[Stem cell-based in vitro models as alternative methods for toxicity and efficacy tests in animals]. / Stammzellbasierte In-vitro-Modelle als Ersatz für Tiermodelle bei Toxizitäts- und Wirksamkeitsprüfungen.

Regarding toxicity and efficacy tests of pharmacological and chemical substances (REACH legislation in Europe), there is a strong need to develop alternative methods for animal in vivo studies, in particular for human in vitro models. Here we present results from early phases of projects exploring the potential of embryonic stem cell models, with a special emphasis on embryo toxicity and neuronal stress.We have been able to demonstrate key functional read-outs of neural hESC models, in addition to representing mechanistic aspects which are characteristic for ischemia or excitotoxicity. There is agreement that these mechanisms underlie a variety of human neurodegenerative diseases. We discuss the possibilities to develop more precise endpoints on the molecular level and present an example of a protein biomarker signature emerging from a European FP6 project about embryo toxicity (www.reprotect.eu), employing murine and human embryonic stem cell models.

Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture.

Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of PO(2) distributions with microelectrodes, and determination of cellular oxygen uptake rates (Qc(O(2))) have been applied. In the ras-transfected, tumorigenic spheroid types, the size dependencies of some of the measured parameters exhibited sharp breaks at diameters of approximately 830 microm for Rat1-T1 and approximately 970 microm for MR1 spheroids, respectively, suggesting that some fundamental change in cell metabolism occurred at these characteristic diameters (denoted as "metabolic switch"). Qc(O(2)) decreased and lactate concentration increased as functions of size below the characteristic diameters. Concomitantly, glucose and lactate turnover rates decreased in MR1 spheroids and increased in Rat1-T1. Spheroids larger than the characteristic diameters (exhibiting cell quiescence and lactate accumulation) showed an enhancement of Qc(O(2)) with size. Systematic variations in the ATP and glucose levels in the viable cell rim were observed for Rat1-T1 spheroids only. Proliferative activity, Qc(O(2)), and ATP levels in small, nontumorigenic Rat1 and M1 aggregates did not differ systematically from those recorded in the largest spheroids of the corresponding ras transfectants. Unexpectedly, respiratory activity was present not only in viable but also in the morphologically disintegrated core regions of M1 aggregates. Our data suggest that myc but not ras transfection exerts major impacts on cell metabolism. Moreover, some kind of switch has been detected that triggers profound readjustment of tumor cell metabolism when proliferative activity begins to stagnate, and that is likely to initiate some other, yet unidentified energy-consuming process.

Precapillary servo control of blood pressure and postcapillary adjustment of flow to tissue metabolic status. A new paradigm for local perfusion regulation.

BACKGROUND: There are several shortcomings in current understanding of how the microvasculature maintains tissue homeostasis. Presently unresolved issues include (1) integration of the potentially conflicting needs for capillary perfusion and hydrostatic pressure regulation, (2) an understanding of signal transmission pathways for conveying information about tissue energetic status from undersupplied tissue sites to the arterioles, (3) accounting for the experimentally observed interrelations between precapillary and postcapillary resistances, and (4) an explanation of how precise local adjustment of perfusion to metabolic demands is achieved. METHODS AND RESULTS: A novel conceptualization of how local microvascular control mechanisms are coordinated is proposed, according to which blood flow is adjusted to the metabolic needs of the tissue by the venules. Arteriolar action is merely called on for controlling capillary pressure through myogenic response and shear stress-induced vasodilation. A mathematical model of this theory is introduced and evaluated using well-established experimental data from the literature on regulating mechanisms of microvessel diameters exclusively. The model results demonstrate the suggested mode of microvascular operation to be functional and efficient under conditions present in vivo. Moreover, the predicted vascular responses are large enough to cover the entire range observed in exercising skeletal muscle during adjustment of perfusion to higher performance levels. CONCLUSIONS: Precapillary pressure regulation combined with postcapillary adjustment of perfusion to tissue metabolic status is suitable to resolve the above shortcomings in our current understanding of microvascular control. With mathematical modeling based on experimental data, this mode of microvascular operation is shown to be functional and effective in controlling muscle microcirculation.

Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation.

To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo. To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3-dimensional growth under well-defined conditions. The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-transfected clone of each (Rat1-T1 and MR1). Spheroid volume growth curves and [3H]thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to monolayer cultures. Studies on Rat1 and M1 aggregates showed that the potential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture. pO2 measurements confirmed that neither cell quiescence nor cell death in the pseudo-normal cell aggregate types is due to an oxygen deficiency. In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures. Cell-line-specific attributes in 3D culture that were not specifically related to ras transfection of the cells included histological structure, development of necrosis and thickness of viable cell rim. However, growth behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformation.

On the relation between size of necrosis and diameter of tumor spheroids.

PURPOSE: In many previous experimental studies on multicellular tumor spheroids, the spheroid diameter at which central necrosis develops has been determined to be twice the thickness of the viable cell rim measured at a later stage of spheroid growth. This procedure tacitly assumes that there is a linear relation between the diameter of necrosis and that of the whole spheroid over the entire range of emergence and growth of necrosis. However, some experimental investigations have demonstrated that necroses do not grow gradually with spheroid diameter, but show a rapid initial increase, once a few cells have died. The present article offers an explanation for this phenomenon, which is derived from basic diffusion theory. METHODS AND MATERIALS: A theoretical relation between sizes of spheroids and of their central necroses is developed, which is based on the assumption that formation of necrosis is caused by depletion of substrates or accumulation of metabolic waste products. In a second part, the theoretical model is fitted to experimental data from the literature, and oxygen consumption rate as a function of spheroid size is determined. RESULTS: It turns out that the model closely mimics the experimentally observed behavior described above. These experimental results, therefore, do not furnish any evidence for assuming other hypotheses of necrosis formation. Resulting O2 consumption rates are well in the range of previously published data. In all cases, approximations to the measured data are better than the corresponding linear squares fits. CONCLUSION: At least in some tumor cell lines, depletion of substrates or accumulation of waste products can explain formation of necrosis without the assumption of any additional mechanisms. Moreover, the model presented in this article offers an alternative way of determining the turnover rate of a substrate or metabolic waste product provided that depletion/accumulation of this substance represents the cause for necrosis development.

Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids.

To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters +/- SD: 867 +/- 20 microns), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/I; 445 microCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20-60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a metabolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements.

An easy-to-use model for O2 supply to red muscle. Validity of assumptions, sensitivity to errors in data.

An easy-to-use capillary cylinder model of O2 supply to muscle is presented that considers all those factors that are known to be most important for realistic results: (1) red blood cell (RBC) O2 unloading along the capillary, (2) effects of the particulate nature of blood, (3) free and hemoglobin-facilitated O2 diffusion and reaction kinetics inside RBCs, (4) free and myoglobin-facilitated O2 diffusion inside the muscle cell, and (5) carrier-free region separating RBC and tissue. In a first approach, a highly simplified yet reasonably accurate treatment of the complex three-dimensional oxygen diffusion field in and next to capillaries is employed. As an alternative, a more realistic description using RBC/capillary diffusing capacity has been included. Model development proceeds step by step and is designed to be easily comprehensible for a broad readership. In spite of the number of features accounted for, the model is simple to apply, even for scientists not specialized in the field of modeling. PO2 distributions calculated by the model are in good qualitative agreement with experimental data and with former modelling results. By means of suitable extensions to the model that are also developed it is shown for a wide range of muscle performances that quite generally the following complication may be neglected safely: (1) complexity of O2 diffusion field near capillaries, (2) deviations of capillary domain cross sections from the circular shape, (3) O2 diffusion parallel to the capillary direction, and (4) PO2 dependence of O2 consumption rate. Finally, a sensitivity analysis is performed in which propagation of errors in the input data into the results is investigated. The interpretation of the calculated sensitivities gives insights in the specific dependencies of muscular O2 supply on the various input parameters. Moreover, basic interrelations governing carrier-facilitated diffusional O2 transport to muscle become apparent and are discussed.

Capillary blood transit time in muscles in relation to body size and aerobic capacity.

The mean minimal transit time for blood in muscle capillaries (tc) was estimated in six species, spanning two orders of magnitude in body mass and aerobic capacity: horse, steer, dog, goat, fox and agouti. Arterial (CaO2) and mixed venous (CvO2) blood O2 concentrations, blood hemoglobin concentrations ([Hb]) and oxygen uptake rates were measured while the animals ran on a treadmill at a speed that elicited the maximal oxygen consumption rate (VO2max) from each animal. Blood flow to the muscles (Qm) was assumed to be 85% of cardiac output, which was calculated using the Fick relationship. Total muscle capillary blood volume (Vc) and total muscle mitochondrial volume were estimated by morphometry, using a whole-body muscle sampling scheme. The tc was computed as Vc/Qm. The tc was 0.3-0.5 s in the 4 kg foxes and agoutis, 0.7-0.8 s in the 25 kg dogs and goats, and 0.8-1.0 s in the 400 kg horses and steers. The tc was positively correlated with body mass and negatively correlated with transcapillary O2 release rate per unit capillary length. Mitochondrial content was positively correlated with VO2max and with the product of Qm and [Hb]. These data suggested that Qm, Vc, maximal hemoglobin flux, and consequently tc, are co-adjusted to result in muscle O2 supply conditions that are matched to the O2 demands of the muscles at VO2max.

Glucose diffusion coefficients determined from concentration profiles in EMT6 tumor spheroids incubated in radioactively labeled L-glucose.

A method for performing and evaluating autoradiography of diffusible 14C labeled substances in multicellular tumor spheroids is presented that allows one to obtain a diffusion coefficient of the substance investigated from each individual spheroid. Application of the method with 14C labeled L-glucose resulted in a glucose diffusion coefficient of 5 x 10(-6) cm2/s. It also revealed problems of the method at very short incubation times of about 10 s or less. These problems are most likely caused by the large penetration depth of beta particles irradiated by 14C labels (as compared to 3H labels) which tends to transform steep 14C concentration gradients into much more shallow optical density gradients during exposure. This transformation can be corrected for by deconvolution of the recorded optical density distributions. Basic data and mathematical tools necessary for the process of deconvolution are presently being developed. It is planned to use this method for determining diffusion coefficients of other substances of interest. One such group of substances are the metabolic waste products, most importantly lactate. Another group consists of larger molecules, e.g. peptides and comprises the various growth factors important in tumor biology. Since for members of this latter group little is known about their velocity of penetration into tissue, model calculations may be applied to predict a range of incubation times suitable for determining diffusion coefficients. Moreover, the algorithms for data analysis will have to be modified to allow for receptor binding of the substance under study.(ABSTRACT TRUNCATED AT 250 WORDS)

Oncogene-associated growth behavior and oxygenation of multicellular spheroids from rat embryo fibroblasts.

The basis of the present investigation was the establishment of an oncogene-dependent, genetically determined two-stage carcinogenesis in vitro model as multicellular spheroids. Spheroid formation was achieved with four rat embryo fibroblast cell lines, two of which represent the first step of malignant transformation, known as stage of immortalization. The ras-transfected counterparts of these two parental cell clones represent fully transformed phenotypes. The data obtained show that spheroid volume growth and cellular viability reflect the degree of tumorigenicity in vivo of the different fibroblast types investigated. In addition, ras-transfection alters not only the growth kinetics but also the cellular oxygen metabolism. Furthermore, the results demonstrate very clearly that different fibroblast clones at the same stage of malignant transformation may be characterized by an entirely different growth behavior, morphology and metabolic activity in spheroid culture. This is true, although these cells originate from the same primary cells, differ only in the step of immortalization, and were cultured as spheroids under identical environmental conditions.

Proliferation and oxygenation status of widr spheroids in different lactate and oxygen environments.

Human colon adenocarcinoma cells WiDr were cultured as monolayers or multicellular spheroids in 5% or 20% (v/v) oxygen and in various external lactate concentrations of 0-20 mM. Doubling times and H-3-thymidine labeling indices of exponential monolayer cells indicated that there was no difference in growth behavior between the two oxygen environments, yet these parameters reflected a growth retardation upon elevation of lactate. Growth of WiDr spheroids was retarded in both low oxygen and high lactate concentrations. There was a tendency towards a decrease in the thickness of the viable cell rim with increasing lactate in 20% O-2, whereas the width of the viable rim increased significantly as a function of external lactate in 5% O-2. Intraspheroidal oxygen tensions (Po-2) measured with microelectrodes were less in 5% O-2 than in 20% O-2, yet did not vary systematically as a function of external lactate. Po-2 values in the spheroid center dropped to 0 mm Hg prior to the emergence of central necrosis under all conditions investigated. Unlike numerous other spheroid types investigated up to now, WiDr spheroids mimic tumor microregions with hypoxia-induced necrosis and with nonproliferating cells at very low oxygen pressure.

Relating measuring signals from PO2 electrodes to tissue PO2: a theoretical study.

Organ surface PO2 measurement by oxygen sensitive electrodes has proved to be an efficient tool for monitoring changes in tissue oxygenation status. A parameter giving more direct information is the PO2 distribution within tissue cells which, however, can only be assessed by more invasive methods. The present study addresses the problem of relating electrode measured surface PO2 to muscle cell PO2. To that end, the magnitude of tissue volumes the PO2 in which electrodes are sensitive to, has been reassessed. It turned out that the measuring signal of current membrane covered PO2 electrodes is only sensitive to the PO2 within the muscle surface and not any deeper, thus rendering the measured PO2 an area weighted average over the surface PO2 rather than a spatial average. The consequences of this finding are illustrated in an example of a maximally working muscle. Under the assumption that there are capillaries located on the muscle surface or immediately beneath it, the PO2 predicted to be measured by a surface electrode is almost four times the average muscle fiber PO2. However, PO2 values actually measured by surface PO2 electrodes are even higher calling for further explanation, in which superficial blood vessels and surface fascia may play an important role. Quantitative information on vascular morphology near muscle surfaces is needed in order to more definitely determine the importance of mechanisms discussed, for experimental results.

Rhabdomyosarcoma spheroids with central proliferation and differentiation.

A novel type of multicellular spheroids was established and characterized with regard to growth behavior, proliferation, and differentiation. The spheroids were grown from clonal rat rhabdomyosarcoma cells using the spinner flask technique. The cell aggregates showed several unique properties that were different from those observed in most of the spheroids investigated to date. These properties include a non-Gompertzian volume growth; the coexistence of undifferentiated mononuclear cells and of differentiated, myotube-like giant cells with numerous nuclei; a relatively homogeneous intraspheroidal distribution of proliferating mononuclear cells with thymidine labeling even in the center of spheroids greater than 1 mm; the absence of necrosis in such large spheroids, and the accumulation of myotube-like cells in the center of these spheroids instead. There was a decrease in the overall proliferative activity of the mononuclear cells with increasing proportion of giant cells in the rhabdomyosarcoma spheroids.