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At least 343 records · Page 19Linked to original sources
PubMed1985

The use of multicellular spheroids in establishing human sarcoma cell lines in vitro.

By a new procedure stable monolayer cultures were derived from spheroids in 8 out of 17 different human sarcomas (16 soft-tissue and I osteogenic sarcoma). Eleven of the sarcomas were obtained from patients undergoing surgery, and 6 from BALB/c nude mice carrying s.c. growing xenografts. The new procedure involves aggregation of single-cell suspensions into spheroids and cultivation of these in agar-coated flasks until the growth rate levels off, at which time the spheroids are transferred to uncoated flasks. Cells proliferating from the rim of adhering spheroids are trypsinized and aggregated to form new spheroids. By 3 to 5 such alternations, monolayer cultures were obtained that have now been subcultured for about 6 months. The cell lines all gave rise to colonies in a clonogenic soft-agar system, and upon s.c. injection into athymic nude mice 3 lines tested formed growing tumors. The histology of spheroids formed from late monolayer passages closely resembled that of the original tumors. That the new procedure is superior to other methods of establishing sarcoma cell lines is indicated by the fact that a stable monolayer culture could be obtained directly from the tumors in only 1/8 cases where the above procedure was successful, and in only 2 instances from soft-agar colonies derived from the tumors.

Adolescent↗
PubMed1995

Integrin expression on colorectal tumor cells growing as monolayers, as multicellular tumor spheroids, or in nude mice.

In this study we compared the expression of integrin alpha chains 2, 3, 4, 5, 6, v and the beta chains 1, 3, 4 in 2 colorectal carcinoma cell lines (HRT-18 and CX-2), growing in confluent and subconfluent monolayer cultures, as multicellular tumor spheroids and in nude mice, using the immunofluorescence technique (confocal microscopy) and flow cytometry. The fast-growing cell line HRT-18 expressed, in confluent and subconfluent monolayer cultures, alpha 2, 3 and beta 1 with a continuous membranous staining pattern, whereas alpha v, alpha 6, and beta 4 were expressed continuously membranous in the intermediate and apical part of the cell layer, and clustered at focal contacts at the base of the cells. In spheroids and tumors of nude mice the focal pattern of alpha v, 6 and beta 4 was changed into a diffuse one. Using flow cytometry, the expression of alpha 3 was found to be reduced in spheroids of HRT-18. The slowly-growing cell line CX-2 expressed, under the same conditions in monolayer culture, alpha 6, beta 1 and beta 4, and very weakly alpha 2, 3, 5 and v. Alpha 3 was expressed in spheroids of CX-2 only at the outer rim where the cells proliferate. In contrast, alpha 2 and 5 were expressed mainly in the quiescent, non-proliferating area. Alpha 6 was reduced in spheroids of CX-2. In the nude mouse tumor of CX-2, alpha 5 was expressed only focally and very weakly, alpha 2 was no longer detectable, but alpha v appeared to be enhanced in a focal pattern. These data indicate that integrin expression of tumor cells depends upon the culture system and that integrin expression in multicellular tumor spheroids is more similar to the in vivo situation in nude mouse tumors.

Animals↗
PubMed1981

Shedding of mitotic cells from the surface of multicell spheroids during growth.

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exponential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytofluorometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.

Animals↗
PubMed1990

A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy.

We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.

Animals↗
PubMed1993

Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.

Normal adult human dermal fibroblasts were cultured at 37 degrees C on a thermoresponsive substratum composed of poly-N-isopropyl acrylamide (PNIPAAm) and type I collagen. The confluent fibroblasts were forced to detach from the substratum as a monolayer cell sheet made of polygonal cells linked together with many microvilli by decreasing an ambient temperature from 37 degrees C to a temperature below the lower critical solution temperature of PNIPAAm (about 30 degrees C). The floating cell sheet shrank and condensed into an aggregate with gap and tight junctions within several hours and finally formed a spheroid by 2 days. Spheroids were covered with squamous cells and contained cuboidal cells inside. Cycloheximide and actinomycin D reversibly inhibited the spheroid formation. Extracellular matrix fibrils deposited among inner cells when the culture period extended over 7 days. A 28-day-old spheroid with a diameter of about 600 microns contained live cells even in the central region. Cellular metabolic activities such as glucose consumption, lactic acid production, and ATP content of spheroids were significantly lower than those of monolayers. The spheroid described in the present study seems to be a useful in vitro model of connective tissues.

Adenosine Triphosphate↗
PubMed2002

Structural polarity and functional bile canaliculi in rat hepatocyte spheroids.

Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).

Animals↗
PubMed1997

Gravitational Settling in the Electrophoresis of Spheroids with Small Potentials in Quiescent Fluid

Electrophoretic motion is analyzed for non-neutrally buoyant, spheroidal particles settling in an unbounded fluid otherwise at rest. The double-layer thickness is arbitrary, and the surface potential of the spheroid is assumed to be small so that the deformation of the double-layer is neglected. Examples are presented for spheroids when the applied electric field is perpendicular to the gravity. The electrophoretic motions are determined for spheroids of arbitrary aspect ratios at arbitrary orientations. Depending on their orientations the gravitational drift can contribute up to 10% of the observed particle velocity in the direction of the electric field. The gravitational contribution is maximum when the angle between the axisymmetric axis of spheroids and the gravity is pi/4. However, for Brownian spheroids with a uniform orientation distribution the sedimentation effects are averaged out, thus there is no net gravitational drift in the direction of the applied electric field.

Journal Article↗
PubMed1991

High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam.

Adult rat hepatocytes formed spherical multicellular aggregates (spheroids) when they were cultured in the pores of polyurethane foam (PUF). The diameter of the spheroids was within the range 100-200 microns. These spheroids partly attached and immobilized in the PUF pores for at least 2 weeks. The albumin production rate by the spheroids increased up to 17.0 micrograms/10(6) nuclei per day during the first 6 days and maintained at a high level for 2 weeks. In contrast, the albumin production rate by the monolayer markedly decreased after 3 days. The spheroid culture using PUF seems to be a convenient and simple method for maintaining some differentiated functions of hepatocytes and for making a bioreactor using the function of spheroids.

Albumins↗
PubMed1984

Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids.

The kinetics of the penetration and binding of the two commonly used antitumour drugs vinblastine and 5-fluorouracil in nonvascularized tumour tissue were studied. Multicellular human tumour spheroids (glioma U-118 MG and thyroid cancer HTh-7) were used as a model system. Radiolabelled drugs were used in all studies. To avoid disturbances in the distribution of unbound drugs a dry histological technique was used in combination with contact autoradiography. In addition, quantitative measurements of the accumulation and binding of the drugs were made. The results showed that vinblastine penetrated the spheroids less efficiently than 5-fluorouracil. Vinblastine required about 2 h to be isotropically distributed within the studied spheroids, while only a few minutes were required for 5-fluorouracil. Vinblastine seemed to be accumulated in the peripheral parts of the spheroids within 15 min. High concentrations of 5-fluorouracil, isotropically distributed in the spheroids, were observed after 2 h of incubation. Significant amounts (about half) of the accumulated drugs resisted gentle washing for 3 X 20 s plus 15 min in fresh medium. The limited penetration of vinblastine correlated well with a previously observed high resistance of spheroids to treatments of short duration with this drug.

Cell Line↗
PubMed1994

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes.

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic "surface" epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.

Biomarkers↗
PubMed1991

Calcitonin gene-related peptide immunoreactivity in spinal spheroids in motor neuron disease.

The spinal cords from seven autopsy cases of sporadic motor neuron disease (MND) and two controls were immunohistochemically examined using antibodies directed to calcitonin gene-related peptide (CGRP) and to neurofilament proteins (Nf). CGRP immunoreactivity was observed in the posterior horns, especially in the laminae I and II, of all the spinal cords examined. In MND cases, in addition, a considerable number of spheroids in the anterior horns were labelled with the antibody. In some spheroids, their entire area was homogeneously immunostained, whereas in others the immunoreactivity was confined to a focal area(s) within the profile of the spheroids and between these two forms of staining several variations of the staining patterns were seen. The anti-Nf intensely and homogeneously labelled all spheroids and there was no appreciable difference in the Nf-immunoreactive pattern between CGRP-positive and -negative spheroids. It is possible that the accumulation of CGRP in spheroids may result from entrapment of CGRP during the anterograde axonal transport, causing loss of CGRP at the neuromuscular junction and producing weakness and atrophy of the muscles.

Aged↗
PubMed1978

Morphological investigations on axonal swellings and spheroids in various human diseases.

Axonal swellings and spheroids in various human diseases were studied by light and electron microscopy. 4 cases of infantile neuroaxonal dystrophy, 2 of degenerative diseases, 2 brain tumors and 3 of cerebrovascular disease were examined. Ultrastructurally most spheroids in infantile neuroaxonal dystrophy consisted of interconnected tubules, stacked membranotubular profiles, alternating layered membranes and accumulations of neurofilaments. Combinations of these four constituents were seen only in infantile neuroaxonal dystrophy. "Torpedos" (fusiform swelling of the axon of a Purkinje cell) consisted exclusively of neurofilaments. Spheroids in case 6 (mental retardation) and 7 (atypical teratoma) consisted of interwoven skeins of neurofilaments and grouped mitochondria. Spheroids in case 8 (demyelination) and 9 (cerebrovascular disease) consisted of packed complex bodies and mitochondria. Spheroids in cases 10 and 11 (cerebrovascular disease) consisted of degenerating organelles only. The morphological differences between cases 9, 10 and 11 probably depends on the severity and timing of the cerebral injury. Most spheroids show similar histological and histochemical properties, but ultrastructural study may give some clue to the origin of the bodies.

Adult↗
PubMed1990

Clonogenicity of mammalian cells in hybrid spheroids: a new assay method.

A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillary melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.

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↗
PubMed1989

Use of a laser diffraction particle sizer for the measurement of mean diameter of multicellular tumor spheroids.

Increasing use is being made of tumor cell lines cultured as cell aggregates (generally referred to as multicellular spheroids) in in vitro radiosensitivity and/or chemosensitivity tests. Conventional procedures for the determination of mean spheroid diameters for the construction of growth delay curves employ a microscope-image analyzer. However, this approach can prove excessively time consuming when large numbers of samples have to be measured. We have, therefore, been exploring the use of a Laser Diffraction Particle Sizer, the Malvern 2600 long bench model, for the measurement of mean spheroid diameter and size distribution. We report here a direct comparison between measurements carried out by the instrument and under the microscope. Also a comparison of growth curves for six cell lines constructed from measurements by the microscope and by the instrument. A number of factors that might affect the accuracy of spheroid diameter measurement by the instrument have been investigated: The effect of stirring to maintain the spheroids in suspension during measurement. Sampling error due to removal of a series of spheroid samples from culture flasks for measurement. Optimum numbers of scans to be carried out by the instrument to reach a constant value for mean diameter, and minimum SE of the mean.

Animals↗
PubMed1979

Morphological and functional characteristics of cells infiltrating and destroying tumor multicellular spheroids in vivo.

EMT6 mammary sarcoma cells were grown in vitro as multicellular spheroids to model for the heterogeneity of microenvironments and structural changes which develop in many tumors, including micrometastases. Spheroids of 700-900 micron diameter were implanted into and recovered at different times from the peritoneal cavities of sensitized or nonsensitized allogeneic and syngeneic mice. The colony forming efficiency of spheroid tumor cells recovered at 24 and 48 h from sensitized allogeneic mice was markedly decreased as compared with those from nonsensitized allogeneic or syngeneic animals. These recovered spheroids were extensively infiltrated by both lymphocytes and macrophages, which ultrastructurally had very close membrane associations with tumor cells. Host cells recovered from spheroids exhibited cytotoxic activity in an in vitro 51Cr release assay. Thus, multicellular spheroids in vivo provide a unique experimental model to study the functional capacity of host cells within a spheroical tumor. Although lacking the stroma and the vasculature of in vivo solid tumors, this model does have many similarities to in vivo tumors and is thus suitable for studying the tumor cell-host cell interactions within the tumor microenvironment. In addition, the system offers the potential for quantitative study of the effects of treatment modalities on tumor cell-host cell interactions.

Animals↗
PubMed1997

Superoxide dismutase-like immunoreactivity in spheroids in Hallervorden-Spatz disease.

Iron accumulation in the basal ganglia and spheroid formation are pathological hallmarks of Hallervorden-Spatz disease (HS). Since an overaccumulation of iron (iron thesaurosis) that exceeds the binding capacity of ferritin could cause oxidative damage, we studied the possible role of oxidative stress in the pathogenesis of HS. The basal ganglia and spinal cord from patients with HS were investigated at autopsy, using histochemistry for iron and immunohistochemistry for Cu/Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2) and ferritin. SOD1-like immunoreactivity (IR), SOD2-IR and ferritin-IR occurred frequently in spheroids observed in the basal ganglia, and associated iron accumulation indicated the possible existence of increased oxidative stress in HS patients. Spheroids in the spinal cord showed intense SOD1-IR and SOD2-IR in HS, in sharp contrast with the occasional weak SOD1-IR and SOD2-IR observed in spheroids from patients with amyotrophic lateral sclerosis (ALS). Neither increased ferritin-IR nor iron accumulation were observed in spinal spheroids from HS and ALS patients. These data may suggest that, at least in the spinal cord, SOD1-IR and SOD2-IR in spheroids in HS patients do not result from oxidative stress directly related to iron accumulation.

Adult↗
PubMed1984

Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model.

In situ host immunity to the EMT6/Ro mammary sarcoma tumor was evaluated by implanting multicellular spheroids of this tumor into the peritoneal cavity of syngeneic BALB/cKa mice and determining the kinetics of host cell infiltration and tumor cell killing. Spheroids grown in vitro and implanted into unsensitized mice continued to grow resulting in peritoneal tumor masses and eventual death of the animal. However, in mice previously sensitized with a single injection of heavily irradiated EMT6/Ro cells, spheroids implanted intraperitoneally were rapidly infiltrated by host immune cells (macrophages, lymphocytes, and granulocytes), tumor cell killing was detectable within 1 day and by Day 6 essentially no clonogenic tumor cells were recoverable. Despite this marked loss of both total and clonogenic tumor cells, there was little decrease in the diameter of the spheroids recovered during this time period. Physical size thus does not provide a reliable estimation of tumor cell killing. The tumor cell killing was immunologically specific in that little killing was observed when EMT6/Ro spheroids were implanted into mice sensitized with other allogeneic or syngeneic tumor cells. Host cells from within the spheroids were found to be cytotoxic for EMT6/Ro tumor cells in a 51Cr release assay. A major portion of these cytotoxic cells appear to be T lymphocytes. However, other host cell types may also be involved in the in vivo tumor cell killing.

Animals↗