The study of cellular “microexudates” by ellipsometry and their relationship to the cell coat

The use of ellipsometry, an optical technique for the detection of very thin films, to measure the thickness of the ‘microexudate’ deposited by mammalian cells on glass and other solid surfaces is described. Significant differences were found in the thickness and the rate of formation of microexudates in different cell types. Cultivation of cells at low temperatures and in medium supplemented with actinomycin D and cycloheximide inhibited the production of cellular microexudates, indicating that these materials are actively synthesized by the cell and do not result merely from passive leakage of macromolecular material as suggested previously. Evidence is reviewed to show that cellular microexudates have a number of properties in common with the cell coat material.     

Electrospun triple-layered PLLA/gelatin. PRGF/PLLA scaffold induces fibroblast migration

The function of fibroblast cells in wounded areas results in reconstruction of the extra cellular matrix and consequently resolution of granulation tissue. It is suggested that the use of platelet-rich plasma can accelerate the healing process in nonhealing or slow-healing wounds. In this study, a simple and novel method has been used to fabricate an electrospun three-layered scaffold containing plasma rich in growth factor with the aim of increasing the proliferation and migration of fibroblast cells in vitro. First, plasma rich in growth factor was derived from platelet rich plasma, and then a three-layered scaffold was fabricated using PLLA nanofibers as the outer layers and plasma rich in growth factor-containing gelatin fibers as the internal layer. The growth morphology of cells seeded on this scaffold was compared to those seeded on one layered PLLA scaffold. The study of the cell growth rate on different substrates and the migration of cells in response to the drug release of multilayered scaffold was investigated by the cell quantification assay and a modified under agarose assay. Scanning electron microscopy and fluorescence images showed that cells seeded on multilayered scaffold were completely oriented 72 hours after seeding compared to those seeded on PLLA scaffold. The cell quantification assay also indicated significant increase in proliferation rate of cells seeded on three-layered scaffold compared to those seeded on PLLA scaffold and finally, monitoring cell migration proved that cells migrate significantly toward the three-layered scaffold up to 48 to 72 hours and afterwards start to show a diminished migration rate toward this scaffold.     

All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes.

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.     

Clonal interactions in fibroblast proliferation: recognition of self vs. non-self

The proliferation and aging of fibroblast populations has been postulated to include a process of clonal selection. Using carbocyanine dyes to label clonal fibroblast populations, we were able to follow their growth in mixed cultures. Individual fibroblast clones seeded as the minority population (20%) with either another clone or the parent line (differentially labeled) always demonstrated increase relative growth so that, by the end of 4 weeks, approximately equal numbers of both populations were present. Labeled cells of the same clone mixed as the minority population with differentially labeled cells of the same clone maintained their minority status. The results indicate that clonal populations of fibroblasts are able to recognize "self" as different from "non-self" and that this recognition leads to alterations in cellular proliferation.     

Photobiological and thermal effects of photoactivating UVA light doses on cell cultures.

While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (315-400 nm) photoactivating light has been well characterized in short-term cell studies and is now being employed in higher doses to control longer-duration phenomena (e.g. gene expression). Annexin V-Cy5/propidium iodide apoptosis flow cytometry assays were used to determine responses of HeLa cells to doses of UVA light up to 23.85 J cm(-2). Cells seeded at low densities had higher percentages of apoptosis and necrosis and were also more susceptible to UVA damage than cells seeded at higher densities. The dose to induce apoptosis and death in 50% of the cells (dose(1/2)) was determined for two different commercially available UVA light sources: 7.6 J cm(-2) for the GreenSpot photocuring system and 2.52 J cm(-2) for the BlakRay lamp. All BlakRay doses tested had significant cellular responses, whereas no significant cellular responses were found for doses below 1.6 J cm(-2) from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling cell cultures during photoexposure has on minimizing cell damage. Cooling during the BlakRay photoexposure significantly reduced the percentage of necrotic cells, but there was no significant difference for cooling during photoactivation with the GreenSpot. Differences in cell responses to similar UVA doses of different intensities suggest that photoduration should be considered along with total dose and thermal conditions in photoactivation studies.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

Contribution of the extracellular matrix to growth properties of cells from a preneoplastic outgrowth: possible role of hyaluronic acid

Hyaluronic acid (HA) accumulates around actively growing normal and tumorigenic mammary epithelial cells and has been implicated as a modulator of cell proliferation. We have tested the role of exogenous HA presented in several different forms in in vitro growth regulation of a cell line (CL-S1) derived from preneoplastic mouse mammary tissue. This cell line grows slowly and synthesizes very little HA. We first assessed growth of CL-S1 cells seeded onto actual matrix generated by CL-S1 cells themselves (which has a low HA content) or by a related tumorigenic cell line, +SA, that generates an HA-rich matrix. Growth on both these HA-containing substrata was significantly enhanced above control values on plastic. Growth on the +SA biomatrix was over 5 times greater than on tissue culture plastic and significantly greater than that seen with all other treatments. Differences in growth responses of CL-S1 cells seeded atop CL-S1- and +SA-derived matrices could be attributable to differences in matrix HA content. As a more direct test of this possibility, growth responses of CL-S1 cells to HA covalently bonded to tissue culture dishes and to HA dissolved in culture media were tested. Growth on the prepared HA substrata was consistently twice that on plastic. In soluble form, HA at a concentration of 100 micrograms HA/ml culture medium, stimulated CL-S1 growth 196 and 125% of control in monolayer cultures, respectively, seeded at low (approximately equal to 10(2) viable cells/cm2) and high (approximately equal to 10(4) viable cells/cm2) densities on plastic. Higher HA concentrations inhibited growth at low seeding densities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of surface topography on the human epithelial cell response to micropatterned substrates with convex and concave architectures

Background

Understanding the fundamental mechanisms underlying the cellular response to topographical surface features will extend our knowledge regarding the regulation of cell functions. Analyzing the cellular response to different topographical features, over multiple temporal and spatial scales, is central to understanding and guiding several biological functions. We used micropatterned substrates with convex and concave architectures to evaluate the behaviors of human epithelial cells on these substrates.

Results

Pillar and pit substrates caused heterogeneous spatial growth and distribution, with differences in cell density, over 48 h. Regional densities and distribution were significantly increased at pillar sidewalls, and at pit sidewalls and bottoms compared with those on flat unpatterned areas. Time-lapse observations revealed that different mechanisms of cell migration were dependent upon pillar and pit features. Cells on pillar substrate migrated towards the sidewall, whereas cells on pit substrate tended to move towards the sidewalls and bottom. Cytoskeletal staining of F-actin and vinculin showed that this migration can be attributed to difference in spatial reorganization of actin cytoskeleton, and the formation of focal adhesions at various points on the at the convex and concave corners of pillar and pit substrates. Cells cultured on the pillar substrate had stress fibers with extended filopodia and immature focal contacts at the sidewalls and convex corners, similar to those on the flat unpatterned substrate. Cells at the sidewalls and concave corners of pit substrate had more contractile stress fibers and stable focal contacts compared with cells on the pillar substrate. We also found that the substrate structures affect cell-cell contact formation via E-cadherin, and that this was associated with reorganization of the actin cytoskeleton at the sidewall, and at the convex and concave corners of the substrate.

Conclusion

Migration is an important factor affecting spatial growth and distribution. Heterogeneity at various locations was caused by different migratory behaviors at the convex and concave corners of pillar and pit substrates. We propose that this investigation is a valuable method for understanding cell phenotypes and the heterogeneity during spatial growth and distribution of epithelial cells during culture.

     

The nature of substrate-attached materials in human fibroblast cultures: localization of cell and fetal calf serum components.

Human fibroblasts have been used as an in vitro model to examine the morphology and origin of substrate-attached materials. In cultures of subconfluent cells, no ‘tracks’ or ‘pools’ of material could be detected on substrata by anodic oxide interferometry or electron microscopy. However, a continuous layer of densely staining material was present on Falcon plastic tissue culture dishes never exposed to cells or culture medium. Exposure of substrata to culture medium caused the adsorption of fetal calf serum (FCS) components onto the substratum within a few minutes. Although antigenic FCS components remained on the substrata for several days, they were seldom adsorbed to the cells. The hypothesis was formulated that adhesion was mediated by FCS components on the substrata, but not by cellular materials deposited extracellularly. Support for this hypothesis was obtained by studying serum-dependent differences in cell adhesion. Fibroblasts subcultured in the presence of FCS components were usually separated from the substratum by a distance of at least 30 Å. In the absence of FCS components, the cells were more closely adherent, in the range at which the near van der Walls forces were effective. Fibroblasts subcultured in the absence of serum components could be removed readily from the substratum, leaving lsfootprints’ of cell surface material behind. Although this material has been prepared similarly to ‘microexudates’ from other types of cultured cells, its relationship to those microexudates has not been determined.     

Tumor cell haptotaxis on covalently immobilized linear and exponential gradients of a cell adhesion peptide

The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.     

Differential cell behaviour on polylysine and gelatin in primary culture of mouse mammary adenocarcinoma.

Cells dissociated from spontaneous and transplanted tumours of C3HJax mammary gland have been cultured on polylysine and gelatin substrates. The isolated cells proliferated to form monolayers with high degree of organoid structure as indicated by formation of alveolar cavities. Differences were observed in the cell attachment, growth pattern, number and size of alveolar cavities, cells which lined the cavity and cell morphology on polylysine and gelatin substrates as compared to conventional cell culture plastic surface. On polylysine more than 90% cells attached rapidly, within 15-45 min after plating, with or without serum and formed confluent monolayers marked by presence of large and small alveolar cavities. Multiple interacting cell types took part in organization of the cavity. Cells lining the cavity constantly proliferated and rearranged to expand it. On gelatin, 60-70% cells attached over a period of 6-24 hr in presence of serum and formed confluent monolayers dominated by small alveolar cavities. Cells forming the cavities were epithelial in nature and cavities once formed did not increase in size. Upon subculture, the cell morphology on these substrates was strikingly different. On polylysine, the predominant cell type had numerous irregular microvilli whereas on gelatin, cells had smoother boundaries with a few stunted cytoplasmic extensions. The cell attachment on conventional surface was low, 40-50%. When seeded at high cell density, formation of alveolar cavities was suppressed and at low cell density, cultures were marked by contact inhibition of cells and failure to attain confluence. These results suggest differential behaviour and interaction of mammary tumour epithelium with the substrates used.     

Proliferation of human carcinoma cells in calcium-deficient culture medium may depend on autocrine growth factor(s)

The ability to proliferate in media with low calcium concentrations (less than 0.1 mM) at clonal seeding densities is a characteristic of malignant cells. Cells of the carcinoma line C-4I are an exception, and are unable to proliferate in medium with 0.02 mM calcium (LCM) when seeded at low densities. Conditioned medium, derived from a subline of C-4I cells that were adapted to grow in LCM, contained an acid-stable factor of greater than 8 kilodaltons molecular weight that mediated proliferation and colony formation of unadapted C-4I cells in LCM in a concentration-dependent manner. The effect was not related to enhanced cell attachment or spreading, and was maximal when the unadapted C-4I cells were seeded in aggregates of 2-20 cells rather than singly. Thus, calcium independence, a component of the neoplastic phenotype, may be mediated by autocrine tumor-cell-derived factor(s) and may involve long- and short-range cell interactions.     

Extracellular matrix density regulates the formation of tumour spheroids through cell migration

In this work, we show how the mechanical properties of the cellular microenvironment modulate the growth of tumour spheroids. Based on the composition of the extracellular matrix, its stiffness and architecture can significantly vary, subsequently influencing cell movement and tumour growth. However, it is still unclear exactly how both of these processes are regulated by the matrix composition. Here, we present a centre-based computational model that describes how collagen density, which modulates the steric hindrance properties of the matrix, governs individual cell migration and, consequently, leads to the formation of multicellular clusters of varying size. The model was calibrated using previously published experimental data, replicating a set of experiments in which cells were seeded in collagen matrices of different collagen densities, hence producing distinct mechanical properties. At an initial stage, we tracked individual cell trajectories and speeds. Subsequently, the formation of multicellular clusters was also analysed by quantifying their size. Overall, the results showed that our model could accurately replicate what was previously seen experimentally. Specifically, we showed that cells seeded in matrices with low collagen density tended to migrate more. Accordingly, cells strayed away from their original cluster and thus promoted the formation of small structures. In contrast, we also showed that high collagen densities hindered cell migration and produced multicellular clusters with increased volume. In conclusion, this model not only establishes a relation between matrix density and individual cell migration but also showcases how migration, or its inhibition, modulates tumour growth.     

Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.     

Fibrin gel enhanced trilayer structure in cell-cultured constructs

Efficient cell seeding and subsequent support from a substrate ensure optimal cell growth and neotissue development during tissue engineering, including heart valve tissue engineering. Fibrin gel as a cell carrier may provide high cell seeding efficiency and adhesion property, improved cellular interaction, and structural support to enhance cellular growth in trilayer polycaprolactone (PCL) substrates that mimic the structure of native heart valve leaflets. This cell carrier gel coupled with a trilayer PCL substrate may enable the production of native-like cell-cultured leaflet constructs suitable for heart valve tissue engineering. In this study, we seeded valvular interstitial cells onto trilayer PCL substrates with fibrin gel as a cell carrier and cultured them for 1 month in vitro to determine if this gel can improve cell proliferation and production of extracellular matrix within the trilayer cell-cultured constructs. We observed that the fibrin gel enhanced cellular proliferation, their vimentin expression, and collagen and glycosaminoglycan production, leading to improved structure and mechanical properties of the developing PCL cell-cultured constructs. Fibrin gel as a cell carrier significantly improved the orientations of the cells and their produced tissue materials within trilayer PCL substrates that mimic the structure of native heart valve leaflets and, thus, may be highly beneficial for developing functional tissue-engineered leaflet constructs.     

The influence of entropic crowding in cell monolayers

Cell-cell interaction dictates cell morphology and organization, which play a crucial role in the micro-architecture of tissues that guides their biological and mechanical functioning. Here, we investigate the effect of cell density on the responses of cells seeded on flat substrates using a novel statistical thermodynamics framework. The framework recognizes the existence of nonthermal fluctuations in cellular response and thereby naturally captures entropic interactions between cells in monolayers. In line with observations, the model predicts that cell area and elongation decrease with increasing cell seeding density—both are a direct outcome of the fluctuating nature of the cellular response that gives rise to enhanced cell-cell interactions with increasing cell crowding. The modeling framework also predicts the increase in cell alignment with increasing cell density: this cellular ordering is also due to enhanced entropic interactions and is akin to nematic ordering in liquid crystals. Our simulations provide physical insights that suggest that entropic cell-cell interactions play a crucial role in governing the responses of cell monolayers.     

Enhancement of the growth of human osteosarcoma cells by human interferon-gamma.

It is well known that interferons inhibit cell growth. However, we found that human interferon-gamma (HuIFN-gamma) enhanced the growth of human osteosarcoma cells, HOS-Y1 cells, in a dose-dependent manner. This enhancing effect was found only under the following conditions: when the cells were precultured for 2 or 3 days and then treated with HuIFN-gamma for 2, 3, or 4 days, and when the cells were seeded at a density of 1,000 or 2,000 cells/well. The degree of enhancement of cell growth was maximum when the cells were precultured at a density of 1,000 cells/well for 3 days and then treated with HuIFN-gamma for 2 days. The enhancing effect of HuIFN-gamma disappeared in the presence of anti-HuIFN-gamma antibody. In addition, it was found that the conditioned medium from HOS-Y1 cells enhanced the growth of HOS-Y1 cells, and that the conditioned medium from HOS-Y1 cells cultured with HuIFN-gamma enhanced the cell growth more than that from cells cultured without HuIFN-gamma. Epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1(TGF-beta 1) did not enhance the growth of HOS-Y1 cells. These results suggest that HuIFN-gamma enhanced the cell growth by augmenting the production of unknown growth factor(s) in HOS-Y1 cells via an autocrine mechanism.     

Selective growth of malignant cells by in vitro incubation on Teflon

The selective growth of malignant cells on non-adhesive Teflon substrates is reported. Neoplastically transformed cell lines of human and murine origin proliferated on Teflon whereas similarly seeded normal non-tumorigenic cells were unable to undergo cell division and subsequent growth. This substrate should be especially useful in establishing primary cultures from human tumor material.