Explant Culture System Research Articles

RU486 antagonizes the inhibitory action of progesterone on prostacyclin and thromboxane A2 synthesis in cultured rat myometrial explants.

A myometrial explant culture system was developed to investigate the effect of progesterone and the antiprogestagen RU486 on prostacyclin (PGI2) and thromboxane A2 (TXA2) synthesis by the rat myometrium. After culture, eicosanoid synthesis was stimulated with arachidonic acid (AA) and the calcium ionophore A23187 (A23187). Spontaneous release of eicosanoids was also studied. Progesterone inhibited the spontaneous release of PGI2 and TXA2 release by myometrial explants in a concentration- and time-dependent manner. Adequate inhibition of myometrial eicosanoid synthesis by physiological concentrations was achieved at 18 h of culture: all subsequent studies were carried out after an 18-h culture of explants. A23187- and AA-stimulated PGI2 and TXA2 synthesis were inhibited equipotently by progesterone. 17 beta-Estradiol alone was without effect on spontaneous AA- or A23187-stimulated PGI2 or TXA2 synthesis and was without effect on progesterone-elicited inhibition of eicosanoid synthesis in the myometrial explants. The protein synthesis inhibitors, actinomycin D and cycloheximide, did not block the inhibitory action of progesterone on A23187- or AA-stimulated eicosanoid synthesis by the myometrial explants and alone mimicked the inhibitory action of progesterone. The inhibitory action of progesterone on AA- and A23187-stimulated PGI2 and TXA2 synthesis was antagonized in a concentration-dependent manner by RU486. These data indicate that within this ex vivo system, progesterone probably inhibits myometrial cycloxygenase, that progesterone may exert this action through inhibition of a modulating or permissive protein, and that the antiprogestagen RU486 is an effective in vitro antagonist or progesterone.

Human retinal pigment epithelium in long term explant culture

Explants of human retinal pigment epithelium were maintained in culture in various types of media, and examined by light and transmission electron microscopy. After one month in vitro, the central areas showed a monolayered configuration with distinct polarity and presence of ruthenium red stainable material on the apical surface. On the peripheral areas of Bruch's membrane, multilayered lesions were observed to develop and to extend from the monolayered epithelium and past the cut edge in Bruch's membrane. Cells in these lesions contained little melanin and generally lacked an apico-basal polarity. Ruthenium red staining revealed the presence of electron dense material on the apical surface of the lesions as well as in the extracellular space between cells in the various layers. Development of multilayered lesions with deposition of extracellular material are seen in various chorio-retinal disorders, including senile macular degenerations and also subsequent to laser and cryo-therapy. The findings in the present study point to the explant culture system as a valuable tool in the study of important aspects of chorio-retinal pathology.

Productivity of immunoreactive prolactin (IR-PRL) by the human decidual explants: A new sampling method.

In the present study, we used an explant culture system of the human decidual tissues involving a new sampling method for investigating the productivity of immunoreactive prolactin (IR-PRL) for a 5 day period in labored and nonlabored deliveries. The maximal release of IR-PRL in the incubation medium for each 24 hour interval was achieved from the 2nd to the 3rd day of culture in both groups, which was 145.2 +/- 14.0 ng/ml/10 mg w.w. (mean +/- s.e.; n = 7) in the labor group and 101.5 +/- 16.3 ng/ml/10 mg w.w. (mean +/- s.e.; n = 4) in the nonlabor group. The release of IR-PRL in both groups was not significantly different for the first 3 days. However, the amount of IR-PRL released in the nonlabor group was 50 to 70% of that of the labor group. The tissue content of IR-PRL in both groups ranged between 3.0 and 5.0 ng/ml/10 mg w.w. From these results, it was concluded that 1) our explant culture system for the human decidual tissues produced considerably more IR-PRL than those previously reported and 2) productivity of IR-PRL was lower in nonlabored delivery than in labored delivery and 3) since the tissue content of IR-PRL for each 24 hour interval was very small, it should be strongly emphasized that the production and release of IR-PRL takes place simultaneously in the human decidual tissues.

Effects of Adenosine and 2'-Deoxyadenosine on Epidermal Keratinocyte Proliferation: Its Relation to Cyclic AMP Formation

Although it has been reported that adenosine has an inhibitory effect on keratinocyte proliferation at both G2 and S phases of the cell cycle, its relation to cyclic AMP formation through the adenylate cyclase system has been less well characterized. In order to determine the precise mechanism of the adenosine effect, another physiologic adenine nucleoside, 2'-deoxyadenosine was employed. 2'-Deoxyadenosine was shown to be remarkably different from adenosine in its ability to stimulate the epidermal adenylate cyclase; whereas adenosine markedly increased cyclic AMP levels of pig epidermis, deoxyadenosine had a much weaker effect on the cyclic AMP levels of the skin. Using several parameters of cell proliferation, comparison was made between the effects of these two compounds. Pig keratinocyte explant culture system was employed for the measurement of outgrowth and mitosis. Mitosis was determined after 72-h incubation (to monitor the overall cell proliferation inhibition) and 4-h incubation (to monitor G2 phase inhibition) with the chemicals. Pig skin keratome slice system was employed for [3H]thymidine uptake measurement. Both adenosine and deoxyadenosine were shown to have marked inhibitory effects on keratinocyte out-growth, [3H]thymidine uptake, and keratinocyte mitosis. The effects of deoxyadenosine on outgrowth and [3H]thymidine uptake were greater than that of adenosine. The inhibitory effect of adenosine and deoxyadenosine on mitosis were about the same in both 4-h and 72-h incubation systems. Thus deoxyadenosine, which is a much weaker stimulator of epidermal adenylate cyclase, was also shown to be as potent an inhibitor of keratinocyte proliferation as adenosine. These results further substantiate the view that cyclic AMP elevating agents (such as adenosine and deoxyadenosine) might not necessarily reveal their inhibitory effects on keratinocyte proliferation through their effects of cyclic AMP formation.

High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.

The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.

Agents that Activate Cyclic AMP-Dependent Protein Kinase Inhibit Explant Culture Growth and Mitotic Activity

Epidermal cells contain 4 separate surface receptors which are linked to adenylate cyclase. Activation of any one of these receptors leads to the accumulation of cAMP within the cell which in turn leads to the activation of cAMP-dependent protein kinase. The levels of cAMP accumulation within the cell caused by the 4 activators are not the same. Epinephrine, histamine, adenosine, and prostaglandins of the "E" series cause easily measurable concentrations of cAMP within 5 min of exposure. Prostaglandin F2 alpha causes only a small nonsignificant increase. Similarly, 2 phosphodiesterase inhibitors, which inhibit the breakdown of cAMP formed within the cell, differ in their ability to accumulate cAMP when cells are exposed to these agents alone. Isobutylmethylxanthine causes a measurable increase in cAMP, while theophylline, a weak inhibitor of phosphodiesterase, gives a nonsignificant increase in cAMP. Recently, experiments have shown that agents that give only slight increases in cAMP by biochemical measurements, that is, prostaglandins F2 alpha and theophylline, are equally able to activate protein kinase within the cell. Since activation of protein kinase is the only mechanism for an increase in cAMP to have a physiologic effect, all of these agents that do activate protein kinase should cause physiologic effects. Using an explant culture system, we show in this paper that this supposition is correct and that all agents that activate protein kinase do result in inhibition of mitotic activity regardless of whether or not they are able to raise cAMP to a level that can be biochemically measured as being significantly different from the baseline value.

Effects of calcium and calcium-ionophore on the outgrowing epidermis--possible activation of epidermal phospholipase A2.

ABSTRACTUsing an in vitro explant culture system of pig skin, the effects of calcium and calcium‐ionophore on the outgrowing epidermis were studied. The optimum concentration of calcium on the rates of epidermal outgrowth was 1.0 mM and that of mitosis was around 1.2 mM. Ionophore A23187 (Ionophore) significantly stimulated both the rates of epidermal outgrowth and the mitosis of the outgrowing epidermis. This stimulation was partially blocked by the addition of hydrocortisone.The cyclo‐oxygenation products, prostaglandins, failed to stimulate the rate of epidermal outgrowth. It appeared that the stimulatory effect of Ionophore on the outgrowing epidermis was due either to the activation of phospholipase A2 or to the activation of the lipoxygenase pathway.

Organ explant culture of adult Syrian golden hamster pancreas.

An organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden hamster. Gastric and duodenal lobe explants of up to 0.5 cm2 size were placed in tissue culture dishes (60 mm2) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum, L-glutamine, hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed with 45% O2, 50% N2, and 5% CO2 and incubated at 36.5 degrees C. Viability of the tissues was determined by light and electron microscopy as well as by [3]thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells and mucus-containing cells characteristic of ductal cells were present throughout this period. However, endocrine cells were only present for the 1st wk in culture.

Biochemical evidence for aromatization of androgen to estrogen in the pituitary

We previously reported that substantial quantities of estrogen were present in the pituitary of the marine teleost ( Myoxocephalus) following perfusion of the isolated head with radiolabeled androgen. In order to determine whether this estrogen was synthesized in situ or was derived from that formed in brain areas with high levels of aromatase activity, we used an explant culture system. Pituitary and brain (preoptic area/anterior hypothalamus) were incubated separately for 20 hr with [ 3H]androstenedione. Alternatively, the two tissues were cultured together, either as an intact unit or after stalk section. [ 3H]Estrone and estradiol-17β were measured in the media and tissue fragments after ether extraction, thinlayer chromatography, and phenolic partition. The authenticity of the products was validated by reverse isotope dilution and recrystallization. Sculpin brain and pituitary in isolated cultures were independently capable of converting radiolabeled androgen to estrogen (estradiol > estrone). Aromatase activity, as indicated by estrogen production per unit wet weight or per unit protein, was greater in pituitary than in brain cultures. Both tissue types, when compared to the medium, retained estrogen against a concentration gradient. Culturing the brain and pituitary together, with or without stalk section, did not alter total estrogen yields or tissue concentration of formed estrogen. It is unlikely that aromatase activity in isolated pituitary cultures could be ascribed to fiber terminals originating in the hypothalamus; however, the cell type responsible for estrogen production remains to be determined. We conclude that, in this teleost, a significant portion of the estrogen reaching target cells in the pituitary may be synthesized in situ from circulating androgen. Hence, control of the aromatization reaction would be one means of regulating gonadotropin secretion. A reexamination of aromatase activity in the pituitary of mammals and other vertebrates is warranted.

Organotypic culture of embryonic electromotor system tissues from Torpedo marmorata

An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system. The myotubes in tissue expiants, taken from the electric organ primordia of 33–38 mm body-length embryos prior to electrocyte differentiation, contract spontaneously on explantation and have electrogenic membranes. The myotubes subsequently lose these properties in vitro and can differentiate in the absence of neural tissue into immature electrocytes which have morphologically characteristic postsynaptic membranes. Isolated expiants of differentiated electric organ tissue from 60–100 mm body-length embryos can be maintained for 3 to 4 weeks in vitro but cellular outgrowth is minimal. In contrast, a rapid, dense outgrowth of cells and a subsequent regeneration of myotubes occurs when differentiated electric organ explants are co-cultured with electric lobe tissue from embryos of the same stage. Cellular outgrowth from differentiated electric-organ tissue expiants can be stimulated by spinal cord, medulla, cerebellum and heart tissues but a subsequent regeneration of myotubes has not been observed. Myotube regeneration in the presence of electric lobe tissue is maximal with tissue from 60–80 mm body-length embryos. The myotubes that regenerate from differentiated electric organ expiants have not been observed to differentiate into electrocytes. Neuritic outgrowth in vitro occurs with electric lobe tissue taken at two different embryonic stages. The first stage corresponds to a period when most of the neuroepithelial cells in the lobe anlagen are withdrawing from the mitotic cycle and projecting axons into the branchial arches. The second, later stage is when the electromotorneurones are normally generating axon collaterals that are invading the interelectrocyte space of electrocyte columns. Maximum neuritic outgrowth at this second, later stage is obtained with tissue from 60–80 mm body-length embryos. Although neuritic invasion of electrocyte column expiants can be obtained in electric organ—electric lobe co-cultures at this later stage, synapses similar to those observed during the early stages of synaptogenesis in the electric organs in vivo have not been observed in vitro.

Retinoid stimulates epidermal outgrowth of pig skin explants.

ABSTRACTUsing a pig explant culture system, the effects of retinoids on pig epidermal cells were studied. Ro 10‐9359 slightly stimulated epidermal outgrowth, but this effect was not significant. Ro 10‐1670 (a metabolite of Ro 10‐9359) significantly stimulated epidermal outgrowth. Cytochalasin B and colchicine markedly inhibited the stimulatory effect of Ro 10‐1670, but hydroxyurea or cycloheximide did not completely block this stimulatory effect. During a migratory period, cytochalasin B completely blocked it. Neither Ro 10‐1670 nor Ro 10‐9359 effected a change in mitotic index. Histological studies revealed that Ro 10‐1670‐treated epidermal cells did not form any keratin layers. These results suggest that Ro 10‐1670 stimulated outgrowth, particularly migratory outgrowth, of epidermal cells, resulting in reduced keratin formation.

Retinoid inhibits keratin formation of pig skin explants.

ABSTRACTUsing a pig explant culture system, the effects of retinoids on keratin formation in pig epidermal cells were studied. The SDS‐PAGE patterns of keratin extracted from the outgrowing epidermis in vitro were different from those of pig epidermis in vivo. The whole pig epidermis has four major keratin bands (M.W. 65,000, 60,000, 56,000 and 51,000), while the outgrowing epidermis has three major keratin bands (M.W. 63,000, 59,000 and 52,000). The SDS‐PAGE patterns of keratin proteins extracted from the outgrowing epidermis treated with Ro 10‐1670 were markedly altered. The highest molecular protein (M.W. 63,000) found in control explant was almost entirely absent in Ro 10‐1670‐treated explants and the second band (M.W. 59,000) found in control explants was split into two minor bands (M.W. 59,000 and 58,000).These results indicated that Ro 10‐1670 strongly modified keratin formation in vitro explant culture of pig skin.

62] Cultured avian liver explants for studies of lipogenic enzymes

Publisher Summary This chapter describes the cultured avian liver explants for the studies of lipogenic enzymes. Most primary liver cell culture systems for the study of lipid metabolism involve disassociated hepatocytes and utilize serum that contains many hormones, unknown growth factors, and lipids, and thus makes it difficult to evaluate the role of hormones and lipids in the regulation of lipogenic enzymes. The characterization of an embryonic avian liver explant culture system maintained in a chemically defined culture medium, not containing serum, has made it possible to probe into the mechanism of hormonal induction of lipogenic enzymes, such as fatty acid synthase, stearoyl-CoA desaturase, and malic enzyme. The explants cultured in control medium lacking hormones maintained their initial specific activities of [U- 14 C]glucose oxidation to CO 2 , [ 3 H]uridine incorporation into RNA, and [ 3 H]leucine incorporation into cellular proteins. These results indicate that the functional viability of the liver can be maintained in explant culture for at least 120 h. The liver explant culture system is well suited for the investigation of mechanism by which insulin induces lipogenic enzymes.

Antimycoplasmal activity of dimethylphenols in a tracheal explant culture system.

Mycoplasma pneumoniae induces pneumonia-like symptoms in hamsters and causes ciliostasis and cytonecrosis in hamster tracheal explants. 2,4-Dimethylphenol and, to a lesser extent, its 2,3-, 2,5-, and 2,6-dimethylphenol isomers protected tracheal explants from these changes after exposure to virulent M. pneumoniae strain PI 1428. The effect was concentration, time, and isomer dependent. At concentrations of 10(-9) M or greater, 2,4-dimethylphenol completely prevented the morphological (loss of ciliated cells) and biochemical (decreased dehydrogenase activity) changes normally observed after exposure to M. pneumoniae. Apparently, 2,4-dimethylphenol interfered with an early event in the infection process. Complete protection required that it be present during the first 2 h of exposure of the explants to the infecting mycoplasmas. These xylenols may prove to be useful tools for helping to define the mechanisms of pathogenesis in certain respiratory infections.

Chapter 16 Human Esophageal Organ Culture Studies

Publisher Summary The chapter provides a detailed morphological, culture, and transplant study of the conditions necessary for maintaining normal human esophageal epithelium in long-term organ culture. First successful attempt to maintain normal human esophagus epithelium in a defined organ culture system for an extended period is presented in this chapter. Successful initiation of esophagus explant cultures are found to be dependent upon placing the epithelial mucosa uppermost when the tissue was placed in the petri dish. This assured that the epithelial tissue was bathed in culture media and permitted adequate diffusion of the nutrients to the cells. The proper orientation in the culture dish is also important in maintaining the normal epithelial polarity. Another important factor in the successful maintenance of esophageal epithelium culture is the inclusion of insulin and hydrocortisone in the chemically defined medium. These two hormones used in the culture system are successfully utilized singly and in combination in a variety of explant culture systems to maintain mammalian epithelia. It is possible to follow the neoplastic process in vitro using suspected organotrophic chemical carcinogens by using morphologic and ultrastructural information on normal human esophagus.

Gamma-Aminobutyric acid receptors visualized in spinal cord cultures by [3H]muscimol autoradiography.

gamma-Aminobutyric acid (GABA) receptors were visualized in mouse spinal cord explant cultures by [3H]muscimol autoradiography over certain small neurons of the dorsal horn grey matter, in the interneuronal neuropil of dorsal and ventral horns, and (rarely) in small clusters on processes of anterior horn cells. Specificity was indicated in control experiments by inhibition of binding by [3H]muscimol after pretreatment with GABA or a GABA analogue, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, isoxazole 16), or with receptor antagonists bicuculline and picrotoxin. Unlabeled muscimol exchanged effectively with [3H]muscimol and produced autoradiographic label in locations indistinguishable from those found in experiments with [3H]muscimol alone. Pretreatment with the GABA uptake and transport inhibitors (-)-nipecotic acid and guvacine did not affect binding with [3H]muscimol. These experiments indicate that explant culture systems can be used for demonstration of functional receptors.

Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens.

An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlled-atmosphere chamber which was gassed with 95% O2 and 5% CO2. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30 degrees C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[alpha]pyrene, 7,12-dimethylbenz[alpha]anthracene, aflatoxin B1, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.