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PubMed1996

The restoration of the functions of serially passaged calf hepatocytes by spheroid formation.

A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers-including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate dehydrogenase, and ammonia-metabolizing activities-have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.

Animals↗
PubMed1998

[In vitro studies of PDR brachytherapy].

BACKGROUND: Calculations on the basis of the LQ-model have been focussed on the possible radiobiological equivalence between common continuous low dose rate irradiation (CLDR) and a superfractionated irradiation (PDR = pulsed dose rate) provided that the same total dose will be prescribed in the same overall time as with the low doserate. A clinically usable fractionation scheme for brachytherapy was recommended by Brenner and Hall and should replace the classical CLDR brachytherapy with line sources with an afterloading technique using a stepping source. The hypothes is that LDR equivalency can be achieved by superfractionation was tested by means of in vitro experiments on V79 cells in monolayer and spheroid cultures as well as on HeLa monolayers. MATERIALS AND METHODS: Simulating the clinical situation in PDR brachytherapy, fractionation experiments were carried out in the dose rate gradient of afterloading sources. Different dose levels were produced with the same number of fractions in the same overall incubation time. The fractionation schedules which were to be compared with a CLDR reference curve were: 40 x 0.47 Gy, 20 x 0.94 Gy, 10 x 1.88 Gy, 5 x 3.76 Gy, 2 x 9.4 Gy given in a period of 20 h and 1 x 18.8 Gy as a "single dose" exposition. As measured by flow cytometry, the influence of the dose rate in the pulse on cell survival and on cell cycle distribution under superfractionation was examined on V79 cells. RESULTS: V79 spheroids as a model for a slowly growing tumor, reacted according to the radiobiological calculations, as a CLDR equivalency was achieved with increasing fractionation. Rapidly growing V79 monolayer cells showed an inverse fractionation effect. A superfractionated irradiation with pulses of 0.94 Gy/h respectively 0.47 Gy/0.5 h was significantly more effective than the CLDR irradiation. This inverse fractionation effect in log-phase V79 cells could be attributed to the accumulation of cycling cells in the radiosensitive G2/M phase (G2 block) during protected exposure which was drastically more pronounced for the pulsed scheme. HeLa cells were rather insensitive to changes of fractionation. Superfractionation as well as hypofractionation yielded CLDR equivalent survival curves. CONCLUSIONS: The fractionation scheme, derived from the PDR theory to achieve CLDR equivalent effects, is valid for many cell lines, however not for all. Proliferation and dose rate dependend cell cycle effects modify predictions derived from the sublethal damage recovery model and can influence acute irradiation effects significantly. Dose rate sensitivity and rapid proliferation favour cell cycle effects and substantiate, applied to the clinical situation, the possibility of a higher effectiveness of the pulsed irradiation on rapidly growing tumors.

Animals↗
PubMed2003

The expression levels of three raft-associated molecules in cultivated vascular cells are dependent on culture conditions.

Relaying a signal across the plasma membrane requires functional connections between the partner molecules. Membrane microdomains or lipid rafts provide an environment in which such specific interactions can take place. The integrity of these sites is often taken for granted when signalling pathways are investigated in cell culture. However, it is well known that smooth muscle and endothelial cells undergo cytoskeletal rearrangements during monolayer culturing. Likewise affected--and with potentially important consequences for signalling events--is the organization of the plasma membrane. The expression levels of three raft markers were massively upregulated, and raft-associated 5'-nucleotidase activity increased in conventional monolayer cultures as compared with a spheroidal coculture model, shown to promote the differentiation of endothelial cells. Our data point to a shift of raft components in monolayer cultures and demonstrate potential advantages of the spheroid coculture system for investigation of raft-mediated signalling events in endothelial cells.

5'-Nucleotidase↗
PubMed2006

In vivo matrix-guided human mesenchymal stem cells.

Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation.

Bone Marrow Cells↗
PubMed2003

Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes.

Chondrocyte ex vivo expansion currently performed to replace damaged articular surfaces is associated with a loss of telomeric repeats similar to decades of aging in vivo. This might affect the incidence or time of onset of age-related disorders within transplanted cells or tissues. This study examined whether more immature progenitor cells, such as mesenchymal stem cells (MSC), which can be expanded and subsequently differentiated into chondrocytes is advantageous regarding telomere-length related limitations of expansion protocols. Primary chondrocytes and bone-marrow-derived MSC were isolated from 12 donors, expanded separately to 4 x 10(6) cells, and (re-)differentiated as three-dimensional chondrogenic spheroids. Cells were collected during expansion, after three-dimensional culturing and chondrogenic differentiation, and sequential analyses of telomere length and telomerase activity were performed. Surprisingly, telomeres of expanded MSC were significantly shorter than those from expanded chondrocytes from the same donor (11.4+/-2.5 vs. 13.4+/-2.2 kb) and tended to remain shorter after differentiation in chondrogenic spheroids (11.9+/-1.8 vs. 13.0+/- kb). While telomere lengths in native chondrocytes and MSC were not related to the age of the donor, significant negative correlations with age were observed in expanded (136 bp/year), three-dimensionally reconstituted (188 bp/year), and redifferentiated (229 bp/year) chondrocytes. Low levels of telomerase activity were found in MSC and chondrocytes during expansion and after (re-)differentiation to chondrogenic spheroids. In terms of replicative potential, as determined by telomere length, ex vivo expansion followed by chondrogenic differentiation of MSC did not provide a benefit compared to the expansion of adult chondrocytes. However, accelerated telomere shortening with age during expansion and redifferentiation argues for an "age phenotype" in chondrocytes as opposed to MSC and suggests an advantage for the use of MSC especially in older individuals and protocols requiring extensive expansion

Age Factors↗
PubMed1998

[Treosulfan displays cytotoxic effect on spheroids of primary cell cultures of renal cell carcinoma independent of p-glycoprotein expression].

Therapy of advanced renal cell carcinoma remains difficult. New therapeutic schemes besides cytokine treatment should be evaluated. The following study analyzes the in vitro toxicity of treosulfan on spheroids of 8 primary cultures of renal cell carcinoma cells. these data were compared to the toxicity of vinblastine. All investigations were performed in regard to the P-glycoprotein (Pgp) expression of the cells, which is one of the main causes of multidrug resistance. Four Pgp positive and four Pgp negative spheroids were incubated with the drugs in increasing doses. Toxicity was measured using the MTT toxicity assay as well as trypan blue exclusion. Significantly higher toxicity of treosulfan compared to vinblastine could be demonstrated. In addition, the effects of treosulfan were not related to Pgp expression. These results are encouraging and a phase II study analyzing the efficacy of treosulfan in patients with advanced renal cell carcinoma has been initiated in our institution.

Antineoplastic Agents, Alkylating↗
PubMed2006

Time of transplantation and cell preparation determine neural stem cell survival in a mouse model of Huntington's disease.

Cell replacement therapies for neurodegenerative diseases, using multipotent neural stem cells (NSCs), require above all, a good survival of the graft. In this study, we unilaterally injected quinolinic acid (QA) into the striatum of adult mice and transplanted syngeneic NSCs of enhanced green fluorescent protein-transgenic mice into the lesioned striatum. The injection of QA leads to an excitotoxic lesion with selective cell death of the medium sized spiny neurons, the same cells that are affected in Huntington's disease. In order to investigate the best timing of transplantation for the survival of donor cells, we transplanted the stem cells at 2, 7 and 14 days after injury. In addition, the influence of graft preparation prior to transplantation, i.e., intact neurospheres versus dissociated cell suspension on graft survival was investigated. By far the best survival was found with the combination of early transplantation (i.e., 2 days after QA-lesion) with the use of neurospheres instead of dissociated cell suspension. This might be due to the different states of host's astrocytic and microglia activation which we found to be moderate at 2, but pronounced at 7 and 14 days after QA-lesion. We also investigated brain derived neurotrophic factor (BDNF)-expression in the striatum after QA-lesion and found no significant change in BDNF protein-level. We conclude that already the method of graft preparation of NSCs for transplantation, as well as the timing of the transplantation procedure strongly affects the survival of the donor cells when grafted into the QA-lesioned striatum of adult mice.

Animals↗
PubMed2007

Histone deacetylase inhibitors increase neuronal differentiation in adult forebrain precursor cells.

Chromatin modification plays a key role in fate decision of neural stem cells. Here, we explored the impact of epigenetic remodelling onto neuronal fate determination using specific inhibitors of histone deacetylases (iHDAC). Adult subventricular zone (SVZ) precursor cells were expanded as neurospheres and treated in vitro with second generation iHDAC MS-275, M344 and suberoylanilide hydroxamic acid (SAHA). All tested compounds revealed a significant increase of betaIII-tubulin positive neurons (ranging from 258 to 431%) in a concentration-dependent manner. The number of oligodendrocytes was decreased by almost 50%, accompanied by a reduction of Olig2 mRNA expression. In contrast, astrocyte quantity remained unaffected after iHDAC treatment. Both control and iHDAC treated cells expressed markers of mature GABAergic and dopaminergic neurons. Increased expression levels of NeuroD, Cyclin D2 and B-lymphocyte translocation gene 3 (Btg3) point to a shift towards neuronal fate determination targeted by HDAC inhibitors.

Age Factors↗
PubMed2001

Fractal dimension of K1735 mouse melanoma clones and spheroid invasion in vitro.

An in vitro tumour-host confrontation method to investigate the invasion behaviour of cancer has been applied to K1735 mouse melanomas. Fluorescently labelled spheroids of cancer cells and host cells were confronted and the temporal course of cancer invasion into the host was investigated using confocal laser scanning microscopy. To improve the quantitative data of this method, the boundary images of the fluorescently labelled confrontation pairs were treated as fractals. The physical and mathematical framework for determination of the fractal capacity dimension is widely used in biology and medicine and has proved to be a very useful tool for describing the cancer invasion process. The fractal capacity dimension determination was carried out by dilation of the binary boundaries of the objects, which were treated as an estimate of the Minkowski-Bouligand dimension. The fractal dimension correlated well with the degree of invasion of the K1735-M2 clone. Control experiments, with host-host confrontations and various K1735 clones with reduced invasiveness, support these results.

Animals↗
PubMed2001

Monocyte and monocyte-derived macrophage secretion of MCP-1 in co-culture with autologous malignant and benign control fragment spheroids.

This study was performed in order to determine how monocytes and macrophages in co-culture with autologous head and neck squamous cell carcinoma (HNSCC) tumor tissue regulate the secretion of monocyte chemotactic protein-1 (MCP-1). The levels of MCP-1 were measured when autologous monocytes or monocyte-derived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from HNSCC tumors or benign mucosa serving as control. MCP-1 secretion from co-culture stimulated monocytes and MDMs was increased compared to spontaneous MCP-1 secretion. With prolonged co-culture, MDMs showed a steady-state MCP-1 secretion above background levels for up to 96 h, even with change of co-culture media every 24 h. Addition of an anti-MCP-1 antibody to the medium decreased co-culture-induced monocyte IL-6 secretion. Addition of lipopolysaccharide (LPS) (1 [microg/ml) reduced MCP-1 secretion compared to spontaneous secretion in monocyte cultures. F-spheroids also secrete MCP-1, but at insignificant levels compared to the MCP-1 secretion from monocytes and MDMs. MCP-1 secretion from monocytes/MDMs is regulated differently when co-culture stimulation is compared to LPS-stimulation. Monocytes and MDMs expressed MCP-1 mRNA at a high level in all tested conditions: stimulated in co-culture, not stimulated or stimulated with LPS, indicating post-transcriptional regulation of MCP-1 secretion. The secretion of MCP-1 from tumor-derived F-spheroids, and the maintenance of co-culture MCP-1 secretion from MDMs in vitro, suggests that tumor-associated macrophages are a source of MCP-1 in HNSCC tumors.

Antibodies, Monoclonal↗
PubMed2003

The role of stress in the growth of a multicell spheroid.

Rather recent experimental results demonstrate the non-negligible role of mechanical stress in the growth of a multicell spheroid. In this paper we discuss a theoretical framework for volumetric growth suitable for modeling the growth of soft tissues exhibiting the properties of a solid. After a proper kinematic decomposition, balance equations for mass, momentum and energy are discussed together with constitutive relationships. The mathematical model is then applied to avascular tumor growth. We show by numerical simulation that, under assumption of spherical symmetry, the mathematical model is able to reproduce the experimental data with a satisfying qualitative agreement.

Algorithms↗
PubMed2004

Residual stress generation and necrosis formation in multi-cell tumour spheroids.

We consider how cell proliferation and death generate residual stresses within a multi-cell tumour spheroid (MCTS). Previous work by Jones and co-workers [8] has shown that isotropic growth in a purely elastic MCTS produces growth induced stresses which eventually become unbounded, and hence are physically unrealistic. Since viscoelastic materials show stress relaxation under a fixed deformation we consider the effect of the addition of a small amount of viscosity to the elastic system by examining formation of equilibrium stress profiles within a Maxwell type viscoelastic MCTS. A model of necrosis formation based upon that proposed by Please and co-workers (see [16] [17] [18]) is then presented in which necrosis forms under conditions of adverse mechanical stress rather than in regions of extreme chemical stress as is usually assumed. The influence of rheology on necrosis formation is then investigated, and it is shown that the excessive stress generated in the purely elastic tumour can be relieved either by the addition of some viscosity to the system or by accounting for an inner necrotic interface with an appropriate stress boundary condition.

Cell Growth Processes↗
PubMed2001

A new mathematical model for avascular tumour growth.

The early development of solid tumours has been extensively studied, both experimentally via the multicellular spheroid assay, and theoretically using mathematical modelling. The vast majority of previous models apply specifically to multicell spheroids, which have a characteristic structure of a proliferating rim and a necrotic core, separated by a band of quiescent cells. Many previous models represent these as discrete layers, separated by moving boundaries. Here, the authors develop a new model, formulated in terms of continuum densities of proliferating, quiescent and necrotic cells, together with a generic nutrient/growth factor. The model is oriented towards an in vivo rather than in vitro setting, and crucially allows for nutrient supply from underlying tissue, which will arise in the two-dimensional setting of a tumour growing within an epithelium. In addition, the model involves a new representation of cell movement, which reflects contact inhibition of migration. Model solutions are able to reproduce the classic three layer structure familiar from multicellular spheroids, but also show that new behaviour can occur as a result of the nutrient supply from underlying tissue. The authors analyse these different solution types by approximate solution of the travelling wave equations, enabling a detailed classification of wave front solutions.

Cell Division↗
PubMed2001

The influence of growth-induced stress from the surrounding medium on the development of multicell spheroids.

A mathematical model is adapted to the description of the growth of an avascular tumour spheroid embedded in a deformable medium (gel). Attention is focused on the influence that the mechanical properties of the gel have on the tumour's growth dynamics. Following the work of Landman and Please (2000), the tumour is treated as a two phase material, whereby the tumour cells and the extracellular fluid form distinct phases. Its growth is modelled by combining mass conservation and force balances for each phase with the usual concepts of diffusion limited growth in response to an externally supplied nutrient. The mechanical properties of the gel are characterised by a strain energy function. The stress induced in the gel by the tumour's expansion is incorporated into the force balance equations, thereby linking it to the tumour's growth. Numerical simulations of the model equations show that as the stiffness of the gel increases, the tumour's growth rate and equilibrium size decrease and the time at which necrosis is initiated is delayed (if it occurs at all). Similar results are obtained when the initial size of the tumour is reduced whilst the mechanical properties of the surrounding gel are held fixed. Such results, which are in good qualitative agreement with available experimental data, suggest that mechanical interactions between a tumour and the tissue or medium in which it is located can significantly influence its growth dynamics.

Animals↗
PubMed2003

Differentiation of neurospheres from the enteric nervous system.

The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm(2) culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.

Animals↗
PubMed2005

Autosomal dominant leukodystrophy with axonal spheroids and pigmented glia: clinical and neuropathological characteristics.

We report two autopsy cases of siblings with adult-onset autosomal dominant leukodystrophy characterized by destruction of cerebral white matter, large numbers of axonal spheroids and pigmented glia in the fronto-temporal lobes. Both patients presented with motor and cognitive symptoms and aphasia, 2-3 years before death. At autopsy, the brain showed brown coloration and decreased volume of white matter in the frontal and temporal lobes as well as corpus callosum. Microscopically, marked loss of myelin and axons and abundant axonal spheroids without apparent neuronal loss were observed in the frontal and temporal lobes, which was consistent with hereditary diffuse leukodystrophy with spheroids (HDLS). In addition, glial cells, most consistent with macrophages and containing pigments that were stained by Sudan III and PAS, were found in the white matter lesions. The present cases showed overlapping features with HDLS and pigmentary type of orthochromatic leukodystrophy, suggesting that the pathomechanisms of these two diseases are closely related.

Axons↗
PubMed1998

Pathological study of mice with total deficiency of sphingolipid activator proteins (SAP knockout mice).

Sphingolipid activator proteins (SAPs) A to D are lysosomal factors required in degradation of sphingolipids with short hydrophilic head groups and are derived from a precursor protein. Sap-B deficiency causes a variant of metachromatic leukodystrophy and sap-C deficiency causes a variant of Gaucher disease. Human total SAP deficiency has been reported in two patients in a single family. In these cases, various inclusions were described in the liver, skin, muscle and peripheral nerves ultrastructurally, but there was no report on the pathological study of the central nervous system (CNS). With targeted disruption of the precursor protein gene, we have generated mice with total SAP deficiency. These mice developed progressive neurological symptoms around day 20 and could not survive beyond day 40. Their cardinal pathology is extensive neurovisceral storage. Neuronal storage was already detected in the dorsal root ganglia as early as postnatal day 1 and diffuse neuronal storage was detected in the CNS after day 10. This storage was immunoreactive with anti-ubiquitin antibody and ultrastructurally appeared as inclusions consisting of numerous concentric lamellar and dense granular structures in the perikarya as well as in dendrites and axons. Axonal spheroids containing electron-dense concentric lamellar bodies and neurofilaments were also conspicuous. The extent of neuronal storage, numbers of storage neurons and axonal spheroids increased with age, accompanied with hypomyelination, astrogliosis and increase of macrophages. After day 30, argyrophilic tangle-like structures, which were immunoreactive with an antibody to phosphorylated neurofilaments, were found in the perikarya of many spinal and some neocortical neurons. Inclusions with various ultrastructural features were also noted in the glial cells, choroid plexus epithelial cells, vascular endothelial cells, Schwann cells, macrophages, fibroblasts, hepatocytes, and renal tubular epithelial cells. Some inclusions in the visceral organs were closely similar to those described in human cases of total SAP deficiency. The ultrastructural features of these inclusions in SAP knockout mice appeared unique and were different from those of other known sphingolipidoses.

Animals↗