Aminocentesis cells: Culture,cryogenic storage,isozymes, and finite life in vitro

Summary Forty-six cell cultures established from amniocentesis fluids were preserved in liquid nitrogen and later recovered from the frozen state with little loss of viability as compared to prefreeze viability. Five to 10% glycerol was found to be optimal for preservation in liquid nitrogen, and as few as 5×10⁵ viable cells per frozen ampule could initiate cell growth. Storage in liquid nitrogen did not affect the genetic stability of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, leucine aminopeptidase, acid phosphatase, or 6-phosphogluconic acid dehydrogenase isozymes of the amnion cultures. These studies were supported by Contract NIH-NIGMS-72-2070, Grant CA-04953-13 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3,16, 749 from the National Institutes of Health.     

Use of a low-speed,iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

Summary A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene. This work was supported in part by the National Institutes of Health Biomedical Research Support Program, and National Institute of Environmental Health Services grant (ES-01737) awarded to M.T.S.H., and by National Cancer Institute grants CA-017175, CA-09135, CA-22484 awarded to H.C.P.N.S. was supported by a Cancer Research Campaign Grant (U. K.) through the International Union Against Cancer. This work was presented in part at the 24th Annual Meeting of the Society of Toxicology, 18–22 March 1985, San Diego, CA.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E₂ production as well as a smaller increase in non-cyclooxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E₂ production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F_2α, prostacyclin and thromboxane A₂. Prostaglandins E₂ and F_2α are the principal cyclo-oxygenase products of this interaction.We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F_2α by leukocytes.     

Autoradiographic studies of human chromosomes

Certain differences in the location of chromosomal material completing DNA synthesis late in the S-period of the cell cycle were demonstrated when a comparison was made between human blood lymphocytes and epithelial cells derived from term amnion grown in vitro for short periods of time. The differences in the patterns of synthesis between these two differentiated diploid cells, each from the same species but of different embryonic origins (mesodermal vs. ectodermal), functions in vivo, and appearances and growth characteristics in vitro, may be reflections of distinctive patterns of condensed interphase chromatin, i.e. a characteristic distribution of heterochromatin, and possibly also of different cellular functions in the organisms.Supported by research grants from the U.S. Public Health Service (HD 04134) and the National Science Foundation (GB 6282). These data were presented at the Fourth Basel Colloquium on Mammalian Sex Chromosomes in Differentiation and Development, March, 1967.     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E₂ is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E₂ by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E₂ than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E₂; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E₂ biosynthesis in this tissue.     

The role of the Golgi complex in the formation of secretion in the test cells of the follicle of the tunicate,Ciona

Summary Electron microscope studies were made on the test cells which comprise a part of the follicular envelope in the ovary of the tunicate Ciona. During development the cells become filled with secretory granules. The Golgi complex is well developed and usually centrally located in the cells. The morphological variations shown and described strongly suggest that the Golgi complex is mainly concerned with the origin of the secretion in these cells.Supported by research grants (GM-9229, 9230) from the National Institute of General Medical Science, United States Public Health Service.This investigation was supported by a Public Health Service Research Career Program Award (1-K3-GM-11, 524) from the National Institute of General Medical Sciences.     

Effect of trypsinization on susceptibility of primary human amniotic cells toChlamydia trachomatis TW-3 strain

The effect of trypsinization of human amnion membranes on the susceptibility of amnion cells toChlamydia trachomatis TW-3 infection was examined by infectivity titrations using standard procedures of chlamydial inoculation, and detection of chlamydial inclusions. Epithelial cells derived from freshly trypsinized membranes as well as primary and secondary cultured cells that were freshly removed from monolayers by trypsin treatment were not susceptible to infection at 30 min and at 2 and 6 h after trypsinization. Monolayers grown 18 h and up to 5 or more days after trypsinization were susceptible to infection. Primary 5-day monolayers derived from each of nine placentas inoculated with chlamydiae showed a range of titers from 10⁻³ to 10^−6.5 (SD=1.2 logarithm). Primary monolayers supported the multiplication of chlamydiae to consistently higher titers than secondary and tertiary monolayers from the same amnion.     

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institues of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749. from the National Institutes of Health.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata, andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749 from the National Institutes of Health.     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Initiation and characterization of a diploid cell line from larval tissues ofAedes dorsalis (Meigen)

Summary Mosquito cell cultures were initiated from the minced tissues of newly hatchedAedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n=6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (includingMycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation. These studies were supported in part by funds from the Office of Naval Research, by Research Grant AI03028 from the National Institute of Allergy and Infectious Diseases, and by General Research Support Grant I-SO1-FR-05441 from the National Institutes of Health, U.S. Department of Health, Education and Welfare.     

Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium

Summary Gingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated at 37° C and 5% CO₂ in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages). This work was supported by Biomedical Research grant RR 05346 from the National Institute of Health, Bethesda, MD, and the University of Washington Graduate School Research Fund.     

Uptake of catecholamines and penetration of trypan blue after blood-brain barrier lesions

Summary Blood-brain barrier lesions were produced on rabbits which had been depleted of their endogenous monoamines with a large dose of reserpine. After the lesion, catecholamines and the blood-brain barrier indicator dye trypan blue were injected. After freeze-drying, the cellular distribution of the injected substances was observed in the fluorescence microscope.It was found that, in the injured areas, the monoamines and trypan blue had penetrated into the brain parenchyma, where the monoamines were taken up and concentrated in nerve terminals. Trypan blue was found diffusely in the neuropil, while the nerve cell bodies and axons exhibited no fluorescence of trypan blue. On the control side, this type of fluorescence of catecholamines or trypan blue could not be detected.The lesions applied seem to be quite specific for the blood-brain barrier, as an active and energy-dependent uptake of catecholamines could be demonstrated in central monoamine nerve terminals. Thus the results also show that these terminals have the same reserpine-resistant membrane pump in vivo as earlier demonstrated for peripheral adrenergic neurons, and for central neurons in vitro.This investigation has been supported by research grants (B 66-158 and B 66-257) from the Swedish Medical Research Council and by a Public Health Service Research Grant (NB 05236-02) from the National Institute of Neurological Diseases and Blindness. For generous supplies of drugs we thank the Swedish Ciba, Stockholm, Sweden for reserpine (Serpasil^®), the Swedish Pfizer, Stockholm, Sweden for nialamide (Niamid^®) and Hoechst Anilin AB, Göteborg, Sweden for -methylnoradrenaline (Corbasil^®).     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Non-surgical (laparoscopic) uterine flushing and egg recovery technique in the squirrel monkey (Saimiri sciureus)

The present study involved six female and one male squirrel monkeys (Saimiri sciureus). Seventeen uterine flushings were done using laparoscopy including one trial flushing of trypan blue solution. In each case, 1.5 ml to 2.5 ml of normal electrolyte solution with 5% dextrose was flushed through the uterine lumen with a 25 ga, 5/8″ hypodermic needle, and a 3ml disposable syringe. An average of 73.2% of flushed fluid was recovered through a polyethylene catheter inserted into the cervical canal. Two ova were recovered by this technique. This research was supported by grants from the National Foundation—March of Dimes, the National Institute of Health and an NIH Research Career Development Award. Approved by the Director, Michigan Agricultural Experiment Station, Journal series No. 7466.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Actions of interleukin-2 on amnion prostaglandin biosynthesis

Preterm labor is associated with increased intrauterine prostaglandin (PG) production. Intrauterine infections are frequently associated with preterm labor and increased cytokine production. The cytokine interleukin-2 (IL-2) is a potent T-cell growth factor necessary for effective cell-mediated immunity. In this study we evaluated the effects of IL-2 on PGE₂ biosynthesis by human amnion cells. IL-2 alone modestly but significantly inhibited amnion PGE₂ production. Moreover, IL-2 also attenuated, in a concentration-related fashion, the stimulatory actions of IL-1β on PGE₂ production by amnion cells. These data suggest that IL-2 could potentially represent a negative regulatory element in the mechanisms of preterm labor in association with intrauterine infection.