Cell–surface interactions involving immobilized magnetite nanoparticles on flat magnetic substrates

A new method to affect cells by cell–surface interaction is introduced. Biocompatible magnetic nanobeads are deposited onto a biocompatible magnetic thin layer. The particles are composed of small magnetite crystals embedded in a matrix which can be functionalized by different molecules, proteins or growth factors. The magnetic interaction between surface and beads prevents endocytosis if the setup is utilized for cell culturing. The force acting between particles and magnetic layer is calculated by a magnetostatic approach. Biocompatibility is ensured by using garnet layers which turned out to be nontoxic and stable under culturing conditions. The garnet thin films exhibit spatially and temporally variable magnetic domain configurations in changing external magnetic fields and depending on their thermal pretreatment. Several patterns and bead deposition methods as well as the cell–surface interactions were analyzed. In some cases the cells show directed growth. Theoretical considerations explaining particular cell behavior on this magnetic material involve calculations of cell growth on elastic substrates and bending of cell membranes.     

Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease treatment and cultured for 5 days. Cellular adhesion of each hepatic cell type, gene expression and extracellular matrix deposition were analyzed by immunohistochemistry and immunoblotting. Immunohistochemical analysis demonstrated that the primary culture of fetal liver cells contained at least hepatoblasts, mesenchymal cells, endothelial cells, hemopoietic cells and Kupffer cells. Although hepatoblasts, mesenchymal cells, and endothelial cells aggregated separately in the initial step, they then formed a spheroid together, adhering to the glass slide, which led to the formation of flattened hepatic organoids. Hepatoblasts more preferentially adhered to mesenchymal cells than endothelial cells. Several extracellular matrix depositions were seen in aggregates consisting of at least hepatoblasts and mesenchymal cells within 12 h, but were poor in those lacking hepatoblasts. These data show that the primary culture of fetal liver cells contains most cell types constituting fetal livers, and may be useful for studying cell–cell interactions during liver development.     

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell–cell and cell–matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or “mesenspheres”, of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.     

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress–strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell–PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell–matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9–3.7 and 1.4–2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell–matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.     

A Riemannian manifold analysis of endothelial cell monolayer impedance parameter precision

Endothelial cell adhesion and barrier function play a critical role in many biological and pathophysiological processes. The decomposition of endothelial cell adhesion and barrier function into cell–cell and cell–matrix components using frequency dependent cellular micro-impedance measurements has, therefore, received widespread application. Few if any studies, however, have examined the precision of these model parameters. This study presents a parameter sensitivity analysis of a representative cellular barrier function model using a concise geometric formulation that includes instrumental data acquisition settings. Both model state dependence and instrumental noise distributions are accounted for within the framework of Riemannian manifold theory. Experimentally acquired microimpedance measurements of attached endothelial cells define the model state domain, while experimentally measured noise statistics define the data space Riemannian metric based on the Fisher information matrix. The results of this analysis show that the sensitivity of cell–cell and cell–matrix impedance components are highly model state dependent and several well defined regions of low precision exist. The results of this study further indicate that membrane resistive components can significantly reduce the precision of the remaining parameters in these models. This work was supported by a National Science Foundation CAREER Award (AE), BES-0238905, and in part by the American Heart Association under Grant 0265029B (AE).     

Involvement of cortactin and phosphotyrosine proteins in cell–cell contact formation in cultured bovine corneal endothelial cells

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell–cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including α-catenin, vinculin and cortactin, are localized at cell–cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell–cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y⁴⁶⁶, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY⁴²¹-cortactin was localized at the leading edge and pY⁴⁶⁶-cortactin at a perinuclear area. In confluent cultures both pY⁴⁶⁶- and pY⁴²¹-cortactin isoforms were localized at the cell–cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell–cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y⁴⁶⁶ and associated with the Triton-insoluble fraction.     

The contribution of adhesion signaling to lactogenesis

The mammary gland undergoes hormonally controlled cycles of pubertal maturation, pregnancy, lactation, and involution, and these processes rely on complex signaling mechanisms, many of which are controlled by cell–cell and cell–matrix adhesion. The adhesion of epithelial cells to the extracellular matrix initiates signaling mechanisms that have an impact on cell proliferation, survival, and differentiation throughout lactation. The control of integrin expression on the mammary epithelial cells, the composition of the extracellular matrix and the presence of secreted matricellular proteins all contribute to essential adhesion signaling during lactogenesis. In vitro and in vivo studies, including the results from genetically engineered mice, have shed light on the regulation of these processes at the cell and tissue level and have led to increased understanding of the essential signaling components that are regulated in temporal and cell specific manner during lactogenesis. Recent studies suggest that a secreted matricellular protein, CTGF/CCN2, may play a role in lactogenic differentiation through binding to β1 integrin complexes, enhancing the production of extracellular matrix components and contributions to cell adhesion signaling.     

Cell aggregation on agar as an indicator for cell-matrix adhesion: effects of opioids

The slow aggregation assay is generally used to study the functionality of cell–cell adhesion complexes. Single cells are seeded on a semisolid agar substrate in a 96-well plate and the cells spontaneously aggregate. We used HEK FLAG-MOP cells that stably overexpress the mu opioid receptor and the mu-opioid-receptor-selective agonists DAMGO and morphine to study whether other factors than functionality of cell–cell adhesions complexes can contribute to changes in the pattern of slow aggregation on agar. HEK FLAG-MOP cells formed small compact aggregates. In the presence of DAMGO and morphine, larger and fewer aggregates were formed in comparison to the vehicle control. These aggregates were localized in the center of the agar surface, whereas in the vehicle control they were dispersed over the substrate. However, in suspension culture on a Gyrotory shaker, no stimulation of aggregation was observed by DAMGO and morphine, showing that opioids do not affect affinity. A dissociation experiment revealed that HEK FLAG-MOP aggregates formed in the absence or presence of opioids are resistant to de-adhesion. We demonstrated that the larger aggregates are neither the result of cell growth stimulation by DAMGO and morphine. Since manipulations of the substrate such as increasing the agar concentration or mixing agar with agarose induced the same changes in the pattern of slow aggregation as treatment with opioids, we suggest that cell–substrate adhesion may be involved in opioid-stimulated aggregation.     

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Stability validation of seeding cell control parameters in large-scale hybridoma cell culture

Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075–0.5×10⁶ cells ml⁻¹, “timepost” after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1–0.3×10⁶ cells ml⁻¹, “timepost” after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036–0.047 h⁻¹ and 0.577–0.747 pg cell⁻¹ h⁻¹, with the positivity rate of antigen-binding activity within 97–99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.     

Regulation of concanavalin A agglutination by the extracellular matrix

The agglutinability of rat C₆ glioma cells by concanavalin A (Con A) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maximum amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which they are affected by density and the point at which they become insensitive to further density increases. Both trypsin and EDTA reduce cell agglutinability. The similarity in recovery kinetics between low density cells and cells dissociated with EDTA or trypsin suggests that low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Density-dependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrix inhibitor of Con A agglutinability, while high density cultures produce a matrix promotor.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Epidermal morphogenesis in an <Emphasis Type="BoldItalic">in-vitro</Emphasis> model using a fibroblasts-embedded collagen scaffold

Summary A novel culture system included a self-designed bi-layer 3-D collagen scaffold with different pore size on both sides and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-β1), in the cell–cell and cell–matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air–liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10 ng per ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10 ng per ml of EGF addition in the air-lifted stage resulted in uneven cell–matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-β1).     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Expansion of human mesenchymal stem cells on microcarriers

The effects on human mesenchymal stem cell growth of choosing either of two spinner flask impeller geometries, two microcarrier concentrations and two cell concentrations (seeding densities) were investigated. Cytodex 3 microcarriers were not damaged when held at the minimum speed, N_JS, for their suspension, using either impeller, nor was there any observable damage to the cells. The maximum cell density was achieved after 8–10 days of culture with up to a 20-fold expansion in terms of cells per microcarrier. An increase in microcarrier concentration or seeding density generally had a deleterious or neutral effect, as previously observed for human fibroblast cultures. The choice of impeller was significant, as was incorporation of a 1 day delay before agitation to allow initial attachment of cells. The best conditions for cell expansion on the microcarriers in the flasks were 3,000 microcarriers ml⁻¹ (ca. 1 g dry weight l⁻¹), a seeding density of 5 cells per microcarrier with a 1 day delay before agitation began at N_JS (30 rpm), using a horizontally suspended flea impeller with an added vertical paddle. These findings were interpreted using Kolmogorov’s theory of isotropic turbulence.     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

CD44 in rheumatoid arthritis

CD44 is a multistructural cell-surface glycoprotein that can theoretically generate close to 800 isoforms by differential alternative splicing. At present, several dozen isoforms are known. The polymorphic nature of CD44 might explain its multifunctionality and its ability to interact with many cell-surface and extracellular ligands, the principal one being hyaluronic acid (HA). Of the many CD44 functions, our review focuses on its involvement in cell–cell and cell–matrix interactions, as well as on its implication in the support of cell migration and the presentation of growth factors to their cognate receptors. Cells involved in pathological activities such as cancer cells and destructive inflammatory cells, and also normal cells engaged in physiological functions, use cell-surface CD44 for their localization and expansion at extravascular sites. This article reviews the evidence that the joint synovium of patients with rheumatoid arthritis (RA) contains considerable amounts of various CD44 isoforms as well as the HA ligand. The review also shows that anti-CD44 monoclonal antibody (mAb) directed against constant epitopes, shared by all CD44 isoforms, can markedly reduce the inflammatory activity of arthritis induced by collagen or proteoglycans in mice. Anti-CD44 mAb also interferes with the migration of RA synovial-like fibroblasts in vitro and is able to disturb the destructive interaction between RA synovial-like fibroblasts and the cartilaginous matrix. However, the transition from the experimental model to the patient's bedside is dependent on the ability to target the CD44 of cells engaged in RA pathology, while skipping the CD44 of normal cells.     

Kinetic analysis of the effect of cell density on hybridoma cell growth in batch culture

The effect of cell density on cell growth was investigated in a suspension batch culture of hybridoma cells. The specific growth rate was found to increase with increasing initial cell density and then to decrease with further increases in initial cell density. In order to quantitatively describe the dependence of specific growth rate on cell density, a kinetic model is proposed, which satisfactorily represents the experimental data.