Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia

2001 ◽  
Vol 281 (4) ◽  
pp. L1021-L1027 ◽  
Author(s):  
Corrie B. Allen ◽  
B. Kelly Schneider ◽  
Carl W. White
Keyword(s):  
Tissue Culture ◽  
Time Course ◽  
Culture Medium ◽  
Cultured Cells ◽  
Long Term Effects ◽  
Temporal Precision ◽  
Cellular Events ◽  
Solubility Of Oxygen

Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired Po 2 within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.

In vitro interactions between epithelial cells and Gyrodactylus derjavini

2000 ◽  
Vol 74 (3) ◽  
pp. 203-208 ◽  
Author(s):  
K. Buchmann ◽  
C.V. Nielsen ◽  
J. Bresciani
Keyword(s):  
Tissue Culture ◽  
Epithelial Cells ◽  
Cell Cultures ◽  
Culture Medium ◽  
Epithelioma Papulosum Cyprini ◽  
Skin Responses ◽  
Enzyme Reactivity ◽  
In Vitro Interactions

AbstractSkin responses of fish to various parasites have been shown to involve various immunologically competent cells producing factors which guide the reactions of epithelial cells. However, the present study has demonstrated that a monoculture of epithelial cells has the ability to encapsulate and partially degrade ectoparasites without involvement of leukocytes. The ectoparasitic monogeneanGyrodactylus derjavini was kept on a monolayer of Epithelioma Papulosum Cyprini (EPC) cells in 24-well multidishes supplied with tissue culture medium. Gyrodactylus derjavini did not reproduce but survived an incubation period of up to139 h in the system. Due to sterile conditions, dead gyrodactylids were not subjected to microbial degradation and remained intact for several weeks. However, at 40 days G. derjavini was overgrown by EPC-cells and became partly degraded during the following 15 days. Analysis of enzyme reactivity in EPC-cells showed reactions for ten enzymes including esterases, amidases, phosphatases and phosphohydrolases. No marked differences for the ten enzymes between cell cultures with and without the ectoparasites were found but it cannot be excluded that some of these enzymes took part in parasite degradation. The study showed the in vitro capability of epithelial cells to interact, encapsulate and degrade G. derjavini without the involvement of leukocytes. This response probably is non-specific and will not exclude that various immunocompetent cells and their products normally optimize and accelerate elimination of invading parasites in vivo.

Short-term effects of dopamine on photoreceptors, luminosity- and chromaticity-horizontal cells in the turtle retina

1995 ◽  
Vol 12 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Josef Ammermüller ◽  
Reto Weiler ◽  
Ido Perlman
Keyword(s):  
Receptive Field ◽  
Time Course ◽  
Direct Action ◽  
Horizontal Cells ◽  
Field Size ◽  
Long Term Effects ◽  
Short Term ◽  
Short Term Effects

AbstractThe effects of dopamine on luminosity-type horizontal cells have been documented in different vertebrate retinas, both in vivo and in vitro. Some of these effects may reflect direct action of dopamine onto these cells, but indirect effects mediated by presynaptic neurons cannot be ruled out. Furthermore, direct effects of dopamine on horizontal cells may affect other, postsynaptic neurons in the outer plexiform layer. To test these possibilities, we studied the effects of dopamine on photoreceptors and all types of horizontal cells in the turtle (Pseudemys scripta elegans) retina. Receptive-field properties, responsiveness to light, and time course of light responses were monitored with intracellular recordings. Dopamine at a concentration of 40 μM exerted effects with two different time courses. “Short-term” effects were fully developed after 3 min of dopamine application and reversed within 30 min of washout of the drug. “Long-term” effects were fully developed after about 7–10 min and could not be washed out during the course of our experiments. Only the “short-term” effects were studied in detail in this paper. These were expressed in a reduction of the receptive-field size of all types of horizontal cells studied; L1 and L2 luminosity types as well as Red/Green and Yellow/Blue chromaticity types. The L1 horizontal cells did not exhibit signs of reduced responsiveness to light under dopamine, while in the L2 cells and the two types of chromaticity cells responsiveness decreased. None of the rods, long-wavelength-sensitive, or medium-wavelength-sensitive cones exhibited any apparent reduction in their receptive-field sizes or responsiveness to light. The present results suggest that the “short-term” effects of dopamine are not mediated by photoreceptors and are probably due to direct action of dopamine on horizontal cells.

scholarly journals Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

2003 ◽  
Vol 369 (2) ◽  
pp. 399-406 ◽  
Author(s):  
Robert G. KEYNES ◽  
Charmaine GRIFFITHS ◽  
John GARTHWAITE
Keyword(s):  
Tissue Culture ◽  
Superoxide Dismutase ◽  
Culture Medium ◽  
Superoxide Radical ◽  
Cultured Cells ◽  
Soluble Guanylate Cyclase ◽  
Subsequent Reaction ◽  
Biological Media ◽  
State Concentration

NO functions ubiquitously as a biological messenger but has also been implicated in various pathologies, a role supported by many reports that exogenous or endogenous NO can kill cells in tissue culture. In the course of experiments aimed at examining the toxicity of exogenous NO towards cultured cells, we found that most of the NO delivered using a NONOate (diazeniumdiolate) donor was removed by reaction with the tissue-culture medium. Two NO-consuming ingredients were identified: Hepes buffer and, under laboratory lighting, the vitamin riboflavin. In each case, the loss of NO was reversed by the addition of superoxide dismutase. The effect of Hepes was observed over a range of NONOate concentrations (producing up to 1μM NO). Furthermore, from measurements of soluble guanylate cyclase activity, Hepes-dependent NO consumption remained significant at the low nanomolar NO concentrations relevant to physiological NO signalling. The combination of Hepes and riboflavin (in the light) acted synergistically to the extent that, instead of a steady-state concentration of about 1μM being generated, NO was undetectable (<10nM). Again, the consumption could be inhibited by superoxide dismutase. A scheme is proposed whereby a ‘vicious cycle’ of superoxide radical (O2•-) formation occurs as a result of oxidation of Hepes to its radical species, fuelled by the subsequent reaction of O2•- with NO to form peroxynitrite (ONOO-). The inadvertent production of ONOO- and other reactive species in biological media, or the associated loss of NO, may contribute to the adverse effects, or otherwise, of NO in vitro.

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions

2009 ◽  
Vol 10 (6) ◽  
pp. 513-521 ◽  
Author(s):  
W. Mark Erwin ◽  
Facundo Las Heras ◽  
Diana Islam ◽  
Michael G. Fehlings ◽  
Robert D. Inman
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Sodium Alginate ◽  
Cultured Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Notochordal Cells ◽  
Collagen Ii

Object The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O2 concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change. Methods The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. Results Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition—robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.

scholarly journals Feedback regulation of granulopoiesis: polymerization of lactoferrin abrogates its ability to inhibit CSA production

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 108-112 ◽  
Author(s):  
GC Jr Bagby ◽  
RM Bennett
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Feedback Regulation ◽  
Tissue Culture Medium ◽  
Mononuclear Leukocytes ◽  
Gel Chromatography ◽  
Polymeric Form ◽  
Higher Doses

Neutrophil extracts were prepared from the peripheral blood of 40 normal volunteers and tested for their ability to inhibit CSA production by mononuclear leukocytes. Highly dilute neutrophil extracts inhibited CSA production/release, while extracts selectively depleted of lactoferrin by antibody affinity chromatography did not. In addition, higher concentrations of neutrophil extracts and higher doses of lactoferrin (10(-9)-10(-6) M) failed to inhibit CSA production/release. We found no evidence of CSA or CSA-enhancing factors in either our lactoferrin or our neutrophil extracts. However, using gel chromatography and rate zonal density sedimentation, we noted that lactoferrin undergoes concentration-dependent polymerization at 10(-9)-10(-10) M in tissue culture medium and that while monomeric lactoferrin effectively inhibits CSA production/release in vitro, the polymeric form does not. Thus, while we have confirmed that lactoferrin is the activity in neutrophil extracts that inhibits CSA production, we have also found that lactoferrin undergoes reversible polymerization at physiologic concentrations and that the polymerized molecule is inactive. The tendency of lactoferrin to polymerize in tissue culture medium and in vivo should be taken into account in any studies on its potential role as a physiologically relevant regulator of granulopoiesis.

Long-term survival and aldosterone secretion pattern of aldosteronoma in culture

1983 ◽  
Vol 104 (4) ◽  
pp. 495-501 ◽  
Author(s):  
Tetsuro Okabe ◽  
Hiroshi Hidaka ◽  
Nakaaki Ohsawa ◽  
Toshio Tsushima
Keyword(s):  
Angiotensin Ii ◽  
Primary Aldosteronism ◽  
Culture Medium ◽  
Cultured Cells ◽  
Aldosterone Secretion ◽  
Term Survival ◽  
Eosinophilic Cytoplasm

Abstract. In an attempt to obtain an in vitro experimental model for aldosteronoma, primary culture was initiated with adenomas from 3 patients with primary aldosteronism. The cells grown in culture retained the morphology and functional properties characteristic of aldosteronoma cells well for periods of up to 200 days. The cells formed monolayer cell colonies and showed an epithelioid morphology with small nuclei containing prominent nucleoli. The cells possessed a clear, eosinophilic cytoplasm resembling that of aldosteronoma cells in vivo. The cultured cells continued to secrete large amounts of aldosterone throughout the culture period. The cells responded to angiotensin II and III by increased release of aldosterone into the culture medium. They also responded to Db-cAMP and ACTH by increased secretion of the hormone.

scholarly journals Background Synaptic Conductance and Precision of EPSP-Spike Coupling at Pyramidal Cells

2005 ◽  
Vol 93 (6) ◽  
pp. 3248-3256 ◽  
Author(s):  
Veronika Zsiros ◽  
Shaul Hestrin
Keyword(s):  
Time Course ◽  
Pyramidal Cells ◽  
Spike Timing ◽  
Synaptic Activity ◽  
Synaptic Conductance ◽  
Temporal Precision ◽  
Voltage Dependent ◽  
Cortical Slices

The temporal precision of converting excitatory postsynaptic potentials (EPSPs) into spikes at pyramidal cells is critical for the coding of information in the cortex. Several in vitro studies have shown that voltage-dependent conductances in pyramidal cells can prolong the EPSP time course resulting in an imprecise EPSP-spike coupling. We have used dynamic-clamp techniques to mimic the in vivo background synaptic conductance in cortical slices and investigated how the ongoing synaptic activity may affect the EPSP time course near threshold and the EPSP spike coupling. We report here that background synaptic conductance dramatically diminished the depolarization related prolongation of the EPSPs in pyramidal cells and improved the precision of spike timing. Furthermore, we found that background synaptic conductance can affect the interaction among action potentials in a spike train. Thus the level of ongoing synaptic activity in the cortex may regulate the capacity of pyramidal cells to process temporal information.

scholarly journals 13C tracer analysis identifies extensive recycling of endogenous CO2 in vivo

2021 ◽  
Author(s):  
Likun Duan ◽  
Daniel E. Cooper ◽  
Grace Scheidemantle ◽  
Jason W. Locasale ◽  
David G. Kirsch ◽  
...  
Keyword(s):  
Time Course ◽  
Cultured Cells ◽  
Tca Cycle ◽  
Data Interpretation ◽  
Carboxylase Activity ◽  
Intact Cells ◽  
Time Course Study ◽  
Metabolic Activities

Abstract13C tracing analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. We have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by liquid chromatography coupled with high-resolution mass spectrometer (LC-HRMS). As expected, citrate M+2 or M+4 is the dominant mass isotopologue in vitro. However, citrate M+1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion. One plausible explanation is that 13CO2 produced from the oxidation of 13C tracers in vitro is negligible due to the dilution of HCO3- supplemented to cell culture when sodium bicarbonante is used and diffusible volume of CO2 in the culture incubator, while endogenous 13CO2 in vivo is substantial and is fixed into the TCA cycle, purine, and serine, resulting in M+1 isotopologues. A time course study shows the generation of high abundance citrate M+1 early in plasma, which may serve as a potent non-invasive biomarker of tissue pyruvate carboxylase activity. Altogether, our results show that recycling of endogenous CO2 is substantial in vivo and provides important insights into the experimental design and data interpretation of 13C tracing assays.

scholarly journals Feedback regulation of granulopoiesis: polymerization of lactoferrin abrogates its ability to inhibit CSA production

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 108-112 ◽  
Author(s):  
GC Jr Bagby ◽  
RM Bennett
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Feedback Regulation ◽  
Tissue Culture Medium ◽  
Mononuclear Leukocytes ◽  
Gel Chromatography ◽  
Polymeric Form ◽  
Higher Doses

Abstract Neutrophil extracts were prepared from the peripheral blood of 40 normal volunteers and tested for their ability to inhibit CSA production by mononuclear leukocytes. Highly dilute neutrophil extracts inhibited CSA production/release, while extracts selectively depleted of lactoferrin by antibody affinity chromatography did not. In addition, higher concentrations of neutrophil extracts and higher doses of lactoferrin (10(-9)-10(-6) M) failed to inhibit CSA production/release. We found no evidence of CSA or CSA-enhancing factors in either our lactoferrin or our neutrophil extracts. However, using gel chromatography and rate zonal density sedimentation, we noted that lactoferrin undergoes concentration-dependent polymerization at 10(-9)-10(-10) M in tissue culture medium and that while monomeric lactoferrin effectively inhibits CSA production/release in vitro, the polymeric form does not. Thus, while we have confirmed that lactoferrin is the activity in neutrophil extracts that inhibits CSA production, we have also found that lactoferrin undergoes reversible polymerization at physiologic concentrations and that the polymerized molecule is inactive. The tendency of lactoferrin to polymerize in tissue culture medium and in vivo should be taken into account in any studies on its potential role as a physiologically relevant regulator of granulopoiesis.

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