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Results for “Diffusion Chambers, Culture”

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PubMed1989

[Methodology of cultivating human tissue in diffusion chambers].

The proliferative activity of human pulmonary carcinoma cells has been examined by autoradiography in 48 cultures in diffuse chambers, incubated with 3H thymidine under various conditions. 3H thymidine has been injected intraperitoneally to one group of animals, in another group the diffuse chambers have been removed from the animals and incubated in culture medium with 3H thymidine in the same concentration. The proliferation intensity of the cell cultures from the same patients has been virtually the same in both modes of incubation. This permits simplifying the autoradiography technique for such experiments, thus helping to avoid the difficulties connected with radioactive waste utilization.

Adenocarcinoma↗

PubMed1994

Comparison of outer-membrane proteins of Pasteurella haemolytica expressed in vitro and in vivo in cattle.

Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolytica were compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitro under iron-sufficient and -deficient conditions, (b) in vivo in the lungs of experimentally infected calves and (c) in vivo in diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivo environment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells.

Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 +/- 160 vs. 387 +/- 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10(-12) to 10(-8) M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10(-12) to 10(-8) M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish the hypothalamic-pituitary in series-perifusion model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.

Animals↗

PubMed2002

A simple flow-through micro-chamber for handling fragile, small tissue explants and single non-adherent cells.

The flow-through chambers described are easily assembled in the laboratory with no need for sophisticated or expensive equipment. They allow for treating micro-explants or single non-adhesive cells with different solutes for defined times by easy and quick changes of the medium in the chambers. The specimen can be fixed, immunostained and observed microscopically within the chamber. During these procedures the cells are not exposed to undue mechanical stress and fragile intercellular structures are well preserved.

Animals↗

PubMed1979

[Lymphocyte acid phosphatase activity in lymphocytic leukemia studied in tissue culture with the use of diffusion chambers].

The reported investigations showed that lymphocytes in chronic lymphocytic leukaemia with low acid phosphatase activity had a rise of this enzyme level during culture in diffusion chamber. This rise is caused by increased number of lymphocytes with activity grades 1 degree and 2 degrees, and from the 6th day of culture also the number of lymphocytes with 3 degrees and 4 degrees activity score rose as well, while at the same time the proportion of cells without acid phosphatase activity fell. In the lymphocytes of healthy subjects a rise in acid phosphatase activity was observed as well under the same culture conditions.

Acid Phosphatase↗

PubMed1991

Microvascular A-V shunts and the growth of autologous tissue flaps in Millipore chambers.

An animal model was developed using the male Sprague-Dawley rat to establish a protocol and design criteria for the growth of autologous tissue based on a microvascular pedicle. It became apparent that growth within the chamber depended heavily on membrane porosity and the presence of the microarteriovenous shunt. Different membrane porosities ranging from 0.0, 0.25, 1.2, to 8.0 microns were evaluated (n = 48). Optimal growth occurred with the 1.2-microns and the 8.0-microns micropore Millipore. Growth within the chamber consisted of a radial deposition of collagen and neovascularization originating from the arteriovenous (A-V) anastomosis. In contrast, control chambers (no A-V anastomosis), with the preceding range in membrane porosity, experienced little to no cell growth. In addition, the majority of A-V shunts did not remain patent in chambers having 0.0-microns porosity or 0.25-microns porosity. Thus it is apparent that a strong relationship exists between membrane porosity, patency, and in situ vascularization allowing for the proliferation of cells and collagen.

Animals↗

PubMed1967

The circumfusion system for multipurpose culture chambers. I. Introduction to the mechanics, techniques, and basic results of a 12-chamber (in vitro) closed circulatory system.

A self-contained mechanical system for circulating nutrient fluid through 12 tissue culture chambers is described in detail. This system utilizes nonperforated cellophane membranes in the chambers which separate the circulating nutrient from the tissue culture environments. The nutrient, therefore, is dialyzed through the cellophane of each chamber; some cell products are retained in the microenvironment between the closely apposed cellophane and cover slip, whereas the other cell products move from chamber to chamber in the circulating nutrient. The resultant environmental conditions directed by the circumfusion systems are highly favorable for maintaining the differentiation of chick embryo tissues over protracted periods; a number of micrographs are shown.

Adrenal Glands↗

PubMed1990

A two-compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds.

A new bioreactor for animal cell cultivation employs two compartments for cells and medium respectively. The two chambers are separated by an ultrafiltration membrane. Cells and solution of collagen or collagen/chitosan mixture were loaded to the cell chamber and were allowed to form gel inside. Contraction of the cell-laden gel occurred subsequently to create a new zone in the cell chamber. In such a bioreactor cells are retained in the reactor, the high molecular product(s) accumulate in the cell chamber, while the small molecular weight nutrients and metabolites are replenished and removed from the medium chamber. By adjusting the flow rates for cell and medium chambers, the resident time for cells, high and low molecular weight components of the system can be manipulated separately. The new bioreactor, in both flat-bed and hollow-fiber configurations, was used to cultivate recombinant human cell, 293, for Protein C production over 60 to 90 days.

Biological Products↗

PubMed2005

Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery.

PURPOSE: To investigate the in vitro, ex vivo, and in vivo transscleral-retinal pigment epithelium (RPE) permeability of PEDF and structurally related proteins for the exploration of novel routes of protein delivery to the retina. METHODS: Monkey RPE cells were cultured on permeable supports to separate apical and basal compartments. Porcine scleral tissue was placed in Ussing chambers to separate uveal and orbital compartments. Transepithelial resistance and voltage were measured by an electrical resistance system, and paracellular tracer flux was evaluated with trypan blue. Subconjunctival administration in rat eyes was by injections of soluble protein or by implantation of polyvinyl alcohol devices containing protein. Fluorescein-conjugated (Fl-) PEDF and ovalbumin were determined by spectrofluorometry, laser scanning, immunoblotting, epifluorescence, and confocal microscopy. Permeability was assessed by fluoresceinated-protein flux. RESULTS: Transepithelial resistance, impermeability to trypan blue, and confocal microscopy confirmed functional and structural tight junction formation of RPE cells cultured on permeable supports. Full-length Fl-PEDF and Fl-ovalbumin proteins diffused through RPE cell monolayers from either the apical or basal side. Fl-ovalbumin diffused through scleral tissues at constant rates. Subconjunctival Fl-PEDF or Fl-ovalbumin administration in vivo revealed movement of full-length protein into the choroid and retina as early as 1 hour. CONCLUSIONS: The sclera and RPE were permeable in vitro, ex vivo, and in vivo to PEDF and ovalbumin proteins. These large proteins can traverse through the sclera-RPE to reach the retina. In addition, these data prompt the proposal that subconjunctival protein delivery may represent a feasible and minimally invasive route for PEDF administration in the clinic.

Animals↗

PubMed1999

Expression of osteopontin in Kupffer cells and hepatic macrophages and Stellate cells in rat liver after carbon tetrachloride intoxication: a possible factor for macrophage migration into hepatic necrotic areas.

Activated Kupffer cells and macrophages accumulate in necrotic areas in the liver. Osteopontin, an extracellular matrix with RGD sequence, has been shown to act as a chemokine that can induce monocyte migration. The possibility that osteopontin can play a role in infiltration of both cells into hepatic necrotic areas was investigated in rats. Northern blot analysis revealed that osteopontin mRNA expression was minimal in Kupffer cells and hepatocytes immediately after isolation from normal rats, but slight in hepatic stellate cells assumed nearly quiescent in function after 3 days of culture on plastic dishes. When rat received carbon tetrachloride, liver necrosis developed between 1 and 3 days following the intoxication. In these rats, osteopontin mRNA expression assessed by quantitative competitive RT-PCR was increased in the liver later than 1 day with its peak at 2 days following the intoxication. Kupffer cells and hepatic macrophages and hepatic stellate cells isolated from such liver showed marked expression of osteopontin mRNA on Northern blotting. Immunohistochemical examination disclosed that osteopontin was stained in macrophages including Kupffer cells and stellate cells in the necrotic areas. On electron microscopy, osteopontin stains were present in the Golgi apparatus in these cells. Recombinant human osteopontin promoted migration of Kupffer cells isolated from normal rats and cultured in a Transwell cell culture chamber in a dose-related manner. We conclude that activated Kupffer cells and hepatic macrophages and stellate cells express osteopontin. These cells might contribute to the infiltration of Kupffer cells and macrophages into hepatic necrotic areas by expressing osteopontin.

Animals↗

PubMed1998

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) increases iodide trapping, inhibits thyroperoxidase and antagonizes the TSH-induced apical iodide efflux in porcine thyroid cells.

4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC50 of 5 x 10(-5) M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC50 being also of 5 x 10(-5) M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10(-3) M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10(-3) M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

A new perfusion cell chamber system for determination of heat shock effects by means of video-enhanced microscopy.

A user friendly microscope perfusion chamber which allows real-time observation of individual cells at high magnification has been designed. An integrated multisensor was used to monitor the cell culture conditions. To prove the potential of the system heat shock experiments were performed. By means of video-enhanced contrast microscopy (VECM) the mitochondria morphology of cultured astrocytes was demonstrated to change from a rod-like to an annular shape after heat shock. For further analyses mitochondria were stained on the microscope stage.

Animals↗

PubMed1989

A compartmentalized device for the culture of animal cells.

A simply-constructed device for the batch culture of animal cells is described that takes advantage of gas-permeable polymer films and continuous dialysis of nutrient fluids. The design separates a cell-growth chamber from a nutrient-medium reservoir with a dialysis membrane, effectively compartmentalizing growth and feeding functions. Resulting culture environment is extraordinarily stable since frequent medium exchanges are not required and macromolecules biosynthesized by cells are retained within the cell-growth compartment. Culture experiments with a variety of mammalian cells (epithelioid, fibroblastic, hybridoma, primary murine) are described that demonstrate unattended culture for up to 30 days. Immunoglobulin (IgG) secreted by cultured hybridoma cells concentrated in the growth compartment more than 25-fold over levels attained at confluence in conventional flasks. Applications for the device in various areas using animal cell culture and potential advantages in scale are discussed.

Animals↗

PubMedDate not supplied

Hormonogenesis in thyroid cells cultured on porous bottom chambers.

Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers. This system allowed to reproduce the functional bipolarity. Cells form a tight and polarized monolayer. Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media. A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level. These TSH effects were the consequence of adenylcyclase stimulation. Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH. These steps occurred at the apical pole of cells. In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%). Iodide concentrations higher than 0.5 microM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis. Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red. The culture system appears to be interesting for pharmacological and toxicological studies.

Animals↗

PubMed1991

Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers.

Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (less than or equal to 1 x 10(8) c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 x 10(1) c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.

Animals↗

PubMed1989

Development of an osteogenic bone-marrow preparation.

The osteogenic effect of bone marrow was tested in rabbits, using chambers that had been implanted in the peritoneal cavity (ectopic site) and in a delayed-union model (orthotopic site). Osteogenesis was accelerated in both sites after concentration of marrow elements by centrifugation, but not after unit gravity sedimentation. Chambers that were implanted with marrow that had been processed by simple and isopyknic centrifugation demonstrated a more pronounced increase in deposition of calcium compared with whole-marrow implants of equal volume (101 compared with 193 per cent). Orthotopic grafting of a rabbit delayed-union model with whole marrow and marrow that had been processed with simple centrifugation significantly increased osteogenesis, as measured biomechanically and biochemically. Significantly improved healing was evident radiographically at five weeks after grafting with bone marrow that had been concentrated by simple centrifugation.

Animals↗

PubMed2006

Automated fast perfusion of Xenopus oocytes for drug screening.

Fast ('concentration jump') applications of neurotransmitters are crucial for screening studies on ligand-gated ion channels. In this paper, we describe a method for automated fast perfusion of neurotransmitters (or drugs) during two-microelectrode voltage-clamp experiments on Xenopus oocytes. The oocytes are placed in a small bath chamber that is covered by a glass plate with two channels for the microelectrodes that are surrounded by a quartz funnel serving as a reservoir for test solutions. The oocytes are perfused in a vertical direction via the two channels in the plate. Automation of compound delivery is accomplished by means of a programmable pipetting workstation. A mean rise time for 10-90% current increase through muscle-type nACh channels of 55.0+/-1.3 ms (30 muM acetylcholine) was estimated. Automation, fast perfusion rates, and economical use of compounds ( approximately 100 mul/data point) make the system suitable for screening studies on ligand- and voltage-gated ion channels.

Animals↗

PubMed2004

LEGO bricks used as chemotactic chambers: evaluation by a computer-assisted image analysis technique.

One of the main techniques used to explore neutrophil motility, employs micropore filters in chemotactic chambers. Many new models have been proposed, in order to perform multiple microassays in a rapid, inexpensive and reproducible way. In this work, LEGO bricks have been used as chemotactic chambers in the evaluation of neutrophil random motility and chemotaxis and compared with conventional Boyden chambers in a "time-response" experiment. Neutrophil motility throughout the filters was evaluated by means of an image-processing workstation, in which a dedicated algorithm recognizes and counts the cells in several fields and focal planes throughout the whole filter; correlates counts and depth values; performs a statistical analysis of data; calculates the true value of neutrophil migration; determines the distribution of cells; and displays the migration pattern. By this method, we found that the distances travelled by the cells in conventional chambers and in LEGO bricks were perfectly identical, both in random migration and under chemotactic conditions. Moreover, no interference with the physiological behaviour of neutrophils was detectable. In fact, the kinetics of migration was identical both in random migration (characterized by a gaussian pattern) and in chemotaxis (characterized by a typical stimulation peak, previously identified by our workstation). In conclusion, LEGO bricks are extremely precise devices. They are simple to use and allow the use of small amounts of chemoattractant solution and cell suspension, supplying by itself a triplicate test. LEGO bricks are inexpensive, fast and suitable for current diagnostic activity or for research investigations in every laboratory.

Cell Culture Techniques↗