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R M Sutherland

Publications and source records attributed to R M Sutherland.

At least 127 records · Page 7Linked to original sources
PubMed1985

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth.

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.

Animals↗
PubMed1985

Changes in O2 consumption of multicellular spheroids during development of necrosis.

Using O2-sensitive microelectrodes oxygen tension profiles were recorded in EMT6-spheroids either showing no necrosis or having developed a small necrotic area in the center. The profiles obtained were in accordance with those measured in previous investigations under similar conditions. The volume-related O2-consumption rate Q in the viable parts of the spheroids could be determined through theoretical considerations. The results show that there is a steep decrease in Q by a factor of three when the spheroids grow from diameters of 200 micron to diameters larger than 1000 micron. The drop in Q mainly occurs in a size range in which central necrosis develops in EMT6-spheroids cultured under these particular conditions. Among other factors, changes in the proliferative status, in extracellular volume fraction or in physiological parameters of the micromilieu in spheroids, such as accumulation of metabolic waste products or of toxic substances from the necrotic area may contribute to the variation in Q observed.

Animals↗
PubMed1985

Opto-electronic immunosensors: a review of optical immunoassay at continuous surfaces.

Optical techniques for monitoring immunological reactions on continuous surfaces are reviewed. Initially Langmuir-Blodgett film techniques and ellipsometry are discussed, followed by internal reflection spectroscopy (IRS) systems. The latter includes attenuated total reflection (ATR) and total internal reflection fluorescence (TIRF). Finally, light scattering and surface plasmon resonance methods are presented. Overall, it was considered that the IRS systems and ellipsometric approaches offered the most promise for the design of a specific immunosensor device. Of these two, the ellipsometric methods are the most sensitive but also the most vulnerable to non-specific signal interference. Although lacking in extreme sensitivity, the IRS approaches reviewed were more specific in signal generation and were considered to have considerable potential for the future.

Antibodies↗
PubMed1985

The interaction between bleomycin and radiation on cell survival and DNA damage in mammalian cell cultures.

Chinese hamster ovary (CHO) cells were exposed to various concentrations of bleomycin (BLM) for 0.5, 1, 2, 4, 6, or 24 hours, followed with graded doses (0 to 800 rad) or radiation. The response to this chemotherapy-radiation combination treatment was measured by cell survival studies and DNA damage as determined by alkaline elution assay. Isobolograms for 1 and 2 log cell kills showed that cytotoxicity from BLM and from radiation were additive at 4, 15 and 75 milliunit/ml (mu/ml) for 1 or 4 hour exposure. When the exposure time of BLM was extended to 24 hours, slightly supraadditivity of lethality was found for the combination treatment with pharmacologically pertinent concentrations of 4 or 15 mu/ml. Therefore 4, 15 and 75 mu/ml concentrations of BLM all interacted positively with all doses of radiation to give enhanced cell kill. The alkaline elution patterns from the BLM-radiation combination also showed an enhancing effect on single strand breaks of DNA from treated cells. A human oat cell carcinoma cell line (MEMAR cells) was also studied and found to be more sensitive to BLM than were CHO cells.

Animals↗
PubMed1985

Regrowth and radiation sensitivity of quiescent cells isolated from EMT6/Ro-fed plateau monolayers.

A quiescent [denoted as Q(G0/G1)] subpopulation was isolated from EMT6/Ro-fed plateau monolayers by centrifugal elutriation. The median Coulter volume of these cells was significantly smaller than that of the original population from which they were elutriated. Using two-step acridine orange staining and dual parameter flow cytometric analysis, over 95% of quiescent cells were found to have G1 DNA content, and 80% of the cells had a decreased RNA content as compared to rapidly proliferating exponential G1 cells. After labeling for 24 hr (two doubling times) with [3H]thymidine, less than 2% of the quiescent cells incorporated [3H]thymidine as measured by autoradiography. The colony-forming efficiency of these cells was not significantly different from that of exponential cells. When such Q(G0/G1) cells were replated in fresh medium at a lower density, there was a lag time of 30 hr before any increase in cell number was detected, after which the cell-doubling rate matched that of exponential culture. Results obtained from the radiation dose-response curves showed that quiescent (G0/G1) cells were more radiosensitive than exponential G1 or unseparated fed plateau cells.

Animals↗
PubMed1985

Further characterization of 4-bromomisonidazole as a potential detector of hypoxic cells.

[14C]Bromomisonidazole was prepared by direct bromination of [ring-2] [14C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 microM [14C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 microM [14C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [14C]misonidazole or [14C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind [14C]bromomisondiazole. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.

Animals↗
PubMed1984

Immunoassays at a quartz-liquid interface: theory, instrumentation and preliminary application to the fluorescent immunoassay of human immunoglobulin G.

The theoretical basis and instrumental requirements of an optical detection technique for monitoring antibody-antigen reactions at a quartz-liquid interface are described. The antibody is covalently immobilized on the optical surface of a planar, fused-quartz waveguide and reacted with antigen solution. A light beam is internally reflected within the waveguide and penetrates into the solution only a fraction of the wavelength of the incident light. This is the evanescent wave which interacts optically with the growing number of antigen-antibody complexes but minimally with the bulk solution. A two-site immunofluorescent assay for human IgG measurement is described using fluorescein as the label. The assay detection limit is approximately 0.8 micrograms/ml and individual fluorescence measurements are completed within 10 min. It is expected that this evanescent wave immunoassay should have wide applicability in both routine and research fields.

Antigen-Antibody Complex↗
PubMed1984

In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth.

The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system.

Animals↗
PubMed1984

Oxygen consumption and oxygen diffusion properties of multicellular spheroids from two different cell lines.

A recently developed method for evaluating pO2 gradients in multicellular spheroids enabled the determination of the volume-related O2 consumption rate Q and of Krogh' diffusion constant KS in EMT6- and in V79-spheroids on the basis of pO2 measurements with microelectrodes. In addition, estimates for the weight-related water content W, for the O2 diffusivity D and for the O2 solubility in spheroids could be derived from KS. The results show that O2 consumption and O2 diffusion properties of multicellular spheroids are greatly influenced by the cell line chosen. On the average, Q is larger in V79-spheroids than in EMT6-spheroids, whereas KS, W, D and alpha are lower in V79-spheroids than in EMT6-spheroids. There is a decrease in Q with increasing spheroid diameter in either spheroid type, and Q in both spheroid types is lower than the respective value in single cells. Using the data obtained, theoretical pO2-profiles can be calculated that are in good agreement with the measured steady state pO2 distribution in all spheroids investigated.

Animals↗
PubMed1984

Growth and cellular characteristics of multicell spheroids.

The data reviewed here demonstrate that there are many similarities in growth and cellular characteristics for different types of tumor cells grown as multicell spheroids. Furthermore, where comparisons have been made many of the features of spheroids also occur in tumors in vivo. However, as for tumors, there are also many characteristics of individual types of spheroids which are relatively specific and cannot be generalized as properties of all spheroid model systems. The results also demonstrate the marked influence which cellular microenvironments regulated by a supply of oxygen and nutrients may have on the development of cellular heterogeneity. Furthermore, using spheroids it was shown that dynamic cellular and metabolic interactions exist in regulating the development of cellular subpopulations and microenvironments. Spheroids are more sensitive to alterations in culture environment than are monolayer or single-cell suspension cultures. Consequently, researchers who use this model system must characterize, optimize, and standardize the growth conditions for the spheroid cell type being investigated. This information then provides a base from which to undertake detailed studies, which are not possible in experimental tumors, of controlled manipulation of microenvironments in spheroids. The ranges of cellular microenvironments and cellular heterogeneity which exist at different stages of spheroid growth provide a model, at least in part, for coexisting size ranges of microregions in many solid tumors. Thus, spheroids provide a model, which at different stages of growth is readily manipulated and controlled experimentally, to facilitate studies of contributions of individual environmental factors, or concomitant changes in these, on cellular phenotypic expression. It is probable that the cellular changes which can be demonstrated to occur during spheroid growth, also occur in vivo. Modulation of cellular characteristics revealed by research with spheroids requires much more study to determine the mechanisms and effects on tumor cell behavior, as well as response to therapeutic agents and their relevance to tumors in vivo.

Animals↗
PubMed1984

Optical detection of antibody-antigen reactions at a glass-liquid interface.

We describe an optical technique for detecting and monitoring antibody-antigen reactions at a solid-liquid interface. The antibody is covalently immobilized onto the surface of either a planar (microscope slide) or cylindrical (fibre optic) waveguide made of fused quartz. The reaction of immobilized antibody with antigen in solution is detected through use of the evanescent wave component of a light beam, which has a characteristic depth of penetration of a fraction of a wavelength into the aqueous phase, thus optically interacting primarily with substances bound (or located very close) to the interface and only minimally with the bulk solution. This resulting in-situ spatial separation of the antibody-bound from free antigen precludes a formal separation step and allows the reaction to be monitored kinetically. An immunoassay for methotrexate by absorption spectrometry achieved a detection limit of about 270 nmol/L; binding of methotrexate by immobilized antibody was monitored by the decrease in transmittance at 310 nm. A two-site immunofluorometric assay for human IgG could detect as little as 30 nmol/L; binding of fluorescein-labeled antibody was monitored by the increase in signal above 520 nm (lambda ex = 495 nm). With both immunoassays the signal-generating phase was monitored kinetically and was completed within 15 min.

Antigen-Antibody Reactions↗
PubMed1983

Frequency distribution histograms of oxygen tensions in multicell spheroids.

Recessed oxygen sensitive microelectrodes have been used to measure oxygen tensions in spheroids from EMT6/Ro mouse mammary carcinoma cells and from V-79-171B hamster lung fibroblasts. During the experiments the spheroids were exposed to conditions similar to those prevailing during growth and previous experiments with radiation and/or drugs. Frequency distribution curves were generated from 386 steady state readings in EMT6 (N = 20) spheroids with diameters ranging from 386 to 1900 micron and from 289 steady state readings in V79 spheroids (N = 20) with diameters between 376 and 1052 micron. Small spheroids exhibit higher pO2 values than spheroids of medium and large size. Oxygen tensions in small and medium size V79 spheroids are lower than in EMT6 spheroids of the equivalent size ranges. The pO2 histograms in large spheroids of both cell types are similar to pO2 frequency distributions in solid tumors. It is concluded that spheroids may represent a valuable model to provide evidence for the way pO2 histograms are influenced by the density of capillaries, by the cellular O2 consumption and by the sampling technique.

Animals↗
PubMed1983

Use of a sedimentation column to obtain uniformly sized populations of multicell spheroids.

Multicell spheroids are useful as in vitro models for certain nodular tumors. Spheroids may contain subpopulations of cells that are necrotic, hypoxic, and redistributed through the cell cycle, and the relative fractions of these subpopulations can change as the spheroids grow. As a result, spheroids of different sizes may respond differently to any given treatment. For experimental studies it is important to use populations of spheroids of homogeneous size. Here we report the design and use of a sedimentation column that uses precision woven screen to obtain homogeneously sized populations of multicell spheroids quickly, accurately, aseptically, and reproducibly. This device may be applied in other biological fields also where separation of cell aggregates of a specific size is required.

Animals↗
PubMed1983

Recommendations for using the standardized terminology of respiratory physiology in radiation research.

A succinct review of the terminology and of correlations between basic quantities in respiration physiology is given with special regard to oxygen as the gas of topical interest in radiation research and tumor pathophysiology. The role of the oxygen partial pressure gradient as the driving force of O2 diffusion is emphasized. It is generally recommended that the O2 partial pressure be considered when investigating oxygen diffusion and distribution problems. During those studies the physical boundary conditions, in particular the temperature, the barometric pressure, and the water vapor saturation, have to be considered and should be indicated. During equilibration of suspensions containing oxygen-consuming sites the impact of geometry and fluid agitation on the efficiency of gas exchange has to be taken into account. Reviews on solutions of diffusion equations, on numerical data for relevant constants to be considered, and on terminology and units in respiration physiology are included.

Animals↗