Institute of Physiology and Pathophysiology, Johannes Gutenberg-University Mainz, D-55099 Mainz, Germany
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ABSTRACT |
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This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.
tumor spheroids; tissue modeling; embryoid bodies
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INTRODUCTION |
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THREE-DIMENSIONAL (3-D) cell cultures have been widely used in biomedical research since the early decades of this century. Although preceded by work of other scientists, Holtfreter (62) and later Moscona (104-107) pioneered the field by their systematic research on morphogenesis using spherical reaggregated cultures of embryonic or malignant cells. Numerous subsequent studies were based on these investigations with regard to techniques and strategies for understanding organogenesis or expression of malignancy. The spectrum of research on cell aggregates was enlarged considerably by the fundamental studies of Sutherland and associates (64, 145-147), who inaugurated multicellular tumor spheroids as an in vitro model for systematic studies on tumor cell response to therapy. As a consequence, therapeutically oriented studies became the major domain of research with cell spheroids, although such investigations also triggered a number of studies on basic biological mechanisms, such as the regulation of proliferation, differentiation, cell death, invasion, angiogenesis, or immune response (for reviews see Refs. 9, 74, 108, 144).
At present, research on 3-D cell systems is more vital and productive than ever. The potential of 3-D cell cultures is currently being exploited in so many areas of biomedical research that it is impossible to review all aspects of these studies completely. Nevertheless, one reason for the recent progress in research on multicell systems may be the increasing interaction between researchers working in different fields of biomedical science and using similar 3-D culture techniques. Over the past 5 years, there has been an increasing number of publications with molecular and cellular biologists, cellular and neurophysiologists, as well as clinical scientists as joint authors, and work on 3-D cell systems has been published in scientific journals of outstanding ranking. Such a research effort mirrors the common need for improved and more refined in vitro models as a link between cell-free systems or single cells and organs or whole organisms in vivo.
One major advantage of 3-D cell cultures is their well-defined geometry, which makes it possible to directly relate structure to function and which enables theoretical analyses, e.g., of diffusion fields. Consequently, the most promising data on these cultures may be obtained with techniques allowing for spatial resolution. Combining such approaches with molecular analysis has clearly demonstrated that, in comparison with conventional cultures, cells in 3-D cultures more closely resemble the in vivo situation with regard to cell shape and cellular environment, and shape and environment can determine gene expression and the biological behavior of the cells. One impressive example demonstrating the significance of the environment is the finding that ectopic implantation of embryonic cells can transform them to malignancy and gives rise to cancer, whereas the same cells lead to normal embryogenesis in the uterus; conversely, teratocarcinoma cells may undergo normal development when implanted into an embryo (97). From a critical point of view, it should be kept in mind that the complexity of 3-D cell systems is not only an advantage but also a limitation. There will always be a number of questions that can only be answered by investigations using single cells or cell-free systems. At the same time, 3-D cultures cannot completely replace the testing of biological mechanisms for their relevance in vivo, e.g., in knockout animals.
The present review is based on a selection of publications, mainly from the past 5 years, dealing with 3-D cell cultures. Besides "classical" topics of research on multicellular tumor spheroids, new developments and efforts in experimental tissue modeling and in the generation of artificial organs are considered. Additionally, recent progress in organogenesis using embryoid bodies is discussed.
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EXPERIMENTAL THERAPEUTICS |
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After intensive research on radiation effects in multicellular tumor spheroids in the 1970s and 1980s, the number of systematic studies in this field has decreased considerably during the first half of the present decade. A detailed and systematic report has been issued by Stuschke and colleagues (143), who identified the degree of differentiation as an important determinant of radioresponsiveness in various spheroid types of human origin. Thus spheroids mirror the radiosensitivity of differentiating tumors in vivo more closely than conventional cell cultures. In another recent example for the exploitation of specific properties of spheroids in radiobiology, Fritz and associates (43) have shown the benefit of certain fractionation schemes in a study on cell cycle effects of dose rate and superfractionation using V79 cells grown as monolayers or spheroids. Regrowth has been demonstrated by Durand (32) to be a dominant variable during fractionated radiotherapy using V79 spheroids.
In a cooperative research effort, the groups of Kerbel and Teicher (52, 75) demonstrated a multicellular-mediated resistance to alkylating drugs. Drug resistance induced in EMT6 tumors in mice was completely lost when the cancer cells were isolated and grown in monolayers but could be fully recapitulated when cells were cultured as multicellular spheroids. This has implications both for tumor treatment in the clinic and for the design of drug testing in vitro. A similar resistance to ionizing radiation was identified more than two decades ago by Durand and Sutherland (33) who created the term "contact effect" for this phenomenon. Considering the recent demonstration of a contact effect for drugs, it seems important and challenging to investigate the molecular mechanisms underlying such a resistance. Recent studies of drug and radiation resistance in spheroids have been reviewed by Olive and Durand (117). As a synopsis of the mechanisms possibly involved in the contact effect, the authors conclude that intercellular communication via gap junctions may not contribute significantly to this effect, even though, in the literature, this mechanism has been assumed to cause contact resistance for some time. Predominantly on the basis of work from their own groups, the authors favor the specific nuclear shape and DNA packing in spheroids with a more efficient DNA repair to be the major reason for multicellular resistance. Although expression and constitutive activity of repair enzymes seem to be unaffected by multicellular growth conditions, the change in the accessibility of the substrate to these enzymes appeared to be improved in spheroids compared with single cells, which may explain an increased activity of these enzymes in situ.
Hypoxia poses many medical problems in a variety of biological tissues, including the induction of radioresistance in solid tumors. With the use of techniques that are applicable in the clinic, the detection of hypoxic cells is associated with problems that have not yet been sufficiently solved. Recent progress has been made by the development of a new hypoxic marker, on the basis of the well-known oxygen-dependent metabolic activation of nitroheterocyclic drugs (88). The major improvement with the new drug EF5 is a considerable reduction of oxygen-independent adduct formation, which had generated a large variability in background labeling in different tissues when previous designs of nitroimidazoles were used. The authors demonstrated the hypoxia specificity of EF5 in multicellular spheroids. Furthermore, it was shown that EF5 accumulated in the inner hypoxic regions of HT-29 human colon adenocarcinoma spheroids and colocalized with elevated expression of vascular endothelial growth factor (VEGF) (159). This work illustrates the potential of using spheroids in studies on the regulation of genes that can be induced by hypoxia or other specific environmental factors. Spheroids were also used for in situ calibration of nitroimidazole binding by the combined use of autoradiographic labeling and oxygen microelectrodes (54). The authors suggest from their results that such calibration curves of grain density vs. oxygen tension can be used for measuring oxygen pressure in tissues, if the drug binding level of fully oxygenated regions can be determined. From these recent investigations with hypoxic markers, one may conclude that channeling research efforts into this field may still be worthwhile. Thus the generation of calibration curves in spheroids to be used for oxygen pressure measurements in tissues and the detection of novel markers such as EF5 [possibly with 19F labels using nuclear magnetic resonance (NMR) or with 18F labels using positron emission tomography] should all be tested for suitability and practicability.
Another recent approach to quantification of hypoxia in tissue samples, which has been applied intensively to multicellular spheroids, is the so-called comet assay that was reviewed by Fairbairn et al. (39). The method with several different versions allows for the determination of DNA strand breaks in individual cells and may be used to measure the radiobiologically hypoxic fraction in tumors and normal tissues (118).
Spheroids have been involved in recent efforts to increase the efficiency of tumor-targeted radionuclide therapy, as reviewed by Wheldon (166). The efficiency and specificity of radionuclide treatment for certain tumor entities have been documented in spheroids from human neuroblastoma NB1-G cells (162), human glioma U-343MgaC12 cells, and human prostatic adenocarcinoma DU 145 cells (37). In these studies, 125I- or 131I-labeled antibodies were used that were directed against neurectodermal antigens of neuroblastomas or were conjugates based on epidermal growth factor (EGF). Radiation doses were achieved that were associated with a substantial sterilizing effect, the extent of which could be predicted on the basis of theoretical considerations. In similar spheroid studies, Mairs et al. (92) arrived at the conclusion that metaiodobenzylguanidine with no carrier added would achieve a significant therapeutic gain in neuroblastoma treatment compared with conventional radiopharmaceuticals. These conclusions could be confirmed by in vivo experiments with immunodeficient animals. The penetration of the EGF-based antibodies into spheroids was very sensitive to antibody binding and could be enhanced by saturation of peripheral binding sites or by increasing the external antibody concentration. This finding is in accordance with the "binding site barrier" phenomenon that was demonstrated, for example, in guinea pig micrometastases (129) and that is considered a major obstacle to the satisfactory monoclonal antibody treatment of bulky cancers. Using anti-carcinoembryonic antigen antibodies and respective Fab fragments in human colon adenocarcinoma spheroids, Langmuir and colleagues (81) demonstrated very clearly and systematically that antibody size and receptor density were the main determinants of antibody penetration, whereas tumor cell architecture appeared to have only a minor impact.
Several investigators have used glioma spheroids for studying the interaction of cancerous tissue with defense cells and in particular with lymphokine-activated killer (LAK) cells that have been generated by incubating peripheral blood lymphocytes with interleukin-2 (65). The clinical background of these studies is the general resistance of malignant gliomas to treatment with LAK cells or recombinant interleukin-2. Penetration of LAK cells into multicell spheroids from glioma cell lines was relatively poor, although cellular damage was documented at a distance from the invading LAK cells, which may be interpreted as an effect of membrane-damaging agents released by these cells (65, 66). Penetration of LAK cells was much better when "organotypic" glioma spheroids were used that were obtained from continuous cultures of tissue specimens (67). The authors were able to demonstrate a substantial difference in both penetration and toxicity between LAK and peripheral blood cells, which makes their model attractive for mechanistic studies with immunotherapy. This finding also shows possible limitations of spheroids in comparison with established cell lines: stromal elements, such as extracellular matrix, may be synthesized by the aggregated tumor cells or may be induced by cocultures with fibroblasts or by reversible implantation in animals. However, such an "extracellular matrix" or "microvasculature" may still not adequately mimic the situation of the respective stroma in vivo at least in some specific experiments.
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METABOLISM AND METABOLIC ENVIRONMENT |
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One "classical" incentive for using multicellular tumor spheroids was their similarity with the initial, avascular growth stage of malignancies, with micrometastases, and with intercapillary tumor microregions (reviewed in Refs. 108 and 144). The concentric arrangement of heterogeneous cell populations in spheroids with proliferative cells at the periphery, an intermediate zone with viable and clonogenic, yet quiescent cells, and a necrotic core in the center is clearly suggestive of a diffusion-limited tissue model. It is therefore not surprising that for many years the development of central necrosis in spheroids was attributed to an insufficient oxygen supply; similarly, it was anticipated that the emergence of cellular quiescence was a consequence of hypoxia. Early experimental evidence suggesting that these assumptions may not be true (21, 110, 111) has often been ignored. Currently, a large amount of data from various laboratories shows that proliferation arrest in spheroids is not elicited by depletion of substrates for energy metabolism and that the same is true, with a few exceptions, for the emergence of necrosis. Some of these data are briefly reviewed here. Investigations by Acker et al. (2) and Carlsson and Acker (20) indicate that there is no single factor, such as hypoxia, lack of nutrients, accumulation of waste products, or low pH, that alone is responsible for the development of necrosis. The authors speculate that a combination of some or all of these factors may lead to a "toxic environment," in which cells will no longer be able to maintain their intracellular homeostasis. The results of recent measurements of oxygen tensions and concentrations of glucose, lactate, and ATP in multicellular spheroids (17, 152, 161) argue against the hypothesis of Acker and colleagues. Obviously, most of the spheroids that are commonly used for such studies do not have a very hostile metabolic milieu at the time of the first emergence of necrosis, although hypoxia may develop during a further increase in spheroid diameter at a later stage of cultivation. It has been demonstrated previously that cells can adapt their metabolism to the specific environmental conditions in a 3-D arrangement by reducing their metabolic turnover rates (41, 109). This may explain the relatively shallow metabolic gradients seen in spheroids before the development of necrosis. Such shallow gradients are reflected by relatively small differences between external and central concentrations of metabolites or between external and central oxygen tensions in smaller spheroids. The ATP content in central regions of EMT6 spheroids decreased from levels of 1-3 µmol/g, commonly observed in single cells, to 0 µmol/g during spheroid cultivation (161). However, this drop was preceded by the incidence of massive cell death in the spheroid center, which suggests that ATP is hydrolyzed as a consequence of cell death and that a critical decrease in cellular ATP levels can be excluded as a mechanism involved in the induction of necrosis. These observations agree well with 31P-NMR measurements by Freyer et al. (42), who found a relatively constant level of energy-rich phosphates in EMT6 spheroids until cells were very close to death. Similarly, these studies confirmed that the emergence of cellular quiescence was not related to a decrease in nucleotide triphosphate content or pH.
There are certain types of spheroids that are different from those described above with regard to mechanisms involved in the induction of cell death. Monz et al. (103) have shown that the first emergence of necrosis and hypoxia coincide during growth of WiDr human colon adenocarcinoma spheroids. In this case, hypoxia may be the predominant necrotizing factor, but this may be only coincidental. Theoretical considerations suggest that one single limiting factor, such as lack of oxygen, can explain the kinetics of the development of central necrosis during spheroid growth (53), which would support the former interpretation. On the other hand, Kunz-Schughart et al. (78) have demonstrated a remarkable tolerance of hypoxia in spheroids from myc/ras-cotransfected embryonic rat MR1 fibroblasts where central oxygen pressure dropped to 0 mmHg, which is more than for 24 h before the first appearance of necrosis. This result argues in favor of the latter interpretation of the WiDr data or indicates that the development of necrosis in spheroids is a multifactorial event.
Knowledge of processes that are involved in the development of necrosis has been expanded considerably by the generation of spheroids from clonal rhabdomyosarcoma cells (69). These spheroids consist of a mixture of undifferentiated, mononuclear cells and of myotube-like, multinucleated giant cells that are formed by fusion of undifferentiated cells, as illustrated in Fig. 1. Although giant cells occur preferentially in the spheroid center, central necrosis does not emerge even at spheroid sizes of 1 mm and larger. Moreover, proliferating mononuclear cells can be identified by autoradiographic [3H]thymidine labeling in the center of such big spheroids, and the difference in the thymidine labeling index between peripheral and inner regions is much smaller than in all other spheroid types investigated up to now. Obviously, the incidence of differentiation is inversely correlated with the emergence of necrosis in these aggregates. This is true despite the fact that central oxygen pressure is very variable and very low in some of these spheroids as illustrated in Fig. 2. The large variability in central oxygen tension that is not found in undifferentiated spheroids can be explained by the pronounced variability in the proportion of giant cells in central spheroid areas, since there is an inverse relationship between these two quantities (see Fig. 3). Because myotube-like differentiation can be induced in monolayer cultures of these cells by an acidic rather than a hypoxic environment (unpublished data), the correlation shown in Fig. 3 may be the result of an increased induction of myotube-like differentiation by the acidic environment in central spheroid regions. This may be associated with a higher volume density of oxygen-consuming sites, higher local oxygen consumption, and lower steady-state oxygen tensions. The relevance of the numerical volume density of mitochondria for the local oxygen consumption rate and the steady-state oxygen pressure distribution in spheroids has been clearly demonstrated in previous studies (17). The data obtained in rhabdomyosarcoma spheroids illustrate how environment can determine the expression of a certain phenotype and how this expression can in turn determine the microenvironment. Such spheroids may therefore represent excellent models for studying the interaction between gene expression and micromilieu.
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When the rhabdomyosarcoma spheroids are grown for extensive lengths of time, necrosis may eventually arise in central portions of the aggregates. This event is preceded by the occurrence of apoptosis mainly of the giant cells. This is illustrated in Fig. 4, showing a histological section through a rhabdomyosarcoma spheroid containing multinucleated giant cells, some of which are apoptotic. Apoptoses are visualized by immunostaining of DNA fragments with the TdT-mediated dUTP nick end labeling (TUNEL) technique (47). Such a sequence of events can also occur in undifferentiated spheroids, as shown for aggregates of V79 hamster lung cells in Fig. 5, A and C, where apoptoses were also labeled with the TUNEL technique in comparison with a general DNA staining with 4',6-diamidino-2-phenylindole dihydrochloride (Fig. 5, B and D). In this case it is obvious that apoptotic death is a single cell event that is distributed more or less uniformly within small spheroids and that can hardly be detected in V79 monolayers. With increasing spheroid size, apoptoses accumulate in the spheroid center and eventually "blend" together to form secondary necroses. Fragmentation of DNA in V79 spheroids can also be demonstrated by "laddering" in gel electrophoresis, as illustrated in Fig. 6. Because DNA fragmentation is not entirely specific for apoptosis, ultrastructural investigations with electron microscopy are required to supplement these studies on spheroids in the future. The signal for the induction of apoptosis in spheroids is not known to date, but induction of massive apoptosis by 3-D growth conditions has been demonstrated recently by Rak et al. (125) in spheroids of intestinal epithelial cells. Chemosensitization and induction of apoptosis by the retroviral wild-type p53 expression vector have been reported for p53-defective human lung cancer spheroids (44, 45). Recent data from our own laboratory indicate that oxidative stress may be involved in the induction of apoptosis in V79 spheroids, since glutathione is transiently increased during accumulation of apoptosis in central portions of spheroids (128).
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In summary, the development of central areas in spheroids with metabolically inactive and/or structurally disintegrated cells is a complex process that is not well understood. For many years, cell death in spheroids has been designated "necrosis," and hypoxia has been presumed to terminate the life of cells in the spheroid center. This may be true for some spheroid types, such as those derived from WiDr colon adenocarcinomas, but there is a variety of spheroid types that develop necrosis in the absence of hypoxia or nutrient deprivation. Currently, evidence is accumulating for the occurrence of apoptosis with increasing frequency in an early stage of spheroid growth and for this to be involved in the emergence of central necrosis in the spheroid center. This is undoubtedly a controversial area, particularly due to methodological problems with the distinction between apoptosis and necrosis. Multicellular spheroids appear to be appropriate models for mechanistic studies in this field, and scientists may be encouraged by this discussion to use spheroids for the clarification of this critical issue.
Molecular aspects of growth regulation in spheroids of A-431 cells have
been investigated intensively by Mansbridge et al. (95, 96), who
demonstrated a reduced expression of EGF cell surface receptors and an
enhancement of tyrosine phosphatases in spheroids compared with
monolayers. Using the same squamous carcinoma cell line, Laderoute et
al. (80) showed that transforming growth factor- (TGF-
) synthesis
is more pronounced in these spheroids than in monolayer cells.
Microenvironment and cell shape appear to be responsible for these
changes.
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MATHEMATICAL MODELING |
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A traditional field of research on spheroids is mathematical modeling, and advances in this area are still being made. This field is not extensively covered by this review but rather is exemplified by a few recent approaches. Using theoretical considerations, Chaplain and associates (22) have analyzed the impact of nonlinearity in either diffusion or production of a growth-inhibiting factor secreted by the spheroid cells on spheroid growth. Duechting et al. (30, 31) have used spheroids for the simulation of different treatment protocols for irradiation of tumors. From their theoretical considerations, the authors were able to predict many clinical observations and to give recommendations on ways of optimizing the therapy with regard to differential effectiveness of radiation in malignant and normal tissue. Tumor treatment with immunoconjugates has been modeled by Kwok et al. (79). They were able to derive macroscopic binding, dissociation, and diffusion constants for monoclonal antibodies in spheroids from experimental data that may be exploited in experimental studies for the improvement of targeting tumor cells with immunoconjugates.
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INVASION AND METASTASIS |
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One of the classical assays for studying invasion in vitro is the coculture of tumor spheroids with embryonic chick heart fragments, a model that is still used intensively in many laboratories. Recent advances elucidating molecular mechanisms involved in the invasive properties of cancer cells come from Mareel and colleagues (14, 119), who studied the invasion suppressor function of E-cadherin in MCF7 human mammary carcinoma spheroids. Using the same model system, Tritthart and colleagues (58, 139, 140) have investigated the anti-invasive properties of anticancer agents employing quantitative image analysis. The authors have developed an intriguing way of analyzing various aspects of cellular motility, such as directional migration, area and number of ruffling sites, and so forth, in mouse melanoma spheroids, and they were able to show in a quantitative manner how different anti-invasive agents were directed toward these different aspects of cellular invasiveness (60). An interesting study that used Rous sarcoma virus-infected rat cerebellar cells invading chick heart fragments showed that expression of the neural cell adhesion molecule NCAM is essential for spheroid growth but not for invasion (15) and that invasiveness of hamster glial cells is not necessarily linked to morphological differentiation induced by dibutyryl adenosine 3',5'-cyclic monophosphate (10). Makiyama et al. (93) grew spheroids from high- and low-metastatic clones of mouse sarcoma cells and demonstrated that the propensity of these cells to metastasize was correlated with their adhesiveness and invasiveness in the chick heart assay. To elucidate mechanisms that contribute to the low incidence of cancer in the lens of the eye, Messiaen et al. (101) have demonstrated that isolated lens cells acquire certain malignant properties under long-term culturing for >25 wk; thus cells became invasive in the chick heart fragment assay and tumorigenic in syngeneic animals. Even so, the stage of metastasis was never reached. Brauner and Hulser (16) have demonstrated that invasiveness may be associated with tumor cell-host cell interaction via gap junctions. Recently, this group has initiated very intriguing studies on the role of different connexins in cell-cell interaction and invasion at the single cell level (34, 156).
Recent developments of novel in vitro systems for invasion studies include 3-D organ cultures of human endometrium, which can be invaded by choriocarcinoma spheroids (55), and fetal rat brain aggregates as targets for the invasion by glioma spheroids either from cell lines (91, 121) or from primary material (36). The latter group of researchers has studied the influence of various growth factors on invasion and differentiation and was able to demonstrate a growth- and invasion-promoting effect of EGF on gliomas. Also, a barrier function of leptomeningial tissue against brain tumor cell invasion could be shown using 3-D culture systems (121). The potential of coculturing fibroblasts with human bladder cancer spheroids for mimicking invasion in vivo has been shown by Schuster et al. (133).
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CELL ADHESION |
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There is a vast amount of literature dealing with cell adhesion molecules and their role in cell aggregation. The majority of this work concentrates on defense cells and their homotypic and heterotypic aggregational behavior. It is not the purpose of this article to review even parts of these investigations, although some aspects of this area may be contained in some of the referenced papers. A second domain of research on adhesion molecules is the field of developmental biology, an area that is partially addressed in EXPERIMENTAL TISSUE MODELING and EMBRYOID BODIES. At this point, only some recent data on adhesion molecules in tumor spheroids and their relation to invasion and metastasis are briefly discussed.
In an elegant, quantitative study, Byers et al. (19) demonstrated how
tumor spheroids can be used to elucidate the role of adhesion molecules
in the dissemination of cancer cells from the primary lesion. Under the
influence of physiological shear forces, cell-cell adhesion in breast
and colon carcinoma spheroids was strong, if the cells expressed intact
E-cadherin, and was reduced in the absence of this molecule or in the
absence of a proper linkage of E-cadherin to the cytoskeleton. Initial
evidence that carcinoembryonic antigen molecules mediate the homotypic aggregation of colorectal carcinoma cells in malignant effusions of
patients comes from studies of Kitsuki et al. (73), which suggest a
possible involvement of the 1
integrin subunit. Various components of extracellular matrix (ECM) and
different integrins have been analyzed by Paulus et al. (120) and
Hauptmann et al. (56). In summary, these investigations show that
synthesis of ECM components and their receptors can be largely
influenced by the culture conditions; in general, expression of these
molecules in 3-D cell cultures reflects the in vivo situation much
better than that in monolayers. The findings of Sutherland and
colleagues (160) support these data by demonstrating a selective
downregulation of integrin receptors in squamous cell carcinoma
spheroids, which mimics the conditions in vivo. An interesting aspect
of manipulating cell adhesion for therapy has been shown by Reith et
al. (126). The authors were able to prevent an invasion of glioma
spheroids into fetal rat brain cell aggregates by incorporation of
exogenous laminin during the formation of these aggregates.
Systematic work on adhesion and ECM molecules in tumor spheroids is rare, which is in contrast to the general knowledge about the importance of cell-cell and cell-matrix interaction in cell biology. Among numerous other problems, the role of ECM synthesis by tumor cells compared with stromal cells is not very well understood and could be studied mechanistically in 3-D cultures. A second example may be the interaction between the expression of adhesion molecules and metabolic gradients, which may be investigated in such culture systems to elucidate the impact of a tumor-typical environment on cellular adhesiveness.
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ANGIOGENESIS |
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For many years, there has been a strong, continuous requirement by scientists working with tumor spheroids for an appropriate in vitro assay of 3-D angiogenesis. Ever since the pioneering work of Folkman, partially reviewed by Mueller-Klieser (108), on angiogenesis in implanted tumor spheroids, there has been little success in the development of a simple assay for vascularization of cell aggregates in vitro. Spheroids seem to be resistant to invasion by endothelial cells in cocultures (116). It is obvious that many of these approaches with negative results have not been published and have come to the knowledge of the author solely by oral communication.
Considerable progress in using spheroids for studies on tumor angiogenesis has been made by Zwi et al. (181, 182), who developed a technique for implantation of spheroids into the peritoneal cavity, where they eventually become vascularized and from where they can be harvested for quantitative assessment of vascularization. Using this technique, some intriguing studies have recently originated from a collaboration between the groups of Keshet and Neeman (1, 137, 141), which were able to show that vascularization in these spheroids can be visualized by magnetic resonance microimaging. The studies impressively demonstrate the role of VEGF as a major determinant of the angiogenesis process in these glioma spheroids, including an upregulation of VEGF in hypoxic or hypoglycemic microenvironments.
Nehls and Drenckhahn (113) have developed a novel 3-D culture system for systematic studies on angiogenesis in vitro. The system consists of endothelial cells seeded on gelatin-coated microcarriers that are entrapped in a 3-D fibrin matrix. Apparently, there is great potential in this new technique for quantitative evaluation of in vivo-like angiogenesis under well-defined in vitro conditions.
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EXPERIMENTAL TISSUE MODELING |
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Various biological tissues can be kept as explants in tissue or organ culture, maintaining cellular function for a limited period of time. Although this technique has been used for many years, considerable progress has been made more recently in generating bioartificial tissues in vitro from single cells of primary material or of cell lines. Some of this recent work is briefly highlighted here. The majority of these investigations was performed to study basic mechanisms of organ development, to develop artificial organs for replacement or support of natural organs after failure, to optimize bioproduction, or to obtain realistic pharmacotoxicological test systems. In principle, 3-D cultures were obtained either by exploiting spontaneous cell aggregation and by generating more or less spherical cellular conglomerates or by culturing cells on artificial substrates that induce cellular differentiation and maintain cellular function.
Over the past few years, intensive research efforts have been focused on the development of an artificial liver, and only a few exemplary studies can be mentioned here. In many of these approaches, hepatocyte spheroids (82, 153, 172, 177) or heterospheroids consisting of hepatocytes and fibroblasts or unspecified parenchymal cells (35, 149, 173) were used. The application of artificial support systems, such as porous gelatin sponges (87), agarose (136), or collagen (115), as well as the induction of aggregation by exogenous adhesion molecules (25), appear to be advantageous for efficient long-term culture of liver cells. Reconstitution of the specific geometry, microenvironment, and metabolism of the hepatic sinusoid has been achieved recently in a complex 3-D culture system by Bader and colleagues (5). The efficiency of liver cell cultures in approximating the in vivo situation has been shown not only for histological architecture and cellular metabolism but also for gene expression (114, 150). Tremendous progress with regard to practicability and effectiveness in the clinic has been made by growing pig hepatocytes in nonparenchymal coculture in an artificial 3-D capillary system, as developed by Gerlach and colleagues (50), who reviewed this field in a recent article (49).
Compared with liver, progress toward the clinical application of
artificial pancreas is not as advanced. So far, spheroids have been
obtained from insulin-secreting recombinant mouse pituitary AtT-20
cells and mouse insulinoma TC3 cells (157). A promising study has
been published by Beattie et al. (8), who achieved long-term viability
and function of human fetal isletlike cell clusters implanted under the
kidney capsule of athymic mice. A number of researchers have used
isolated piscine islets, so-called Brockmann bodies, as "natural"
pancreatic spheroids that can be implanted as xenografts after
microencapsulation with alginate (131). Oxygen supply seems to play a
crucial role for the survival and maintenance of function of such
islets, which is the background of quantitative measurements of the
oxygen tension distributions in Brockmann bodies (131, 132).
Of the hormone-producing tissues, the pituitary gland has been modeled by spheroids most extensively, with research focused mainly on hormone release, such as luteinizing hormone, after stimulation with the appropriate releasing hormone (134, 158). 3-D cultures of melatonin-secreting cells from the pineal gland represent a further hormone-related tissue model that has been established recently (72). In contrast to pineal cell monolayers, 3-D cultures remained functional for >3 wk and secreted melatonin when challenged with isoproterenol. These aggregates may therefore be useful for kinetic studies on the release of indole amines. Apart from some studies on collagen-embedded spheroids from "classical" FRTL-5 thyroid cells (27, 122), there are only a few recent reports on isolated adult thyroid cells that tend to form follicles in aggregation cultures (174). Nevertheless, thyroid cell spheroids allowed for systematic investigations concerning the role of cell motility, cell adhesion, and E-cadherin in the biogenesis of thyroid follicles (175).
Besides organotypic tissue or slice cultures, aggregating brain cell cultures represent an important part of the methodological repertoire in neuroscience and developmental biology. The scientific groups that are actively working with this model are so numerous that only a fragmentary selection of reports demonstrating the potential of 3-D brain cell cultures can be given here. There are a number of publications from the groups of Bjerkvig and Laerum on brain tumor invasion (e.g., see Refs. 9, 36, 91, 121, 126). Brain cell differentiation has been studied intensively in 3-D cultures (7, 51), as has the process of myelination and demyelination (71, 90, 100, 154) and neuronal degeneration (23). Brain cell aggregates have been used for studying the neural toxicity of lead (180), for analysis of neuronal behavior during human immunodeficiency virus infectious disease (76, 176), during Alzheimer's disease (48), or during Parkinson's disease (167). A specific role of the Muller glia for retinal development could be demonstrated in aggregation cultures of retinal cells by Willbold et al. (169). This field has been reviewed recently by Schmid et al. (130).
Homotypic and heterotypic 3-D cultures, including fibroblasts and/or keratinocytes, have been used for studies on the formation of extracellular matrix (3, 25, 40, 148) and skin (63). Pioneering work on many aspects of skin development and carcinogenesis has come from the laboratory of Fusenig, including studies on hair follicle formation (84, 85) or mesenchymal influences on the differentiation of keratinocytes (84 ,86).
During the past 2-4 years, there has been a tremendous increase in the number of publications regarding the use of 3-D cultures for studying chondrogenesis. This includes research on molecular aspects of matrix formation (89, 94, 123) or chondrocyte differentiation (26, 29). Some of these studies in which fetal cells are employed are transitional between aggregation cultures and cultures of embryoid bodies (26), a cell system that is briefly reviewed below.
Another field in which aggregated cell cultures have been used with increasing frequency for the past 2-3 years is the 3-D preparation of heart cells, mainly of fetal origin. Sinoatrial node cell preparations (13), atrial cell preparations (83, 164), embryonic chick heart cell aggregates (24, 124, 178), or ventricular cell aggregates (151) served as potent models for electrophysiological and pharmacological investigations of the role of various ionic channels and electromechanical coupling or of the autonomic nervous system in the regulation of heart activity. Furthermore, myocardial cell aggregates were employed to study specific questions, such as mechanisms underlying defibrillation (155), pacemaker currents (18), or cardiac side effects of anticancer drugs (102).
There are various other tissues that have been modeled or remodeled by aggregation cultures, which are mentioned only briefly here. These include 3-D cultures of mesangial cells (4, 57), urothelial cells (12), and nasopharyngeal cells (11).
Among other factors, one advantage of aggregating cell cultures is that they can be derived from stable established cell lines, from fetal cells, or from primary material. Aggregate composition can be manipulated, e.g., by growing tumor spheroids with or without fibroblasts (68). The disadvantages may be a limited degree of differentiation and a limited availability of appropriate cells for mixed cultures. For example, which type of fibroblast should be used for heterospheroids may be a critical question, since fibroblasts may be different in different tissues of origin. Some of these problems may be solved or circumvented by culturing embryonic stem cells as multicellular aggregates, so-called embryonic bodies.
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EMBRYOID BODIES |
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Aggregating embryonic cell cultures have to be clearly distinguished from embryoid bodies that represent 3-D cultures of pluri- or omnipotent embryonic stem cells. Although embryoid bodies have been used for some time after the pioneering research of Evans and Kaufman (38) and Martin and Evans (97-99), there has been a recent burst of reports on mechanistic and molecular work with these powerful models that is, among other indicators, reflected by reviews in highly ranked journals (for a recent review see Ref. 70). Some basic properties of embryoid bodies and some recent advances in the field are briefly summarized.
Embryoid bodies are derived from embryonic stem cell lines that have
retained their capacity of lineage commitment, i.e., of generating
cells of the hematopoietic, endothelial, muscle, and neuronal lineages.
The majority of publications covers hematopoiesis, a field in which
knowledge of embryogenesis is most advanced and which is nearing the
generation of artificial blood to be used in clinics (61). Much
progress has come from the data of Keller and colleagues (165, 168),
highlighting the potential of targeted mutations in embryoid bodies,
from Nakano et al. (112) on lymphocyte differentiation, or from Zhang
et al. (179), showing similar developmental processes in vitro and in
vivo. The mechanisms of vasculogenesis have been investigated in
embryoid bodies by Wang et al. (163), by Doetschman et al. (28), by
Krah et al. (77), and by Heyward et al. (59), with the latter authors
analyzing the role of cell adhesion molecules for the development of
vessels and their response to inflammatory stimuli. Basic work on
neurogenesis has been published by Strubing et al. (142), dealing with
electrophysiological and immunocytochemical cellular properties, and by
Bain et al. (6), concerning the inducing activity of retinoic acid. A
very exciting field of research is the development of muscle cells. Rohwedel et al. (127) have shown that the development of skeletal muscle myocytes is very similar in embryoid bodies and in vivo with
regard to the activation of muscle-related genes. The role of MyoD
status has been investigated by Shani et al. (135), and the role of
TGF- in muscular development has been studied systematically by
Slager et al. (138). Pioneering research has been reported recently
from the laboratories of Wobus and Hescheler concerning cardiomyogenesis. The authors generated beating embryoid bodies with
cardiospecific receptors, ionic channels, and action potentials (170,
171). Gassmann et al. (46) have studied the oxygenation and
oxygen-regulated gene expression in embryoid bodies in different oxygen
environments, which may be relevant for early embryogenesis where
embryonic cells reside at low oxygen tensions before implantation and
vascularization.
There are many more interesting aspects of this field that cannot be discussed here. In summary, studies on embryoid bodies represent perhaps one of the most exciting fields of research with 3-D culture systems, and much progress with regard to our understanding of embryonic development and carcinogenesis may come from this research area in the near future.
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ACKNOWLEDGEMENTS |
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I thank all my co-workers who contributed significantly to this article, and I thank Dr. Deborah Bickes-Kelleher for assistance in writing the manuscript.
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FOOTNOTES |
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This work was supported in part by the Deutsche Forschungsgemeinschaft (Mu 576/2-4, Mu 576/4-1), the German Israeli Science Foundation (I-368-149.02/94), and the Stiftung Rheinland-Pfalz fuer Innovation (8036-386261/198).
Address for reprint requests: W. Mueller-Klieser, Institute of Physiology and Pathophysiology, Johannes Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany.
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REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
1.
Abramovitch, R.,
G. Meir,
and
M. Neeman.
Neovascularization induced growth of implanted C6 glioma multicellular spheroids: magnetic resonance microimaging.
Cancer Res.
55:
1956-1962,
1995[Abstract].
2.
Acker, H.,
J. Carlsson,
W. Mueller-Klieser,
and
R. M. Sutherland.
Comparative pO2 measurements in cell spheroids cultured with different techniques.
Br. J. Cancer
56:
325-327,
1987[Medline].
3.
Armstrong, M. T.,
J. W. Fenton,
T. T. Andersen,
and
P. B. Armstrong.
Thrombin stimulation of matrix fibronectin.
J. Cell. Physiol.
166:
112-120,
1996[Medline].
4.
Ayo, S. H.,
and
J. I. Kreisberg.
Heparin increases hillock formation in mesangial cell cultures.
J. Am. Soc. Nephrol.
2:
1153-1157,
1991[Abstract].
5.
Bader, A.,
E. Knop,
A. Kern,
K. Boeker,
N. Fruehauf,
O. Crome,
H. Esselmann,
C. Pape,
G. Kempka,
and
K.-F. Sewing.
3-D coculture of hepatic sinusoidal cells with primary hepatocatesdesign of an organotypical model.
Exp. Cell Res.
226:
223-233,
1996[Medline].
6.
Bain, G.,
D. Kitchens,
M. Yao,
J. E. Huettner,
and
D. I. Gottlieb.
Embryonic stem cells express neuronal properties in vitro.
Dev. Biol.
168:
342-357,
1995[Medline].
7.
Barnea, A.,
G. Cho,
G. Lu,
and
M. Mathis.
Brain-derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y-producing neurons.
J. Neurosci. Res.
42:
638-647,
1995[Medline].
8.
Beattie, G. M.,
C. Butler,
and
A. Hayek.
Morphology and function of cultured human fetal pancreatic cells transplanted into athymic mice: a longitudinal study.
Cell Transplant
3:
421-425,
1994[Medline].
9.
Bjerkvig, R.
Spheroid Culture in Cancer Research. Boca Raton, FL: CRC, 1992,
10.
Boghaert, E. R.,
J. Simpson,
R. J. Jacob,
T. Lacey,
J. W. Walsh,
and
S. G. Zimmer.
The effect of dibutyryl camp (dBcAMP) on morphological differentiation, growth and invasion in vitro of a hamster brain-tumor cell line: a comparative study of dBcAMP effects in 2- and 3-dimensional cultures.
Int. J. Cancer
47:
610-618,
1991[Medline].
11.
Boxberger, H. J.,
M. J. Sessler,
B. Maetzel,
and
T. F. Meyer.
Highly polarized primary epithelial cells from human nasopharynx grown as spheroid-like vesicles.
Eur. J. Cell Biol.
62:
140-151,
1993[Medline].
12.
Boxberger, H. J.,
M. J. Sessler,
B. Maetzel,
I. M. Mosleh,
H. D. Becker,
and
T. F. Meyer.
Highly polarized primary urothelial cells from human ureter grown as spheroid-like vesicles.
Epithelial Cell Biol.
3:
85-95,
1994[Medline].
13.
Boyett, M. R.,
I. Kodama,
H. Honjo,
A. Arai,
R. Suzuki,
and
J. Toyama.
Ionic basis of the chronotropic effect of acetylcholine on the rabbit sinoatrial node.
Cardiovasc. Res.
29:
867-878,
1995[Medline].
14.
Bracke, M. E.,
B. M. Vyncke,
E. A. Bruyneel,
S. J. Vermeulen,
G. K. De Bruyne,
N. A. Van Larebeke,
K. Vleminckx,
F. M. Van Roy,
and
M. M. Mareel.
Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro.
Br. J. Cancer
68:
282-289,
1993[Medline].
15.
Brady Kalnay, S. M.,
E. R. Boghaert,
S. Zimmer,
D. R. Soll,
and
R. Brackenbury.
Invasion by WC5 rat cerebellar cells is independent of RSV-induced changes in growth and adhesion.
Int. J. Cancer
49:
239-245,
1991[Medline].
16.
Brauner, T.,
and
D. F. Hulser.
Tumor cell invasion and gap junctional communication. II. Normal and malignant cells confronted in multicell spheroids.
Invasion Metastasis
10:
31-48,
1990[Medline].
17.
Bredel Geissler, A.,
U. Karbach,
S. Walenta,
L. Vollrath,
and
W. Mueller-Klieser.
Proliferation-associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture.
J. Cell. Physiol.
153:
44-52,
1992[Medline].
18.
Brochu, R. M.,
J. R. Clay,
and
A. Shrier.
Pacemaker current in single cells and in aggregates of cells dissociated from the embryonic chick heart.
J. Physiol. (Lond.)
454:
503-515,
1992[Abstract].
19.
Byers, S. W.,
C. L. Sommers,
B. Hoxter,
A. M. Mercurio,
and
A. Tozeren.
Role of E-cadherin in the response of tumor cell aggregates to lymphatic, venous and arterial flow: measurement of cell-cell adhesion strength.
J. Cell Sci.
108:
2053-2064,
1995
20.
Carlsson, J.,
and
H. Acker.
Relations between pH, oxygen partial pressure and growth in cultured cell spheroids.
Int. J. Cancer
42:
715-720,
1988[Medline].
21.
Carlsson, J.,
C. G. Stalnacke,
H. Acker,
M. Haji Karim,
S. Nilsson,
and
B. Larsson.
The influence of oxygen on viability and proliferation in cellular spheroids.
Int. J. Radiat. Oncol. Biol. Phys.
5:
2011-2020,
1979[Medline].
22.
Chaplain, M. A. J.,
D. L. Benson,
and
P. K. Maini.
Nonlinear diffusion of a growth inhibitory factor in multicell spheroids.
Math. Biosci.
121:
1-13,
1994[Medline].
23.
Chatterjee, S. S.,
and
M. Noldner.
An aggregate brain cell culture model for studying neuronal degeneration and regeneration.
J. Neural Transm. Suppl.
44:
47-60,
1994[Medline].
24.
Clay, J. R.,
A. S. Kristof,
J. Shenasa,
R. M. Brochu,
and
A. Shrier.
A review of the effects of three cardioactive agents on the electrical activity from embryonic chick heart cell aggregates: TTX, ACh, and E-4031.
Prog. Biophys. Mol. Biol.
62:
185-202,
1994[Medline].
25.
Dai, W. G.,
and
W. M. Saltzman.
Fibroblast aggregation by suspension with conjugates of poly-ethylene-glycol and RGD.
Biotechnol. Bioeng.
50:
349-356,
1996.
26.
Denker, A. E.,
S. B. Nicoll,
and
R. S. Tuan.
Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cells upon treatment with transforming growth factor- 1.
Differentiation
59:
25-34,
1995[Medline].
27.
Derwahl, M.,
H. Studer,
G. Huber,
H. Gerber,
and
H. J. Peter.
Intercellular propagation of individually programmed growth bursts in FRTL-5 cells. Implications for interpreting growth factor actions.
Endocrinology
127:
2104-2110,
1990[Abstract].
28.
Doetschman, T.,
M. Shull,
A. Kier,
and
J. D. Coffin.
Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders.
Hypertension
22:
618-629,
1993[Abstract].
29.
Dozin, B.,
R. Quarto,
G. Campanile,
and
R. Cancedda.
In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid.
Eur. J. Cell Biol.
58:
390-394,
1992[Medline].
30.
Duchting, W.,
W. Ulmer,
T. Ginsberg,
N. Kikhounga,
and
C. Saile.
Radiogenic responses of normal cells induced by fractionated irradiationa simulation study. II. Late responses.
Strahlenther. Onkol.
171:
525-533,
1995[Medline].
31.
Duchting, W.,
W. Ulmer,
T. Ginsberg,
and
C. Saile.
Radiogenic responses of normal tissue induced by fractionated irradiationa simulation study. I. Acute effects.
Strahlenther. Onkol.
171:
460-467,
1995[Medline].
32.
Durand, R. E.
Contributions of flow cytometry to studies with multicell spheroids.
Methods Cell Biol.
42B:
405-422,
1994[Medline].
33.
Durand, R. E.,
and
R. M. Sutherland.
Effects of intercellular contact on repair of radiation damage.
Exp. Cell Res.
71:
75-80,
1972[Medline].
34.
Elfgang, C.,
R. Eckert,
H. Lichtenberg Frate,
A. Butterweck,
O. Traub,
R. A. Klein,
D. F. Hulser,
and
K. Willecke.
Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.
J. Cell Biol.
129:
805-817,
1995[Abstract].
35.
Endoh, K.,
K. Ueno,
A. Miyashita,
and
T. Satoh.
An experimental model of acute liver injury using multicellular spheroids composed of rat parenchymal and non-parenchymal liver cells.
Res. Commun. Chem. Pathol. Pharmacol.
82:
317-329,
1993[Medline].
36.
Engebraaten, O.,
R. Bjerkvig,
P. H. Pedersen,
and
O. D. Laerum.
Effects of EGF, bFGF, NGF and PDGF(bb) on cell proliferative, migratory and invasive capacities of human brain-tumour biopsies in vitro.
Int. J. Cancer
53:
209-214,
1993[Medline].
37.
Essand, M.,
C. Gronvik,
T. Hartman,
and
J. Carlsson.
Radioimmunotherapy of prostatic adenocarcinomas: effects of 131I-labelled E4 antibodies on cells at different depth in DU 145 spheroids.
Int. J. Cancer
63:
387-394,
1995[Medline].
38.
Evans, M. J.,
and
M. H. Kaufman.
Establishment in culture of pluripotential cells from mouse embryos.
Nature
292:
154-156,
1981[Medline].
39.
Fairbairn, D. W.,
P. L. Olive,
and
K. L. O'Neill.
The comet assay: a comprehensive review.
Mutat. Res.
339:
37-59,
1995[Medline].
40.
Fleischmajer, R.,
A. Schechter,
M. Bruns,
J. S. Perlish,
E. D. Macdonald,
T. C. Pan,
R. Timpl,
and
M. L. Chu.
Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro.
J. Invest. Dermatol.
105:
597-601,
1995[Abstract].
41.
Freyer, J. P.
Rates of oxygen consumption for proliferating and quiescent cells isolated from multicellular tumor spheroids.
Adv. Exp. Med. Biol.
345:
335-342,
1994[Medline].
42.
Freyer, J. P.,
P. L. Schor,
K. A. Jarrett,
M. Neeman,
and
L. O. Sillerud.
Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids.
Cancer Res.
51:
3831-3837,
1991[Abstract].
43.
Fritz, P.,
K. J. Weber,
C. Frank,
and
M. Flentje.
Differential effects of dose rate and superfractionation on survival and cell cycle of V79 cells from spheroid and monolayer culture.
Radiother. Oncol.
39:
73-79,
1996[Medline].
44.
Fujiwara, T.,
E. A. Grimm,
T. Mukhopadhyay,
D. W. Cai,
L. B. Owen Schaub,
and
J. A. Roth.
A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis.
Cancer Res.
53:
4129-4133,
1993[Abstract].
45.
Fujiwara, T.,
E. A. Grimm,
T. Mukhopadhyay,
W. W. Zhang,
L. B. Owen-Schaub,
and
J. A. Roth.
Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene.
Cancer Res.
54:
2287-2291,
1994[Abstract].
46.
Gassmann, M.,
J. Fandrey,
S. Bichet,
M. Wartenberg,
H. H. Marti,
C. Bauer,
R. H. Wenger,
and
H. Acker.
Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.
Proc. Natl. Acad. Sci. USA
93:
2867-2872,
1996
47.
Gavrieli, Y.,
Y. Sherman,
and
S. A. Ben Sasson.
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J. Cell Biol.
119:
493-501,
1992[Abstract].
48.
Gebicke Haerter, P. J.,
K. Appel,
P. Honegger,
and
M. Berger.
Changes of beta-amyloid precursor protein splice patterns in brain cell aggregate cultures.
J. Neurosci. Res.
38:
32-40,
1994[Medline].
49.
Gerlach, J. C.
Use of hepatocyte cultures for liver support bioreactors.
Adv. Exp. Med. Biol.
368:
165-171,
1994[Medline].
50.
Gerlach, J. C.,
N. Schnoy,
J. Encke,
M. D. Smith,
C. Muller,
and
P. Neuhaus.
Improved hepatocyte in vitro maintenance in a culture model with woven multicompartment capillary systems: electron microscopy studies.
Hepatology
22:
546-552,
1995[Medline].
51.
Gorodinsky, A.,
J. Barg,
M. M. Belcheva,
R. Levy,
R. J. McHale,
Z. Vogel,
and
C. J. Coscia.
Dynorphins modulate DNA synthesis in fetal brain cell aggregates.
J. Neurochem.
65:
1481-1486,
1995[Medline].
52.
Graham, C. H.,
H. Kobayashi,
K. S. Stankiewicz,
S. Man,
S. J. Kapitain,
and
R. S. Kerbel.
Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.
J. Natl. Cancer Inst.
86:
975-982,
1994[Abstract].
53.
Groebe, K.,
and
W. Mueller-Klieser.
On the relation between size of necrosis and diameter of tumor spheroids.
Int. J. Radiat. Oncol. Biol. Phys.
34:
395-401,
1996[Medline].
54.
Gross, M. W.,
U. Karbach,
K. Groebe,
A. J. Franko,
and
W. Mueller-Klieser.
Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids.
Int. J. Cancer
61:
567-573,
1995[Medline].
55.
Grummer, R.,
H. P. Hohn,
M. M. Mareel,
and
H. W. Denker.
Adhesion and invasion of three human choriocarcinoma cell lines into human endometrium in a three-dimensional organ culture system.
Placenta
15:
411-429,
1994[Medline].
56.
Hauptmann, S.,
C. Denkert,
H. Lohrke,
L. Tietze,
S. Ott,
B. Klosterhalfen,
and
C. Mittermayer.
Integrin expression on colorectal tumor cells growing as monolayers, as multicellular tumor spheroids, or in nude mice.
Int. J. Cancer
61:
819-825,
1995[Medline].
57.
He, C. J.,
L. J. Striker,
M. Tsokos,
C. W. Yang,
E. P. Peten,
and
G. E. Striker.
Relationships between mesangial cell proliferation and types I and IV collagen mRNA levels in vitro.
Am. J. Physiol.
269 (Cell Physiol. 38):
C554-C562,
1995[Abstract].
58.
Helige, C.,
J. Smolle,
G. Zellnig,
R. Fink Puches,
H. Kerl,
and
H. A. Tritthart.
Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro.
Eur. J. Cancer
29A:
124-128,
1992.
59.
Heyward, S. A.,
N. Dubois Stringfellow,
R. Rapoport,
and
V. L. Bautch.
Expression and inducibility of vascular adhesion receptors in development.
FASEB J.
9:
956-962,
1995
60.
Hofmann Wellenhof, R.,
R. Fink Puches,
J. Smolle,
C. Helige,
H. A. Tritthart,
and
H. Kerl.
Correlation of melanoma cell motility and invasion in vitro.
Melanoma Res.
5:
311-319,
1995[Medline].
61.
Hole, N.,
G. J. Graham,
U. Menzel,
and
J. D. Ansell.
A limited temporal window for the derivation of multilineage repopulating hematopoetic progenitors during embryonal stem cell diffenetiation in vitro.
Blood
88:
1266-1276,
1996
62.
Holtfreter, J.
A study of the mechanism of gastrulation.
J. Exp. Zool.
95:
171-212,
1944.
63.
Ihara, S.,
M. Watanabe,
E. Nagao,
and
N. Shioya.
Formation of hair follicles from a single-cell suspension of embryonic rat skin by a two-step procedure in vitro.
Cell Tissue Res.
266:
65-73,
1991[Medline].
64.
Inch, W. R.,
J. A. McCredie,
and
R. M. Sutherland.
Growth of nodular carcinomas in rodents compared with multi-cell spheroids in tissue culture.
Growth
34:
271-282,
1970[Medline].
65.
Jaaskelainen, J.,
E. Lehtonen,
P. Heikkila,
P. Kalliomaki,
and
T. Timonen.
Damage to multicellular human H-2 glioma spheroids incubated with LAK cells: an ultrastructural study.
J. Natl. Cancer Inst.
82:
497-501,
1990[Abstract].
66.
Jaaskelainen, J.,
A. Maenpaa,
M. Patarroyo,
C. G. Gahmberg,
K. Somersalo,
J. Tarkkanen,
M. Kallio,
and
T. Timonen.
Migration of recombinant IL-2-activated T and natural killer cells in the intercellular space of human H-2 glioma spheroids in vitro. A study on adhesion molecules involved.
J. Immunol.
149:
260-268,
1992
67.
Kaaijk, P.,
D. Troost,
P. K. Dast,
F. Vandenberg,
S. Leenstra,
and
D. A. Bosch.
Cytolytic effects of autologous lymphokine activated killer cells on organotypic multicellular spheroids of gliomas in vitro.
Neuropathol. Appl. Neurobiol.
21:
392-398,
1995[Medline].
68.
Kammerer, R.,
and
S. von Kleist.
Artificial tumor: a novel heterotypic, polymorphic, three-dimensional in vitro model of individual human solid tumors.
Tumour Biol.
16:
213-221,
1995[Medline].
69.
Karbach, U.,
C. D. Gerharz,
K. Groebe,
H. E. Gabbert,
and
W. Mueller-Klieser.
Rhabdomyosarcoma spheroids with central proliferation and differentiation.
Cancer Res.
52:
474-477,
1992[Abstract].
70.
Keller, G. M.
In vitro differentiation of embryonic stem cells.
Curr. Opin. Cell Biol.
7:
862-869,
1995[Medline].
71.
Kerlero de Rosbo, N.,
P. Honegger,
H. Lassmann,
and
J. M. Matthieu.
Demyelination induced in aggregating brain cell cultures by a monoclonal antibody against myelin/oligodendrocyte glycoprotein.
J. Neurochem.
55:
583-587,
1990[Medline].
72.
Khan, N. A.,
V. Shacoori,
R. Havouis,
D. Querne,
J. P. Moulinoux,
and
B. Rault.
Three dimensional culture of pineal cell aggregates: a model of cell-cell co-operation.
J. Neuroendocrinol.
7:
353-359,
1995[Medline].
73.
Kitsuki, H.,
M. Katano,
T. Morisaki,
and
M. Torisu.
CEA-mediated homotypic aggregation of human colorectal carcinoma cells in a malignant effusion.
Cancer Lett.
88:
7-13,
1995[Medline].
74.
Knuechel, R.,
and
R. M. Sutherland.
Recent developments in research with human tumor spheroids.
Cancer J.
3:
234-243,
1990.
75.
Kobayashi, H.,
S. Man,
C. H. Graham,
S. J. Kapitain,
B. A. Teicher,
and
R. S. Kerbel.
Acquired multicellular-mediated resistance to alkylating agents in cancer.
Proc. Natl. Acad. Sci. USA
90:
3294-3298,
1993[Abstract].
76.
Kolson, D. L.,
J. Buchhalter,
R. Collman,
B. Hellmig,
C. F. Farrell,
C. Debouck,
and
F. Gonzalez Scarano.
HIV-1 Tat alters normal organization of neurons and astrocytes in primary rodent brain cell cultures: RGD sequence dependence.
AIDS Res. Hum. Retroviruses
9:
677-685,
1993[Medline].
77.
Krah, K.,
V. Mironov,
W. Risau,
and
I. Flamme.
Induction of vasculogenesis in quail blastodisc-derived embryoid bodies.
Dev. Biol.
164:
123-132,
1994[Medline].
78.
Kunz-Schughart, L. A.,
K. Groebe,
and
W. Mueller-Klieser.
Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation.
Int. J. Cancer
66:
578-586,
1996[Medline].
79.
Kwok, C. S.,
S. K. Yu,
and
S. L. Lee.
Mathematical models of the uptake kinetics of antitumor antibodies in human melanoma spheroids.
Antibody Immunocon. Radiopharmaceut.
8:
155-169,
1995.
80.
Laderoute, K. R.,
B. J. Murphy,
S. M. Short,
T. D. Grant,
A. M. Knapp,
and
R. M. Sutherland.
Enhancement of transforming growth factor- synthesis in multicellular tumour spheroids of A431 squamous carcinoma cells.
Br. J. Cancer
65:
157-162,
1992[Medline].
81.
Langmuir, V. K.,
J. K. McGann,
F. Buchegger,
and
R. M. Sutherland.
The effect of antigen concentration, antibody valency and size, and tumor architecture on antibody binding in multicell spheroids.
Int. J. Radiat. Appl. Instrum. Part B
18:
753-764,
1991.
82.
Li, A. P.,
S. M. Colburn,
and
D. J. Beck.
A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids.
In Vitro Cell. Dev. Biol.
28A:
673-677,
1992.
83.
Li, G. R.,
J. Feng,
A. Shrier,
and
S. Nattel.
Contribution of ATP-sensitive potassium channels to the electrophysiological effects of adenosine in guinea-pig atrial cells.
J. Physiol. (Lond.)
484:
629-642,
1995[Abstract].
84.
Limat, A.,
D. Breitkreutz,
T. Hunziker,
C. E. Klein,
F. Noser,
N. E. Fusenig,
and
L. R. Braathen.
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.
Cell Tissue Res.
275:
169-176,
1994[Medline].
85.
Limat, A.,
D. Breitkreutz,
H. J. Stark,
T. Hunziker,
G. Thikoetter,
F. Noser,
and
N. E. Fusenig.
Experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle outer root sheath and matrix cells.
Ann. NY Acad. Sci.
642:
125-146,
1991[Abstract].
86.
Limat, A.,
T. Hunziker,
D. Breitkreutz,
N. E. Fusenig,
and
L. R. Braathen.
Organotypic cocultures as models to study cell-cell and cell-matrix interactions of human hair follicle cells.
Skin Pharmacol.
7:
47-54,
1994[Medline].
87.
Lin, K. H.,
S. Maeda,
and
T. Saito.
Long-term maintenance of liver-specific functions in three-dimensional culture of adult rat hepatocytes with a porous gelatin sponge support.
Biotechnol. Appl. Biochem.
21:
19-27,
1995[Medline].
88.
Lord, E. M.,
L. Harwell,
and
C. J. Koch.
Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts.
Cancer Res.
53:
5721-5726,
1993[Abstract].
89.
Loty, S.,
N. Forest,
H. Boulekbache,
and
J. M. Sautier.
Cytochalasin D induces changes in cell shape and promotes in vitro chondrogenesis: a morphological study.
Biol. Cell
83:
149-161,
1995[Medline].
90.
Loughlin, A. J.,
P. Honegger,
M. N. Woodroofe,
V. Comte,
J. M. Matthieu,
and
M. L. Cuzner.
Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.
J. Neurosci. Res.
37:
647-653,
1994[Medline].
91.
Lund-Johansen, M.,
K. Forsberg,
R. Bjerkvig,
and
O. D. Laerum.
Effects of growth factors on a human glioma cell line during invasion into rat brain aggregates in culture.
Acta Neuropathol. (Berl.)
84:
190-197,
1992[Medline].
92.
Mairs, R. J.,
J. Russell,
S. Cunningham,
J. A. O'Donoghue,
M. N. Gaze,
J. Owens,
G. Vaidyanathan,
M. R. Zalutsky,
T. Saga,
R. D. Neumann,
T. Heya,
J. Sato,
S. Kinuya,
N. Le,
C. H. Paik,
and
J. N. Weinstein.
Enhanced tumour uptake and in vitro radiotoxicity of no-carrier-added [131I]meta-iodobenzylguanidine: implications for the targeted radiotherapy of neuroblastoma targeting cancer micrometastases with monoclonal antibodies: a binding-site barrier.
Eur. J. Cancer
92:
8999-9003,
1995.
93.
Makiyama, N.,
H. Matsui,
H. Tsuji,
and
K. Ichimura.
Attachment and invasion of high- and low-metastatic clones of RCT sarcoma in a three-dimensional culture system.
Clin. Exp. Metastasis
9:
411-425,
1991[Medline].
94.
Mallein Gerin, F.,
F. Ruggiero,
T. M. Quinn,
F. Bard,
A. J. Grodzinsky,
B. R. Olsen,
and
M. van der Rest.
Analysis of collagen synthesis and assembly in culture by immortalized mouse chondrocytes in the presence or absence of 1(IX) collagen chains.
Exp. Cell Res.
219:
257-265,
1995[Medline].
95.
Mansbridge, J. N.,
W. A. Ausserer,
M. A. Knapp,
and
R. M. Sutherland.
Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: changes in surface receptor expression and protein tyrosine phosphorylation.
J. Cell. Physiol.
161:
374-382,
1994[Medline].
96.
Mansbridge, J. N.,
R. Knuchel,
A. M. Knapp,
and
R. M. Sutherland.
Importance of tyrosine phosphatases in the effects of cell-cell contact and microenvironments on EGF-stimulated tyrosine phosphorylation.
J. Cell. Physiol.
151:
433-442,
1992[Medline].
97.
Martin, G. R.
Teratocarcinomas and mammalian embryogenesis.
Science
209:
768-776,
1980[Medline].
98.
Martin, G. R.
Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.
Proc. Natl. Acad. Sci. USA
78:
7634-7638,
1981[Abstract].
99.
Martin, G. R.,
and
M. J. Evans.
Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro.
Proc. Natl. Acad. Sci. USA
72:
1441-1445,
1975[Abstract].
100.
Matthieu, J. M.,
V. Comte,
M. Tosic,
and
P. Honegger.
Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.
J. Neuroimmunol.
40:
231-234,
1992[Medline].
101.
Messiaen, L.,
S. Qian,
G. De Bruyne,
E. Boghaert,
T. Moens,
M. Rabaey,
F. Van Roy,
and
M. Mareel.
Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro.
In Vitro Cell. Dev. Biol.
27A:
369-380,
1991.
102.
Mitrius, J. C.,
and
S. M. Vogel.
Doxorubicin-induced automaticity in cultured chick heart cell aggregates.
Cancer Res.
50:
4209-4215,
1990[Abstract].
103.
Monz, B.,
U. Karbach,
K. Groebe,
and
W. Mueller-Klieser.
Proliferation and oxygenation status of WiDr spheroids in different lactate and oxygen environments.
Oncol. Rep.
1:
1177-1183,
1996.
104.
Moscona, A.
Cell suspensions from organ rudiments of chick embryos.
Exp. Cell Res.
3:
535-539,
1952.
105.
Moscona, A.
Formation of lentoids by dissociated retinal cells of the chick embryo.
Science
125:
598-599,
1957.
106.
Moscona, A.
The development in vitro of chimeric aggregates of dissociated embryonic chick and mouse cells.
Proc. Natl. Acad. Sci. USA
43:
184-194,
1957.
107.
Moscona, A.
Rotation-mediated histogenetic aggregation of dissociated cells.
Exp. Cell Res.
22:
455-475,
1961.
108.
Mueller-Klieser, W.
Multicellular spheroids. A review on cellular aggregates in cancer research.
J. Cancer Res. Clin. Oncol.
113:
101-122,
1987[Medline].
109.
Mueller-Klieser, W.,
J. P. Freyer,
and
R. M. Sutherland.
Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids.
Br. J. Cancer
53:
345-353,
1986[Medline].
110.
Mueller-Klieser, W. F.,
and
R. M. Sutherland.
Oxygen tensions in multicell spheroids of two cell lines.
Br. J. Cancer
45:
256-264,
1982[Medline].
111.
Mueller-Klieser, W. F.,
and
R. M. Sutherland.
Influence of convection in the growth medium on oxygen tensions in multicellular tumor spheroids.
Cancer Res.
42:
237-242,
1982[Medline].
112.
Nakano, T.,
H. Kodama,
and
T. Honjo.
Generation of lymphohematopoietic cells from embryonic stem cells in culture.
Science
265:
1098-1101,
1994[Medline].
113.
Nehls, V.,
and
D. Drenckhahn.
A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.
Microvasc. Res.
50:
311-322,
1995[Medline].
114.
Nemoto, N.,
and
J. Sakurai.
Activation of Cyp1a1 and Cyp1a2 genes in adult mouse hepatocytes in primary culture.
Jpn. J. Cancer Res.
84:
272-278,
1993[Medline].
115.
Nishikawa, Y.,
Y. Tokusashi,
T. Kadohama,
H. Nishimori,
and
K. Ogawa.
Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix.
Exp. Cell Res.
223:
357-371,
1996[Medline].
116.
Offner, F. A.,
H. C. Wirtz,
J. Schiefer,
I. Bigalke,
B. Klosterhalfen,
F. Bittinger,
C. Mittermayer,
and
C. J. Kirkpatrick.
Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers.
Am. J. Pathol.
141:
601-610,
1992[Abstract].
117.
Olive, P. L.,
and
R. E. Durand.
Drug and radiation resistance in spheroids: cell contact and kinetics.
Cancer Metastasis Rev.
13:
121-138,
1994[Medline].
118.
Olive, P. L.,
C. M. Vikse,
and
R. E. Durand.
Hypoxic fractions measured in murine tumors and normal tissues using the comet assay.
Int. J. Radiat. Oncol. Biol. Phys.
29:
487-491,
1994[Medline].
119.
Parmar, V. S.,
R. Jain,
S. K. Sharma,
A. Vardhan,
A. Jha,
P. Taneja,
S. Singh,
B. M. Vyncke,
M. E. Bracke,
and
M. M. Mareel.
Anti-invasive activity of 3,7-dimethoxyflavone in vitro.
J. Pharm. Sci.
83:
1217-1221,
1994[Medline].
120.
Paulus, W.,
C. Huettner,
and
J. C. Tonn.
Collagens, integrins and the mesenchymal drift in glioblastomas: a comparison of biopsy specimens, spheroid and early monolayer cultures.
Int. J. Cancer
58:
841-846,
1994[Medline].
121.
Pedersen, P. H.,
G. J. Rucklidge,
S. J. Mork,
A. J. Terzis,
O. Engebraaten,
M. Lund-Johansen,
E. O. Backlund,
O. D. Laerum,
and
R. Bjerkvig.
Leptomeningeal tissue: a barrier against brain tumor cell invasion.
J. Natl. Cancer Inst.
86:
1593-1599,
1994[Abstract].
122.
Peter, H. J.,
H. Gerber,
H. Studer,
P. Groscurth,
and
M. Zakarija.
Comparison of FRTL-5 cell growth in vitro with that of xenotransplanted cells and the thyroid of the recipient mouse.
Endocrinology
128:
211-219,
1991[Abstract].
123.
Poliard, A.,
A. Nifuji,
D. Lamblin,
E. Plee,
C. Forest,
and
O. Kellermann.
Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways.
J. Cell Biol.
130:
1461-1472,
1995[Abstract].
124.
Rabkin, S. W.
Comparison of indapamide and hydrochlorothiazide on spontaneous contraction of cardiomyocytes in culture: the effect on alterations of extracellular calcium or potassium.
Gen. Pharmacol.
24:
699-704,
1993[Medline].
125.
Rak, J.,
Y. Mitsuhashi,
V. Erdos,
S. N. Huang,
J. Filmus,
and
R. S. Kerbel.
Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant C-H-ras oncogene expression.
J. Cell Biol.
131:
1587-1598,
1995[Abstract].
126.
Reith, A.,
R. Bjerkvig,
and
G. J. Rucklidge.
Laminin: a potential inhibitor of rat glioma cell invasion in vitro.
Anticancer Res.
14:
1071-1076,
1994[Medline].
127.
Rohwedel, J.,
V. Maltsev,
E. Bober,
H. H. Arnold,
J. Hescheler,
and
A. M. Wobus.
Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents.
Dev. Biol.
164:
87-101,
1994[Medline].
128.
Romero, F. J.,
D. Zukowski,
and
W. Mueller-Klieser.
Glutathione content of V79 cells in two- or three-dimensional culture.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1507-C1512,
1997
129.
Saga, T.,
R. D. Neumann,
T. Heya,
J. Sato,
S. Kinuya,
N. Le,
C. H. Paik,
and
J. N. Weinstein.
Targeting cancer micrometastases with monoclonal antibodies: a binding-site barrier.
Proc. Natl. Acad. Sci. USA
92:
8999-9003,
1995[Abstract].
130.
Schmid, B. P., P. Honegger, and P. Kucera.
Embryonic and fetal development: fundamental research.
Reprod. Toxicol. 7, Suppl. 1: 155-164, 1993.
131.
Schrezenmeir, J.,
L. Gero,
C. Laue,
J. Kirchgessner,
A. Muller,
A. Huls,
R. Passmann,
H. J. Hahn,
L. Kunz,
W. Mueller-Klieser,
and
J. J. Altman.
The role of oxygen supply in islet transplantation.
Transplant. Proc.
24:
2925-2929,
1992[Medline].
132.
Schrezenmeir, J.,
J. Kirchgessner,
L. Gero,
L. A. Kunz,
J. Beyer,
and
W. Mueller-Klieser.
Effect of microencapsulation on oxygen distribution in islet organs.
Transplantation
57:
1308-1314,
1994[Medline].
133.
Schuster, U.,
R. Buttner,
F. Hofstadter,
and
R. Knuchel.
A heterologous in vitro coculture system to study interaction between human bladder cancer cells and fibroblasts.
J. Urol.
151:
1707-1711,
1994[Medline].
134.
Shacoori, V.,
B. Saiag,
A. Girre,
and
B. Rault.
Stimulatory effect of perhexiline maleate on the basal and LHRH-stimulated luteinizing hormone release from rat pituitary cell aggregates in vitro.
Res. Commun. Mol. Pathol. Pharmacol.
87:
115-123,
1995[Medline].
135.
Shani, M.,
A. Faerman,
C. P. Emerson,
S. Pearson White,
I. Dekel,
and
Y. Magal.
The consequences of a constitutive expression of MyoD1 in ES cells and mouse embryos.
Symp. Soc. Exp. Biol.
46:
19-36,
1992[Medline].
136.
Shiraha, H.,
N. Koide,
H. Hada,
K. Ujike,
M. Nakamura,
T. Shinji,
S. Gotoh,
and
T. Tsuji.
Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids.
Biotechnol. Bioeng.
50:
416-421,
1996.
137.
Shweiki, D.,
M. Neeman,
A. Itin,
and
E. Keshet.
Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis.
Proc. Natl. Acad. Sci. USA
92:
768-772,
1995[Abstract].
138.
Slager, H. G.,
W. Van Inzen,
E. Freund,
A. J. Van den Eijnden Van Raaij,
and
C. L. Mummery.
Transforming growth factor- in the early mouse embryo: implications for the regulation of muscle formation and implantation.
Dev. Genet.
14:
212-224,
1993[Medline].
139.
Smolle, J.,
C. Helige,
H. P. Soyer,
S. Hoedl,
H. Popper,
H. Stettner,
H. Kerl,
H. A. Tritthart,
and
H. Kresbach.
Quantitative evaluation of melanoma cell invasion in three-dimensional confrontation cultures in vitro using automated image analysis.
J. Invest. Dermatol.
94:
114-119,
1990[Abstract].
140.
Smolle, J.,
C. Helige,
and
H. A. Tritthart.
An image analysis and statistical evaluation program for the assessment of tumour cell invasion in vitro.
Anal. Cell. Pathol.
4:
49-57,
1992[Medline].
141.
Stein, I.,
M. Neeman,
D. Shweiki,
A. Itin,
and
E. Keshet.
Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.
Mol. Cell. Biol.
15:
5363-5368,
1995[Abstract].
142.
Strubing, C.,
G. Ahnert Hilger,
J. Shan,
B. Wiedenmann,
J. Hescheler,
and
A. M. Wobus.
Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.
Mech. Dev.
53:
275-287,
1995[Medline].
143.
Stuschke, M.,
V. Budach,
G. Stuben,
C. Streffer,
and
H. Sack.
Heterogeneity in the fractionation sensitivities of human tumor cell lines: studies in a three-dimensional model system.
Int. J. Radiat. Oncol. Biol. Phys.
32:
395-408,
1995[Medline].
144.
Sutherland, R. M.
Cell and environment interactions in tumor microregions: the multicell spheroid model.
Science
240:
177-184,
1988[Medline].
145.
Sutherland, R. M.,
W. R. Inch,
and
J. A. McCredie.
Phytohemagglutinin (PHA)-induced transformation of lymphocytes from patients with cancer.
Cancer
27:
574-578,
1971[Medline].
146.
Sutherland, R. M.,
W. R. Inch,
J. A. McCredie,
and
J. Kruuv.
A multi-component radiation survival curve using an in vitro tumour model.
Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
18:
491-495,
1970[Medline].
147.
Sutherland, R. M.,
J. A. McCredie,
and
W. R. Inch.
Growth of multicell spheroids in tissue culture as a model of nodular carcinomas.
J. Natl. Cancer Inst.
46:
113-120,
1971[Medline].
148.
Takezawa, T.,
Y. Mori,
T. Yonaha,
and
K. Yoshizato.
Characterization of morphology and cellular metabolism during the spheroid formation by fibroblasts.
Exp. Cell Res.
208:
430-441,
1993[Medline].
149.
Takezawa, T.,
M. Yamazaki,
Y. Mori,
T. Yonaha,
and
K. Yoshizato.
Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes.
J. Cell Sci.
101:
495-501,
1992[Abstract].
150.
Tamura, T.,
N. Koide,
H. Hada,
H. Shiraha,
and
T. Tsuji.
Gene expression of liver-specific proteins in hepatocyte spheroids in primary culture.
Acta Med. Okayama
49:
161-167,
1995[Medline].
151.
Tatsukawa, Y.,
M. Arita,
T. Kiyosue,
Y. Mikuriya,
and
M. Nasu.
A comparative study of effects of isoproterenol and dihydroouabain on calcium transients and contraction in cultured rat ventricular cells.
J. Mol. Cell. Cardiol.
25:
707-720,
1993[Medline].
152.
Teutsch, H. F.,
A. Goellner,
and
W. Mueller-Klieser.
Glucose levels and succinate and lactate dehydrogenase activity in EMT6/Ro tumor spheroids.
Eur. J. Cell Biol.
66:
302-307,
1995[Medline].
153.
Tong, J. Z.,
P. De Lagausie,
V. Furlan,
T. Cresteil,
O. Bernard,
and
F. Alvarez.
Long-term culture of adult rat hepatocyte spheroids.
Exp. Cell Res.
200:
326-332,
1992[Medline].
154.
Tosic, M.,
S. Torch,
V. Comte,
M. Dolivo,
P. Honegger,
and
J. M. Matthieu.
Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.
J. Neurochem.
59:
1770-1777,
1992[Medline].
155.
Tovar, O. H.,
and
J. L. Jones.
Biphasic defibrillation waveforms reduce shock-induced response duration dispersion between low and high shock intensities.
Circ. Res.
77:
430-438,
1995
156.
Traub, O.,
R. Eckert,
H. Lichtenberg Frate,
C. Elfgang,
B. Bastide,
K. H. Scheidtmann,
D. F. Hulser,
and
K. Willecke.
Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells.
Eur. J. Cell Biol.
64:
101-112,
1994[Medline].
157.
Tziampazis, E.,
and
A. Sambanis.
Tissue engineering of a bioartificial pancreas: modeling the cell environment and device function.
Biotechnol. Prog.
11:
115-126,
1995[Medline].
158.
VanBael, A.,
M. Proesmans,
D. Tilemans,
and
C. Denef.
Interaction of LHRH with growth hormone releasing factor dependent and independent postnatal development of somatotrophs in rat pituitary cell aggregates.
J. Mol. Endocrinol.
14:
91-100,
1995[Abstract].
159.
Waleh, N. S.,
M. D. Brody,
M. A. Knapp,
H. L. Mendonca,
E. M. Lord,
C. J. Koch,
K. R. Laderoute,
and
R. M. Sutherland.
Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.
Cancer Res.
55:
6222-6226,
1995[Abstract].
160.
Waleh, N. S.,
J. Gallo,
T. D. Grant,
B. J. Murphy,
R. H. Kramer,
and
R. M. Sutherland.
Selective down-regulation of integrin receptors in spheroids of squamous cell carcinoma.
Cancer Res.
54:
838-843,
1994[Abstract].
161.
Walenta, S.,
J. Dotsch,
and
W. Mueller-Klieser.
ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence.
Eur. J. Cell Biol.
52:
389-393,
1990[Medline].
162.
Walker, K. A.,
R. Mairs,
T. Murray,
T. E. Hilditch,
T. E. Wheldon,
A. Gregor,
and
I. M. Hann.
Tumor spheroid model for the biologically targeted radiotherapy of neuroblastoma micrometastases.
Cancer Res.
50:
1000s-1002s,
1990[Abstract].
163.
Wang, R.,
R. Clark,
and
V. L. Bautch.
Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development.
Development
114:
303-316,
1992[Abstract].
164.
Wang, Z.,
B. Fermini,
J. Feng,
and
S. Nattel.
Role of chloride currents in repolarizing rabbit atrial myocytes.
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H1992-H2002,
1995
165.
Weiss, M. J.,
G. Keller,
and
S. H. Orkin.
Novel insights into erythroid development revealed through in vitro differentiation of GATA-1 embryonic stem cells.
Genes Dev.
8:
1184-1197,
1994[Abstract].
166.
Wheldon, T. E.
Targeting radiation to tumours.
Int. J. Radiat. Biol.
65:
109-116,
1994[Medline].
167.
Wiese, C.,
M. Cogoli Greuter,
M. Argentini,
T. Mader,
R. Weinreich,
and
K. H. Winterhalter.
Metabolism of 5-fluoro-dopa and 6-fluoro-dopa enantiomers in aggregating cell cultures of fetal rat brain.
Biochem. Pharmacol.
44:
99-105,
1992[Medline].
168.
Wiles, M. V.,
and
G. Keller.
Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture.
Development
111:
259-267,
1991[Abstract].
169.
Willbold, E.,
M. Reinicke,
C. Lance Jones,
C. Lagenaur,
V. Lemmon,
and
P. G. Layer.
Muller glia stabilizes cell columns during retinal development: lateral cell migration but not neuropil growth is inhibited in mixed chick-quail retinospheroids.
Eur. J. Neurosci.
7:
2277-2284,
1995[Medline].
170.
Wobus, A. M.,
T. Kleppisch,
V. Maltsev,
and
J. Hescheler.
Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels.
In Vitro Cell. Dev. Biol. Anim.
30A:
425-434,
1994.
171.
Wobus, A. M.,
J. Rohwedel,
V. Maltsev,
and
J. Hescheler.
Development of cardiomyocytes expressing cardiac-specific genes, action potentials, and ionic channels during embryonic stem cell-derived cardiogenesis.
Ann. NY Acad. Sci.
752:
460-469,
1995[Medline].
172.
Wu, F. J.,
J. R. Friend,
C. C. Hsiao,
M. J. Zilliox,
W. J. Ko,
F. B. Cerra,
and
W. S. Hu.
Efficient assembly of rat hepatocyte spheroids for tissue engineering applications.
Biotechnol. Bioeng.
50:
404-415,
1996.
173.
Yagi, K.,
C. Yamada,
M. Serada,
N. Sumiyoshi,
N. Michibayashi,
Y. Miura,
and
T. Mizoguchi.
Reciprocal regulation of prothrombin secretion and tyrosine aminotransferase induction in hepatocytes.
Eur. J. Biochem.
227:
753-756,
1995[Abstract].
174.
Yap, A. S.,
and
S. W. Manley.
Thyrotropin inhibits the intrinsic locomotility of thyroid cells organized as follicles in primary culture.
Exp. Cell Res.
214:
408-417,
1994[Medline].
175.
Yap, A. S.,
B. R. Stevenson,
J. R. Keast,
and
S. W. Manley.
Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation.
Endocrinology
136:
4672-4680,
1995[Abstract].
176.
Yeung, M. C.,
L. Pulliam,
and
A. S. Lau.
The HIV envelope protein gp120 is toxic to human brain-cell cultures through the induction of interleukin-6 and tumor necrosis factor-.
AIDS
9:
137-143,
1995[Medline].
177.
Yuasa, C.,
Y. Tomita,
M. Shono,
K. Ishimura,
and
A. Ichihara.
Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.
J. Cell. Physiol.
156:
522-530,
1993[Medline].
178.
Zhang, J.,
R. L. Rasmusson,
S. K. Hall,
and
M. Lieberman.
A chloride current associated with swelling of cultured chick heart cells.
J. Physiol. (Lond.)
472:
801-820,
1993[Abstract].
179.
Zhang, R.,
F. Y. Tsai,
and
S. H. Orkin.
Hematopoietic development of vav/
mouse embryonic stem cells.
Proc. Natl. Acad. Sci. USA
91:
12755-12759,
1994
180.
Zurich, M. G.,
F. Monnet Tschudi,
and
P. Honegger.
Long-term treatment of aggregating brain cell cultures with low concentrations of lead acetate.
Neurotoxicology
15:
715-719,
1994[Medline].
181.
Zwi, L. J.,
B. C. Baguley,
J. B. Gavin,
and
W. R. Wilson.
Blood flow failure as a major determinant in the antitumor action of flavone acetic acid.
J. Natl. Cancer Inst.
81:
1005-1013,
1989[Abstract].
182.
Zwi, L. J.,
B. C. Baguley,
J. B. Gavin,
and
W. R. Wilson.
The use of vascularised spheroids to investigate the action of flavone acetic acid on tumour blood vessels.
Br. J. Cancer
62:
231-237,
1990[Medline].