Adaptation of an automated selective dissociation procedure to two novel spheroid types.
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Animals↗
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Bioeffects of diagnostic ultrasound in vitro.
Biological effects induced by ultrasound were frequently reported for continuous wave (cw) mode. Thresholds for the onset of bioeffects of pulsed ultrasound, starting from diagnostic conditions, have not yet been defined by standardized in vitro models. We therefore investigated the effects of pulsed ultrasound on cultured cells using diagnostic ultrasound devices, a selfmade transducer and a sonochemical laboratory reactor tunable from pulsed diagnostic conditions to cw ultrasound. Additionally, we determined physical parameters of the ultrasonic field by different types of hydrophones. Sonochemical reactions and the effects induced by the ultrasonic fields in cultured cells indicated a threshold for bioeffects.
Animals↗
Multiple cell hits by particle tracks in solid tissues.
Relative Biological Effectiveness (RBE) and Quality Factor (Q) at extreme values of Linear Energy Transfer (LET) have been determined on the basis of experiments with single-cell systems and specific tissue responses. In typical single cell systems, each heavy particle (Ar or Fe) passes through a single cell or no cell. In tissue end-point experiments each heavy particle passes through several cells, and the LET can exceed 200 keV/micrometer in every cell. In most laboratory animal tissue systems, however, only a small portion of the hit cells are capable of expressing the end-point of interest to the investigator, such as cell killing, mutation or carcinogenesis. The following question must therefore be addressed: Do RBE's and Q factors derived from single-cell experiments properly account for the increased probability of multiple-cell damage by HZE tracks? A model is offered in which measured radiation effects and known tissue properties are combined to estimate the value of a multiplier of damage effectiveness on the basis of number of cells at risk, p3n, per track containing a hit cell, where n is the number of cells per track, based on tissue and organ geometry, and P3 is the probability that a cell in the track is capable of expressing the experimental end-point.
Animals↗
The effect of type III collagen on migration and invasion of human glioblastoma cell lines in vitro.
The effect of type III collagen, an extracellular matrix protein, on the in vitro migration and invasion of glioblastoma cells was assayed by chemotaxis using four cell lines. Migration and invasion of gliomablastoma cells was observed in the presence of varying concentrations of type III collagen. In contrast to control experiments in which the protein was not added, type III collagen significantly increased migration and invasion of glioblastoma cells in a dose dependent manner up to 10 micrograms/ml; however, higher concentrations of the protein eliminated this affect on migration and invasion as did the presence of a monoclonal type III collagen antibody. Type III collagen was also shown to stimulate the migration of glioblastoma cells from spheroids to monolayers. The results of this study indicate that type III collagen does influence the migration and invasion of human glioblastoma cells in vitro.
Antibodies↗
Detection of heparan sulfate in spinal spheroids of beta, beta'-iminodipropionitrile (IDPN)-treated rats.
Beta,beta'-Iminodipropionitrile (IDPN) is known to produce a massive accumulation of neurofilaments in the proximal portion of axons of spinal anterior horn cells. The spinal cords of 20 Wistar rats treated with IDPN were examined immunohistochemically with a monoclonal antibody against heparan sulfate (HepSS-1). Virtually all axonal swellings were intensely labeled with HepSS-1. This immunoreactivity was almost completely absorbed by the presence of CDSNS-HS (completely desulfated, N-sulfated heparan sulfate). Western blot study revealed that HepSS-1 recognized four distinct bands at the positions of approximately 17 kDa, approximately 20 kDa, approximately 21 kDa, and approximately 25 kDa. The present study suggests that the deposit of heparan sulfate in spheroids is related to the pathomechanism for the formation of axonal swelling.
Animals↗
Radiation response of human melanoma multicellular spheroids measured as single cell survival, growth delay, and spheroid cure: comparisons with the parent tumor xenograft.
The radiation response of multicellular spheroids, initiated from a human melanoma xenograft (E.E.) propagated in athymic mice, was studied using cell survival, growth delay, and spheroid cure as endpoints. The relationship between these endpoints was analyzed, and the radiation response of the spheroids was compared with the parent xenograft. At irradiation, the spheroids were 100 +/- 5 micron in diameter and did not contain radiobiologically hypoxic cells. Growth delay of the spheroids mainly depended on the fraction of surviving cells as measured in soft agar, that is, there was a good correlation between these two endpoints. Moreover, Do-values calculated from spheroid cure curves were similar to those of the cell survival curves measured in soft agar. However, the number of stem cells per spheroid, calculated from SCD50-values (the doses required to cure 50% of the spheroids), was at least a factor of seven lower than the clonogenicity of cells from disaggregated spheroids would indicate. The cellular radiosensitivity of the spheroids was similar to the parent xenograft. An intercellular contact effect was not found for the spheroids, in agreement with observations from studies of xenografted tumors. Moreover, specific growth delays, as well as Do-values calculated from cure curves were similar for spheroids and tumors when the data for the latter were corrected for the presence of hypoxic cells. The high degree of conformity in the results indicates that multicellular spheroids and xenografted tumors may complement one another in studies of human tumor radiobiology.
Animals↗
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.
Least-Squares Analysis↗
Ultrastructure of size-regulated hetero-spheroids composed of human keratinocytes and fibroblasts.
In order to develop a new in vitro skin model multicellular floating hetero-spheroids were prepared by culturing keratinocytes and fibroblasts on hydrophilic culture dishes coated with type I collagen and a thermo-responsive polymer. Upon decreasing the substratum's temperature to an ambient temperature, the spheroids detached from the substratum and were thereafter maintained in either: Medium I, a medium mixture of keratinocyte growth medium (KGM) and supplemented Dulbecco's modified Eagle's medium (DMEM) at a ratio of 1 to 2; or medium II, KGM for the initial 24 h followed by supplemented DMEM for the remainder of the culture periods. The spheroids displayed a typical pattern of an external rim of keratinocytes with an internal core of fibroblasts. A minute space separated the keratinocytes and fibroblasts. The stratification of cells cultured in medium II was more prominent than that of the cells cultured in medium I. Markers of the advanced stages of keratinization such as keratohyalin granules, membrane coating granules and the cornified envelope were not observed. Interestingly, keratinocytes underwent the same differentiation pathway as non-keratinized stratified epithelia such as esophagus. With consideration to keratinocyte-fibroblast interactions, it may be of interest to incorporate the study of such morphological impairments when investigating the effects of growth factors and their ligands.
Cell Aggregation↗
pO2- and pH-gradients in multicellular spheroids and their relationship to cellular metabolism and radiation sensitivity of malignant human tumor cells.
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Humans↗
Enhanced tumour uptake and in vitro radiotoxicity of no-carrier-added [131I]meta-iodobenzylguanidine: implications for the targeted radiotherapy of neuroblastoma.
In vitro and in vivo neuroblastoma models were used to determine whether improvements in tumour targeting in vivo and therapeutic efficacy in vitro could result from the use of no-carrier-added (n.c.a.) [131I]MIBG. Results were compared with use of the conventional therapy MIBG preparation (ex. [131I]MIBG) of lower specific activity which is produced by iodide exchange reaction. The efficacy of n.c.a. [131I]MIBG was compared with that of [131I]MIBG over a range of specific activities by the assessment of neuroblastoma spheroid growth delay. Whereas n.c.a. [131I]MIBG at a radioactivity concentration of 2 MBq/ml prevented the regrowth of 84% of spheroids, toxicity was significantly reduced by the addition of non-radiolabelled MIBG to the incubation medium. The time-dependent biodistribution of n.c.a. [131I]MIBG in nude mice bearing human neuroblastoma xenografts was compared with that of the conventional therapy radiopharmaceutical. The n.c.a. agent gave improved tumour uptake but also significantly greater accumulation in normal tissues known to accumulate MIBG such as heart, adrenal and skin. However, uptake and retention in the blood was unaltered. For all tissues examined, the 3-day calculations were undertaken to predict organ to tumour dose ratios which would result in human neuroblastoma patients with each of the [131I]MIBG preparations. These results suggest that significant therapeutic gain may be achieved by the use of n.c.a. [131I]MIBG as a treatment agent in neuroblastoma. neuroblastoma.
3-Iodobenzylguanidine↗
Cancer: a challenge for control theory and computer modelling.
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Cell Transformation, Neoplastic↗
Effects of sex- and glucocorticoid steroids on breast cancer cells grown as either multicellular tumor spheroids or monolayers.
The effects of estradiol, medroxyprogesterone acetate (MPA) dexamethasone, dihydrotestosterone and the antihormones 4-OH tamoxifen and RU 38486 were studied in two established breast carcinoma cell lines, the estrogen-sensitive ZR-75-1 and the estrogen-independent BT 20 cells applying two different in vitro systems, spheroid and monolayer cell culture in steroid deprived medium. Growth of ZR-75-1 spheroids was dramatically stimulated by the addition of estradiol, an effect which was neutralized by the simultaneous addition of 4-OH tamoxifen. The antiestrogen alone as well as dihydrotestosterone and MPA reduced ZR-75-1 spheroid growth significantly. While growth of BT 20 spheroids was only transiently inhibited by tamoxifen and dihydrotestosterone, a persistent increase in BT 20 spheroid growth was observed under MPA treatment in a concentration of 1 microM. This effect, although statistically significant, was very moderate. With the exception of this finding, growth effects of the different test compounds were similar in both in vitro systems, tumor spheroids and monolayer cell cultures.
Breast Neoplasms↗
In vitro function of porcine hepatocyte spheroids in 100% human plasma.
To assess their functional stability, porcine hepatocyte spheroids, which have great potential in bioartificial liver systems and/or xenogeneic liver transplantation, were cultured in vitro in 100% human plasma, and their ammonium detoxification capacity was measured. In a short-term functional study, collagen-gel entrapped and rotationally cultured porcine hepatocyte spheroids showed almost the same functional expression, which was approximately twice that of monolayers. No significant functional deterioration was observed up to day 10 as compared with those in a synthesized medium without collagen gel entrapment. In a longer term study (4 wk) using rotational culture, morphological and functional deterioration presumably due to immunorejection involving complement proteins and natural antibodies progressed gradually with culture time. On the termination of the culture, the detoxification ability for the residual cells was approximately two-thirds that on day 2.
Animals↗
Quantitative recording of vitality patterns in living multicellular spheroids by confocal microscopy.
Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or intravascular microregions of large tumors. With progressing growth three distinct concentric annular shells develop. A rim of proliferating cells in the periphery is followed towards the center by layers of quiescent cells and at a defined spheroid diameter cell death occurs in the central core. Fluorescein diacetate (FDA) and Calcein/AM were used as vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for dead cells. For loading multicellular spheroids with the esterase substrate dyes we used a two step cold incubation technique to avoid dye accumulation in the most peripheral cell layers. Homogenously stained tissue allowed to describe the fluorescence attenuation in depth as a monoexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 x 10(-3) in vital stained tissue and 17.9 x 10(-3) in lethal stained tissue. Using the respective attenuation coefficient the raw data were corrected for light absorption and scattering in depth. In radial recordings of the vitality status of multicellular glioma spheroids using CLSM-technique we showed that spheroids up to a diameter of 250 microns were homogenously stained with Calcein/AM and FDA. Spheroids larger than 250 microns consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 microns. The thickness of the rim of vital stained cells decreased with increasing diameter of the spheroids to 64 +/- 7 microns in spheroids of a diameter of 550 +/- 25 microns. Thereafter the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93 +/- 9 microns in spheroids of 700 microns in diameter. The first signs of dead cell staining in the central core occurred at a diameter of 400 +/- 25 microns. The radius of the core increased in an exponential way. The cell layer which was stained neither by vital nor by lethal dyes showed a thickness of 150 microns in spheroids of 550 +/- 25 microns in diameter. Our staining technique and the radial recording of mean field fluorescence signals in living multicellular spheroids will be a valuable tool for experimental cancer research providing a non invasive quantification of cell vitality in living multicellular spheroids.
Cell Survival↗
Three-dimensional co-culture of rat hepatocyte spheroids and NIH/3T3 fibroblasts enhances hepatocyte functional maintenance.
Functional maintenance of primary hepatocytes in culture can be improved by several distinct approaches involving optimization of the extracellular matrix microenvironment, media composition and cell-cell interactions, both homotypic and heterotypic. Using a galactose-decorated surface, we have developed a method to combine these two approaches by co-culturing rat primary hepatocyte spheroids with NIH/3T3 mouse fibroblast cells. Spheroids were performed by culturing hepatocytes for 3 days on galactosylated poly(vinylidene difluoride) membrane; NIH/3T3 cells were subsequently seeded and co-cultured with the spheroids. Results showed that although NIH/3T3 cells alone responded poorly to the galactosylated PVDF surface and displayed limited attachment, NIH/3T3 fibroblasts attached to the periphery of the hepatocyte spheroids and proliferated around them. Co-cultured hepatocyte spheroids exhibited significantly higher liver-specific functions as compared to spheroids cultured alone. Albumin secretion level in this co-culture system peaked on day 11, which was 1.8- and 2.9-times higher than the peak expression level in spheroid homo-culture control in serum-free (day 3) and serum-containing media (day 4), respectively. The albumin secretion function was maintained for at least two weeks; it was 5.1 (in serum-free medium) and 17.8 (in serum-containing medium) times higher than spheroid homo-culture on day 13. Similarly, the co-culture system also expressed approximately 5.5- and 3.1-times higher 3-methylcholanthrene-induced cytochrome P450 enzymatic activity on day 14 as compared to the homo-culture control in serum-free and serum-containing medium, respectively. In conclusion, this unique co-culture system demonstrated the synergistic roles of homotypic cell-cell interaction, heterotypic cell-cell interaction, cell-substrate interaction and soluble stimuli in hepatocyte functional maintenance.
Animals↗
Stresses in growing soft tissues.
Biochemical processes of tissue growth lead to production of new proteins, cells, and other material particles at the microscopic level. At the macroscopic level, growth is marked by the change of the tissue shape and mass. In addition, the appearance of the new material particles is generally accompanied by deformation and, consequently, stresses in the surrounding material. Built upon a microscopic toy-tissue model mimicking the mechanical processes of mass supply, a simple phenomenological theory of tissue growth is used in the present work for explaining residual stresses in arteries and studying stresses around growing solid tumors/multicell spheroids. It is shown, in particular, that the uniform volumetric growth can lead to accumulation of residual stresses in arteries because of the material anisotropy. This can be a complementary source of residual stresses in arteries as compared to the stresses induced by non-uniform tissue growth. It is argued that the quantitative assessment of the residual stresses based on in vitro experiments may not be reliable because of the essential stress redistribution in the tissue samples under the cutting process. Concerning the problem of tumor growth, it is shown that the multicell spheroid or tumor evolution depends on elastic properties of surrounding tissues. In good qualitative agreement with the experimental in vitro observations on growing multicell spheroids, numerical simulations confirm that stiff hosting tissues can inhibit tumor growth.
Animals↗
Parallel development of cardiomyocytes and neurons in embryonic stem cell culture.
Recent studies suggest that there are strong parallels between development and patterning of the vertebrate vascular system and the nervous system. While previous observations reported generation of vascular and neuronal progenitors from embryonic stem (ES) cells, the question of parallel development of vascular and neuronal cells in the same culture has not yet been investigated. Mouse D3 ES cells were cultured for 4 days in differentiation medium IMDM with 15% FBS in 100 mm non-adhesive Petri dishes to allow cells to aggregate and form embryoid bodies. At day 5, fibronectin or all-trans retinoic acid with fibronectin was added to the culture. On day 9, the embryoid bodies were seeded on poly-L-ornithine/fibronectin-coated plates. After plating, half of the plates were treated with laminin for 3 days and maintained for 1 week in Neurobasal media with B27. Here we show that ES cells differentiate into interconnected rhythmically contracting aggregates of functional cardiomyocytes and neurons. Double immunofluorescence with anti-phospholamban, anti-SERCA2 antibodies to detect cardiomyocytes and with anti-MAP2 antibodies to detect neurons revealed the cell aggregates consisting entirely of cardiomyocytes with neuronal cells located on the periphery or covering the aggregate's surface. The observed concurrent development of cardiomyocytes and neurons suggests bidirectional communication between both cell types. We propose that crosstalk between cardiovascular and neuronal progenitors is an important mechanism for the development of both systems.
Animals↗
Necrotic and apoptotic features of cell death in response to Foscan photosensitization of HT29 monolayer and multicell spheroids.
Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.
Apoptosis↗