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PubMed1996

Three-dimensional co-culture of human monocytes and macrophages with tumor cells: analysis of macrophage differentiation and activation.

Here we report on an experimental system for generating TAM in vitro by culturing human MO and MO-derived macrophages (MAC) within 3-dimensional multicellular tumor spheroids (MCS). MO as well as MO-derived MAC migrate into tumor spheroids and spread throughout the entire spheroid within 16 hr. In contrast, fibroblast-spheroids were not infiltrated. The regular expression of MAC maturation-associated antigens on infiltrating MO was suppressed within MCS of the undifferentiated bladder carcinoma line J82 with regard to carboxypeptidase M (CPM), MAX.3 antigen and CD105. However, MAC within spheroids of highly differentiated papillary RT4 cells failed only the single antigen CD51, whereas MAC expressed the complete maturation-associated phenotype within non-tumorigenic HCV29 spheroids. Interestingly, the suppressive effect of J82 carcinoma cells could only be observed in 3-dimensional but not in monolayer cultures. The J82-MCS induced suppression of CPM and MAX.3 expression was only seen to be operative on infiltrating blood MO: MO first differentiated for 2 days and subsequently co-cultured with J82-MCS showed normal expression of MAX.3 and CPM within the spheroid. Besides the modulation of MAC phenotype, the cytokine response of intraspheroidal MAC was analyzed: upon co-culture MO secreted high IL-1beta and IL-6 but low amounts of TNF-alpha as compared to MAC. This MO typical cytokine pattern remained constant for up to 8 days in culture, again indicating a disturbed MO to MAC maturation within tumor spheroids. In conclusion, a 3-dimensional interaction with tumor cells in vitro results in significant changes in the phenotype and function of the spheroid-associated MO and MAC.

Antigens, Neoplasm↗
PubMed1996

Comparative targeting of human colon-carcinoma multicell spheroids using one- and two-step (bispecific antibody) techniques.

In the perspective of radioimmunotherapy (RIT) of micrometastases, we compared, in multicell spheroids (MS), the uptake and retention kinetics of 125I-F(ab)'2 F6 anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), and the affinity enhancement system (AES) using an anti-CEA/anti-DTPA-indium bispecific antibody (BsMAb) and a 125I-labeled di-DTPA-In-tyrosine-lysine bivalent hapten. We used MS of colorectal tumor cell lines expressing CEA strongly (LS 174T), weakly (HT-29) or not at all (HRT-18). Uptake and retention kinetics of 125I-F(ab)'2 F6 and 125I-BsMAb used alone gave similar results. The highest uptake values, obtained with LS 174T MS, were slightly lower with AES than with 125I-F(ab)'2 F6. However, effective retention half-lives were longer for AES than for 125I-F(ab)'2 F6 or for 111In-labeled monovalent hapten after pre-incubation of spheroids with BsMAb. Autoradiography showed the same slow and heterogeneous distribution of 125I-F(ab)'2 F6 and 125I-BsMAb. These results indicate that the 2-step technique is more favorable for RIT: uptake values were approximately the same but uptake kinetics were more rapid, and retention half-life was longer than with the one-step technique.

Antibodies, Bispecific↗
PubMed1997

Release of replication-deficient retroviruses from a packaging cell line: interaction with glioma tumor spheroids in vitro.

The present study describes how various growth conditions affect gene expression and virus production from a retroviral packaging cell line (Liz 9), grown as monolayers and as multicellular spheroids. In addition, to study the direct interaction between packaging cells and tumor tissue of glioma origin, Liz 9 spheroids were confronted with tumor spheroids derived from a human glioma cell line, GaMg. The results show a progressive gene transfer into the tumor tissue, with 9% transfection efficacy after 5 days of co-culture. In comparison, no gene transfer was observed when the Liz 9 spheroids were confronted with normal brain-cell aggregates. The Liz 9 spheroids established from early-passage cultures (passages 7-14) showed limited growth during 28 days, whereas those initiated from late-passage monolayer cultures (passages 39-49) showed extensive growth. Flow-cytometric DNA profiles of monolayers and of spheroids indicated no difference in cell-cycle distribution or ploidy between early and late passages. A cell-viability assay using scanning confocal microscopy revealed mostly viable cells in the Liz 9 spheroids, with only a few dead cells scattered within the structures. The lacZ-gene expression was maintained in early- and in late-passage cultures. In comparison, in Liz 9 early-passage monolayers, the virus titer was 3.1 x 10(4) +/- 0.4 x 10(4) CFU/ml, whereas no virus titer was found in late-passage cultures. The virus titer from the Liz 9 spheroids was found to be between 10(3) and 10(4) CFU/ml. It is concluded that the virus production from packaging cells may vary, depending on passage number and tissue-culture conditions. In the present study, this is demonstrated by a complete loss in virus titer during prolonged culture of packaging cells. In addition, the 3-dimensional confrontation system described allows direct visualization of how packaging cells interact with tumor tissue. Thus, the co-culture system represents a model for studying the efficiency of packaging cells in transfecting heterogeneous tumor tissue in vitro.

Animals↗
PubMed1997

Radiation-induced apoptosis in human ovarian carcinoma cells growing as a monolayer and as multicell spheroids.

Response to external gamma irradiation was studied in a human ovarian carcinoma cell line (OVCAR 3) growing as a monolayer and as multicell spheroids. Necrosis and apoptosis were documented using Trypan-blue uptake and acridine-orange staining, respectively, and apoptosis was quantified using a terminal deoxynucleotidyl transferase assay. Exposure of OVCAR 3 cells growing as a monolayer to 137Cs gamma radiation at a dose of 10 Gy produced 30-40% apoptosis 72 hr after irradiation. Cell-cycle analysis of irradiated cells showed an accumulation of cells in G2/M phase 24 hr after irradiation and then a decline at 48 hr in conjunction with apoptosis onset. The loss of G0/G1 cells in irradiated cultures suggested a preferential entry into apoptosis. No increase in apoptotic cell number was observed in OVCAR 3 spheroids after irradiation, and the cells probably died as a result of necrosis. When spheroids were disrupted immediately after irradiation to obtain a cell suspension, minor apoptosis was observed in association with a marked increase in TB-positive cell number after 96 hr of incubation following irradiation. Thus, a relationship was found between radiation-induced apoptosis and the cell cycle. Results with spheroids suggested the possible involvement of cell-to-cell interactions in apoptosis regulation.

Apoptosis↗
PubMed1998

Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids.

Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400+/-70 microm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100+/-50 microm), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 microm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 microm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Cytokine repertoire during maturation of monocytes to macrophages within spheroids of malignant and non-malignant urothelial cell lines.

Terminal maturation of human blood monocytes to macrophages (MAC) in vivo is believed to be important for the morphology, antigen expression and functional activity of the resulting MAC population. This process is modulated by the specific tissue micro-environment to which blood monocytes migrate upon leaving the vasculature. Tumor-associated macrophages (TAM) are a special type of MAC, and little is known about the modulating capacity of the tumor environment on monocyte-to-MAC differentiation. By co-culturing 3-dimensional multicellular spheroids (MCS) of the urothelial-bladder-carcinoma cell lines J82 and RT4 with human monocytes/MAC we generated TAM in vitro. For comparison, monocytes/MAC were co-cultured with the non-tumorigenic urothelial cell line HCV29. The effects on monocyte differentiation were analyzed, particularly with respect to cytokine release. Monocyte maturation was modulated within the tumor spheroid dependent upon the tumor cell type. Monocytes co-cultured with MCS of the poorly differentiated J82 carcinoma spontaneously produced high amounts of IL-1beta and IL-6, but only low amounts of TNF-alpha, which could be further increased by the addition of LPS. This cytokine pattern is characteristic for monocytes and remained constant for up to 8 days in J82-MCS co-cultures. However, in RT4-MCS and HCV29-MCS co-cultures, the initial cytokine pattern changed and after 8 days corresponded well to that of MAC differentiated in vitro without tumor contact. In addition to functional parameters, we analyzed the morphology of J82-MCS-TAM and found that they displayed a monocyte-like morphology. Our data indicate that (1) tumor cells can influence monocyte-to-MAC differentiation, giving rise to TAM with monocyte-specific phenotypic properties; and (2) this capacity is dependent on the type of tumor cell.

Cell Differentiation↗
PubMed1998

Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the "optical probe technique".

Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors. We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm). MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas. A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS. This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery. The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm. A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS. In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained. When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers. Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects. The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms.

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

Animals↗
PubMed1998

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids.

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10-20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo.

Aminoacridines↗
PubMed1999

Differences in metabolism of 5-fluorouracil and 5-fluorouridine and regulation by glucosamine in human colon cancer multicell tumor spheroids.

Glucosamine (GlcN) modulates fluoropyrimidine metabolism and enhances cytotoxicity of 5-fluorouridine (FUrd), but not of 5-fluorouracil (FUra), in human tumor models. To elucidate the underlying metabolic differences between FUra and FUrd, by the use of 19F and 31P NMR spectroscopy we studied these drugs in multicell tumor spheroids (MTS) formed by human colon carcinoma cells HT-29. This experimental system allowed detailed kinetic measurements of anabolic intracellular phosphates and fluorophosphates over periods of up to 2 days. Time-dependent NMR data were reduced and interpreted by the use of nonlinear compartmental models which yielded numerical values for the empirical rate constants characterizing mass transfer among the compartments. An analysis of these rate constants indicated qualitative and quantitative differences in the metabolism of FUra and FUrd and in the effects of GlcN on these drugs. The enhanced generation of FUDP-hexoses was a predicted effect of GlcN, but inhibited formation of fluorouridine diphosphates and fluorouridine triphosphates in FUra-treated MTS, and the magnitude of stimulation of fluoropyrimidine incorporation into macromolecules in FUrd-treated MTS were not predicted.

Body Fluid Compartments↗
PubMed2000

Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: in vitro studies using cell lines.

At the initial phase of embryo implantation, the trophoblast must have acquired competence for adhesion to the uterine epithelium, a condition whose cell biological basis is far from understood. In the present study, trophoblast-type cells (BeWo, JAr, and Jeg-3 choriocarcinoma cell lines) were treated with retinoic acid, methotrexate, dibutyryl-cAMP, or phorbol-12-myristate-13-acetate in order to modulate their ability to adhere to uterine epithelial cells (RL95-2). In an established model, multicellular spheroids of choriocarcinoma cells were transferred onto the surface of monolayer cultures of RL95-2 cells followed by a centrifugal force-based adhesion assay. In controls, about 45% of BeWo and JAr cell spheroids and 75% of Jeg-3 spheroids adhered to uterine monolayers within 30 min. Pretreatment of spheroids with either of the agents stimulated differentiation as indicated by the rate of chorionic gonadotropin secretion, but consistently reduced the adhesion to the endometrial monolayer in all three choriocarcinoma cell lines. While previous investigations had shown that invasiveness of trophoblast cells (into extracellular matrix) does not seem to be linked to the differentiation program in a simple manner, the present data suggest that such an (inverse) link may indeed exist with respect to the ability to initiate an adhesive interaction with the uterine epithelium. These observations support the view that epithelial cell interactions as typical for the initial phase of embryo implantation are regulated in a way that is clearly different from cell-matrix interactions governing later phases of trophoblast invasion into the endometrial stroma.

Cell Adhesion↗
PubMed2000

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids.

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.

Animals↗
PubMed2003

Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels.

We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology.

Animals↗
PubMed2003

Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells.

OBJECTIVE: To compare the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BMSC) and adipose tissue-derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage. METHODS: Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription-polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage. RESULTS: The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage. CONCLUSION: Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage-like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches.

Adipose Tissue↗
PubMed2006

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.

Cell Line, Tumor↗
PubMed2002

Aggregation kinetics of well and poorly differentiated human prostate cancer cells.

Aggregation of attachment-dependent animal cells represents a series of motility, collision, and adhesion events applicable to such diverse fields as tissue engineering, bioseparations, and drug testing. Aggregation of human prostate cancer cells in liquid-overlay culture was modeled using Smoluchowski's collision theory. Using well (LNCaP) and poorly differentiated (DU 145 and PC 3) cell lines, the biological relevance of the model was assessed by comparing aggregation rates with diffusive and adhesive properties. Diffusion coefficients ranged from 5 to 90 microm(2)/min for single LNCaP and PC 3 cells, respectively. Similar diffusivities were predicted by the persistent random walk model and Einstein relation, indicating random motion. LNCaP cells were the most adhesive in our study with reduced cell shedding, 100% adhesion probability, and enhanced expression of E-cadherin. There was an increase in DU 145 cells staining positive for E-cadherin from nearly 20% of single cells to uniform staining across the surface of all aggregates; under 30% of PC 3 aggregates stained positive. Aggregation rates were more consistent with adhesive properties than with motilities, suggesting that aggregation in our study was reaction-controlled. Relative to other assays employed here, aggregation rates were more sensitive to phenotypic differences in cell lines and described size-dependent changes in aggregation at a finer resolution. In particular, model results suggest similar aggregation rates for two-dimensional DU 145 and PC 3 aggregates and upwards of 4-fold higher rates for larger three-dimensional DU 145 spheroids, consistent with expression of E-cadherin. The kinetic model has application to spheroid production, to cell flocculation and as an adhesion assay.

Cell Adhesion↗
PubMed2004

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions.

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.

Albumins↗
PubMed2005

TGF-beta1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte function in 3D microenvironment.

Co-culture of hepatocytes or hepatocyte spheroids with the supporting NIH3T3 in a 3D microcapsule formed with a hybrid natural/synthetic matrix has led to enhanced hepatocyte functions. We investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. The conditioned media from the co-culture induced higher P450 cytochrome oxidase activity (indicated by EROD assay) in the microencapsulated hepatocytes than the conditioned media from the NIH3T3- or the hepatocytes-alone controls. Conditioned media from physically separated co-culture of hepatocytes-NIH3T3 by a membrane insert reduced the functional enhancement. Among the known stimulators of hepatocyte functions, TGF(beta)1 is primarily responsible for the stimulation of hepatocyte functions in this 3D co-culture since the removal of TGF(beta)1 by antibody depletion eliminated the functional enhancement and the reconstitution of TGF(beta)1 restored the functional enhancement. Activation of latent TGF(beta)1 in an extracellular environment were upregulated in co-culture with no observable increase in the TGF(beta)1 expression at transcriptional and translational levels. Our data led to an improved understanding of how co-culture enhances hepatocyte functions in vitro and pave the way for further innovations in liver tissue engineering, drug metabolism studies, and other applications that require functional hepatocytes cultured in vitro.

Animals↗