic rat liver rudiments were grown and maintained in a chemically defined medium for up to 16 weeks. Liver explants grew and developed liver plates, bile canaliculi and ductules. Liver cells differentiated and, by the end of the second culture week, revealed cytological and ultrastructural features of adult rat liver cells. Glycogen synthetase activity appeared and reached a plateau within 2 weeks.Plasma proteins produced by the explants appeared sequentially in the culture medium. Their relative concentrations in the medium increased progressively to a plateau and they persisted throughout the culture period. Albumin was present in trace amounts during early days of culture. Its relative concentration increased and it became the major protein constituent of the medium by the second week.Transferrin, α₁M-globulin, haptoglobin, β₁c- and α₁B-globulin appeared in that order. The three fibrinogen subunits were identified during the first week of culture.Fetal proteins were identified during the early culture period, the most prominent being α-fetoprotein. The relative concentration of fetal proteins declined steadily as the culture progressed.     

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)     

Intrinsic properties inhibit axonal outgrowth from neonatal rat spinal cord explant

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.     

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.     

Mechanical and biochemical characterization of cartilage explants in serum-free culture

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.     

Epidermal growth factor (EGF) influences DNA synthesis in human fetal kidneys maturing in serum-free organ culture

Human fetal kidney explants (13-17 weeks of gestation) were maintained in serum-free organ culture. The influence of epidermal growth factor (EGF) was determined after 2 and 5 days by evaluating DNA and protein synthesis as well as the activities of five brush border hydrolases. During the studied period the overall morphology was preserved and the analysed parameters remained constant. Only DNA synthesis decreased after 2 days. The addition of EGF to the medium did not change any of the cell activities, except DNA synthesis. In fact, the incorporation of [3H]thymidine was significantly stimulated by 105% in 5-day explants cultured in the presence of the growth factor. These results indicate that EGF directly influences proliferation but not maturation of brush border enzymes in fetal human kidneys in culture.     

Dipetalonema vitae: survival of adult females and microfilarial release in both a chemically defined and serum-supplemented medium

Studies were conducted on survival and microfilarial release of afult Dipetalonema viteae in culture, using worms of various ages derived from jirds. In chemically defined NI medium (a 1:1 mixture of NCTC 135 and Iscove's modified Dulbecco's medium) under a gas phase of 5% CO2 in nitrogen (pO2 of medium approximately 40 mm Hg), the peak of microfilarial release of several thougsand microfilariae per female per 24 hr occurred at approximately day 10. Thereafter, microfilarial release declined and generally ended about 1 mo after the start of culture. The adult females moved actively for about 50 days or more and survived up to 82 days in NI medium alone. The females in NI medium supplemented with fetal bovine serum showed serpentine movement for approximately 2 mo. Some of the worms survived more than 83 days. The total number of microfilariae deposited in culture by D. viteae increased as adult females grew in size (volume) over time. Microfilarial deposition continued to increase after worms reached maximum size, deposition reaching a plateau between approximately 300 and 400 days of age. Thereafter, microfilarial deposition decreased as females continued to age. Addition of fetal bovine serum to the NI medium increased the number of microfilariae released and extended the period of release.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture

Fetal rat hepatocytes (day 19 of gestation) multiply in primary culture in arginine-free, hydrocortisone-containing chemically defined medium MX-82 supplemented either with epidermal growth factor (EGF) or insulin or both. In contrast, hepatocytes did not multiply under similar culture conditions using Dulbecco's minimum essential medium (DMEM). Cells underwent two divisions within 10 days in cultures maintained in MX-82 medium without a medium change, and cells grew to increased final cell densities when the medium was renewed every third day. When the medium MX-82 was enriched by the addition of lipids, intermediary metabolites, and trace metals (medium MX-83), cells grew to higher densities. In the absence of the growth factors, cells became quiescent and subsequently could be induced to synthesize DNA in response to EGF. With the increasing numbers of cells per dish, the growth response of the hepatocytes diminished. Levels of hepatocyte-specific albumin and alpha-fetoprotein mRNAs at day 0 were similar to those observed at day 10 in primary fetal rat hepatocyte cultures and were maintained at higher levels in medium MX-83 than in medium MX-82.     

Precocious development of UDP-glucuronyltransferase activity in cultured fetal rat liver brought about by glucocorticoids and requiring amino acid incorporation into protein

Fetal-rat liver explants cultured in a defined protein-free medium containing dexamethasone, corticosterone or cortisol (all 2 microM) exhibit precocious development of UDPglucuronyltransferase activity to o-aminophenol. Transferase activity in 14-day fetal livers cultured with the glucocorticoids for 3 days rises from virtually zero to 5 times the activity seen in fresh 17 day fetal liver. With 15-day fetal livers, precocity was also observed, but to a lesser degree. Precocity always required addition of glucocorticoids, though explants were viable without them. Protein synthesis, not activation, was probably involved, for assays were performed in a range of digitonin concentrations to ensure 'optimal' activation; also, precocious development of transferase activity and uptake of [14C]-leucine into protein exhibited parallel behaviour during inhibition by, and recovery from, cycloheximide-pulsing. This is the first demonstration of a protein-synthesis-dependent stimulation of fetal mammalian UDPglucuronyltransferase by known compounds of endogenous origin. Results with other substrates are discussed.     

Biochemical changes in cultured foetal rat liver explants.

Liver explants from 20-day-old foetuses cultured for 48h in the absence of serum released 70% of their total soluble protein content into the medium. In the presence of serum this loss still amounted to 60%. The concentration of total particulate protein remained unchanged but there was some translocation of mitochondrial enzymes to the cytosol, and enzymes expected to increase during this stage of development failed to do so. The addition of cortisol plus glucagon (to serum-containing media) did not decrease the loss of total soluble protein from the explants but induced considerable tyrosine aminotransferase activity which was not released into the medium. The observations suggest that under the usual culture conditions a minority of the cells retain their functional integrity. The extent of deterioration, not reflected in histologically visible necrosis or cell damage, can be conveniently monitored by the malate dehydrogenase activity released to the medium.     

A simplified method for culture of human fetal heart tissue

Human fetal heart tissue obtained consequent to suction termination of pregnancy between 6 and 12 weeks of gestation were cultured as explants and maintained in a viable state, with spontaneous contractions up to 75 days. Ultrastructural morphology of the explant revealed that the cells remained healthy up to 21 days in culture. The model can therefore be used for experimental studies during the first 3 weeks in culture.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

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.     

Effects of sialagogues on the syntheses of polyamines and DNA in murine parotid gland

When rat parotid explants were cultured on siliconized lens paper floating on chemically defined 199 medium, all of sialagogues tested increased ornithine decarboxylase activity, which was roughly proportional to the amylase released into the culture medium. S-Adenosylmethionine decarboxylase and DNA synthesis were also induced by isoproterenol, methoxamine, carbachol and pilocarpine, but not by serotonin or substance P. The increases of the two decarboxylase activities and DNA synthesis were observed in vivo in mouse parotid gland after repeated injections of carbachol or pilocarpine. These results indicate that both adrenergic and cholinergic sialagogues stimulate the syntheses of polyamines and DNA in murine parotid gland.     

Characterization of excretory-secretory proteins synthesized in vitro by Schistosoma mansoni primary sporocysts

Excretory-secretory (E-S) products released by larval schistosomes have been implicated in the interference of host snail defense systems. Because of the potentially important role that E-S products play in the parasite-host relationship, total and newly synthesized E-S proteins from in vitro-cultured Schistosoma mansoni primary sporocysts were characterized using incorporation of [35S]methionine followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Total E-S protein decreased more than 5-fold from day 1 to day 3 of culture and remained constant until day 8 when protein concentrations began to increase. Release of newly synthesized protein, however, increased from day 1 through day 8. Both silver staining and fluorography of SDS-PAGE-separated E-S products revealed a wide variety of polypeptides ranging in Mr from 13 to greater than 200 kDa. The dynamics of the release of individual polypeptides, both total and newly synthesized, varied over time. Although certain polypeptides decreased in concentration, others remained constant or increased with time in culture. Culture conditions were found to be important for sporocyst viability and growth, and for release of newly synthesized proteins. Sporocysts cultured in medium containing fetal bovine serum (complete) grew significantly larger and had a significantly greater viability than did sporocysts cultured in medium lacking serum (incomplete). Also, sporocysts cultured in complete medium synthesized and released significantly more protein than did sporocysts cultured in incomplete medium. These sporocysts continued to produce a 54-kDa polypeptide, whereas sporocysts in incomplete medium stopped producing this protein by day 3 of culture. The present study has shown that S. mansoni primary sporocysts, cultured in vitro, synthesize and secrete a wide variety of glycoproteins and that the type and quantity of glycoproteins released are dependent on culture conditions.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases.

Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-,erm culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1 M or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellualr changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases

Summary Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-term culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1m or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellular changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

Elastin synthesis during perinatal lung development in the rat

The rate of soluble elastin synthesis was estimated in lung explants from rats of differing ages to better define periods in lung development important to the deposition of lung elastin. Lungs from rat pups at days 1, 3, 7, 9, 12, 15, and 21 post-parturition and from adult rats were incubated in a defined medium containing L-[3H]valine. Following incubation, labelled soluble elastin (tropoelastin) was separated from other soluble proteins by coacervation and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The tropoelastin synthetic rate was then estimated after correcting for differences in recovery of radioactivity as tropoelastin and lung tissue L-[3H]valine specific activity. Maximal rates of elastin synthesis were observed in lung explants from 7-12-day-old rats. The rate of elastin synthesis during this period was 5-8-times the rate observed in adult rat lung (expressed per g of fresh lung) and represented approx. 2% of the total protein synthesis. Moreover, the values derived from lung explant culture for elastin synthesis were consistent with values for lung elastin deposition in the perinatal rat (5-10 micrograms elastin/h per g lung).