SOFT‐MI: A novel microfabrication technique integrating soft‐lithography and molecular imprinting for tissue engineering applications

An innovative approach has been employed for the realization of bioactive scaffolds able to mimic the in vivo cellular microenvironment for tissue engineering applications. This method is based on the combination of molecular imprinting and soft‐lithography technology to enhance cellular adhesion and to guide cell growth and proliferation due to presence of highly specific recognition sites of selected biomolecules on a well‐defined polymeric microstructure. In this article polymethylmethacrylate (PMMA) scaffolds have been realized by using poly(dimethylsiloxane) (PDMS) microstructured molds imprinted with FITC‐albumin and TRITC‐lectin. In addition gelatin, an adhesion protein, was employed for the molecular imprinting of polymeric scaffolds for cellular tests. The most innovative aspect of this research was the molecular imprinting of whole cells for the development of substrates able to enhance the cell adhesion processes. Biotechnol. Bioeng. 2010;106: 804–817. © 2010 Wiley Periodicals, Inc.     

Development of motorized plasma lithography for cell patterning

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.     

Induction of cell fusion by a factor released by the cellular slime mold Polysphondylium pallidum

Heterokaryons and hybrid cells, which are extremely useful for research in cell biology, can be produced artificially by treating cells with either polyethylene glycol or certain inactivated viruses that alter the plasma membrane. We report here a novel cell-fusion inducing factor secreted by CK-8 strain cells of cellular slime mold Polysphondylium pallidum. Treatment of other strains or other species of cellular slime molds, such as NC-4 of Dictyostelium discoideum with the diluted fraction, containing molecules larger than 50 kDa, of the conditioned medium of CK-8 cell culture induces cell fusion at high frequency and produces multinucleated large cells. This cell fusion is inducible between cells of either a single strain or of two different strains of cellular slime molds.Abbreviations BSS Bonner's salt solution - CM conditioned medium - EDTA ethylenediaminetetraacetic acid - F2 fraction containing cell-fusion induction factor - Mr molecular mass     

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.     

Do β-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation

Abstract The extent of short-term adhesion of various suspension-cultured plant cell species to polymer substrates exhibiting a wide range of surface tensions was examined. Adhesion of cells with a relatively low surface tension, suspended in distilled water, to the polymers fluorinated ethylenepropylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), and sulphonated polystyrene (SPS) increased with decreasing substrate surface tension following the sequence SPS < PET < PS < FEP. These results are in agreement with the predictions of a thermodynamic model of particle adhesion which considers the role of the substrate, suspending-liquid, and cellular surface tensions. In contrast, little adhesion of relatively high surface tension cells to any of the polymer substrates was observed. Electrostatic repulsive forces between these cells and the polymer surface prevent adhesion because the magnitude of the attractive van der Waals force is small. A correlation was observed between the general adhesiveness of the various cultured plant cell species, especially to the low surface tension substrates, and the cellular surface tension determined by measuring the water contact angle on smooth layers of the cells. The cellular surface tensions ranged from approximately 42 mJ/m² for Digitalis purpurea cells to approximately 70mJ/m² for Papaver somniferum cells. Adhesion of cells to the polymer substrates increased with decreasing cellular surface tension under otherwise identical conditions. These results are also consistent with thermodynamic model predictions.     

Prowling wolves in sheep's clothing: the search for tumor stem cells

The importance of a subset of cells which have 'stem like' characteristics and are capable of tumor initiation has been reported for a range of tumors. Isolation of these tumor-initiating cells (TICs) has largely been based on differential cell surface protein expression. However, there is still much debate on the functional significance of these markers in initiating tumors, as many properties of tumor initiation are modified by cell-cell interactions. In particular, the relationship between TICs and their microenvironment is poorly understood but has therapeutic implications, as the microenvironment can maintain tumor cells in a prolonged period of quiescence. However, a major limitation in advancing our understanding of the crosstalk between TICs and their microenvironment is the lack of sensitive techniques which allow the in vivo tracking and monitoring of TICs. Application of new in vivo cellular and molecular imaging technologies holds much promise in uncovering the mysteries of TIC behavior at the three-dimensional level. This review will describe recent advances in our understanding of the TIC concept and how the application of in vivo imaging techniques can advance our understanding of the biological fate of TICs. A supplementary resource guide describing TICs from different malignancies is also presented.     

The principles of cellular geometry scaling

Cellular dimensions profoundly influence cellular physiology. For unicellular organisms, this has direct bearing on their ecology and evolution. The morphology of a cell is governed by scaling rules. As it grows, the ratio of its surface area to volume is expected to decrease. Similarly, if environmental conditions force proliferating cells to settle on different size optima, cells of the same type may exhibit size-dependent variation in cellular processes. In fungi, algae and plants where cells are surrounded by a rigid wall, division at smaller size often produces immediate changes in geometry, decreasing cell fitness. Here, we discuss how cells interpret their size, buffer against changes in shape and, if necessary, scale their polarity to maintain optimal shape at different cell volumes.     

Pay attention to membrane tension: Mechanobiology of the cell surface

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Micropatterning and Alignment of Skeletal Muscle Myoblasts Using Microflowed Plasma Process

ObjectivesThe primary objective of the study was to optimize micropatterning environments using the microchannel flowed plasma process for controlling the orientation and behaviour of skeletal muscle cells. We have studied the cellular patterning and alignment of skeletal myoblast cells on the various micropattern widths developed on glass substrates.Materials and MethodsIn this method, we have utilized the microchannel flowed plasma process to create micropatterned self-assembled monolayers of octadecyltrichlorosilane and 3-aminopropyltrichlorosilane for creating cell adhesive widths of 20, 200 and 1000 microns on the glass substrates. The micropatterned substrates were characterized by using fluorescein 5(6)-isothiocyanate. Thereafter, the substrates were used to culture and pattern C2C12 and primary rat skeletal muscle cells. Further, we have studied the spatiotemporal variation in the orientation of the cells by using bright field and fluorescence microscopy. The microscopic images were analysed by using orientation order parameter and orientation distribution analysis.ResultsFITC based characterization of micropatterns reveals that the adopted process for micropatterning can effectively create cell adhesive widths with dimensions comparable to the diameter of myofiber. Microscopic observations and the orientation order parameter analysis reveal the precise alignment and specific orientation of myoblasts along the designated cell adhesive widths that closely mimics the physiological scenario. Both the cells showed immediate alignment within smaller cell adhesive widths of 20 and 200 μm. Actin cytoskeletal staining and its orientation distribution analysis of micropattrned C2C12 cells emphasises the influence of micropatterned environment on cytoskeletal actin orientation.ConclusionThis study corroborates the alignment of the myoblasts using surface cues facilitated by changing surface chemistry of the glass substrates. The study promotes the application of a simple micropatterning technique as a useful tool to regulate the orientation and behaviour of skeletal muscle cells. Also, the study emphasizes the role of spatial topography created by surface modification and its effect on cell adhesion and communication of alignment information across the micropatterns. The microchannel flowed plasma process could be applied to selectively pattern different adherent cell types, which could prove to be a useful platform for the exploration of various cellular processes.     

Effects of the substratum on the migration of primordial germ cells

It is now clear from work on defined cell types on artificial substrates that various chemical and physical inhomogeneities in the substrates can guide cell locomotion. It is also becoming clear that less well defined inhomogeneities in living cell substrates can guide the normal locomotion of embryonic migratory cells in vivo. The primordial germ cells (p.g.cs) of early anuran amphibian embryos are proving a useful model for the study of cell migration. When isolated from the embryo and cultured on living cellular substrate, p.g.cs become oriented by the shapes of the underlying cells or by their stress fibre cytoskeleton, or both. A combination of scanning and transmission electron microscopy in vivo shows a clearly aligned cellular substrate for p.g.c. migration along part of their route. Furthermore, we find that the glycoprotein fibronectin is involved in p.g.c. adhesion, which suggests a link between orientation of the substrate cells and p.g.c. guidance.     

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.     

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.     

Friction-controlled traction force in cell adhesion

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.     

Micro 3D cell culture systems for cellular behavior studies: Culture matrices,devices, substrates,and in‐situ sensing methods

Microfabricated systems equipped with 3D cell culture devices and in‐situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio‐functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near‐cell surface detection of excreted cellular compounds, SERS‐based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development.     

Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.     

Photodynamic inactivation of Candida albicans using bridged polysilsesquioxane films doped with porphyrin

Novel photoactive bridged polysilsesquioxane films were prepared by doped with a porphyrin derivative. The films were formed by acid-catalyzed polycondensation reaction of a precursor of a bridged silsesquioxane, based on the reaction product of (glycidoxypropyl)trimethoxysilane with n-dodecylamine in the presence of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin, followed by solvent evaporation. This procedure allowed obtaining flexible thin films. Absorption and fluorescence spectroscopic analysis showed the characteristic bands of the porphyrin in the visible region indicating that the photosensitizer is mainly embedded as monomer in the films. Photodynamic properties of the polymeric films were studied in solution containing photooxidizable substrates. Singlet molecular oxygen, O(2)((1)Δ(g)), production was observed by the reaction with 9,10-dimethylanthracene and 9,10-anthracenediyl-bis(methylene)dimalonic acid in different media. Also, these films photosensitized the decomposition of l-tryptophan. In vitro investigations showed that these films produce photodynamic inactivation of Candida albicans cells in aqueous suspensions and on their surfaces. These films exhibit a photosensitizing activity causing a ～2.5 log (99.7%) decrease of cellular survival after 60 min of irradiation with visible light. Also, the photocytotoxicity of the surfaces was tested under condition of microbial growth. Yeast cells exposed to the film and illuminated showed growth delay compared with controls. Studies of photodynamic action mechanism showed that the photoinactivation increased in D(2)O, while cells were protected in the presence of azide ion. In contrast, the addition of mannitol produced a negligible effect on the cellular phototoxicity. These results provide evidence that O(2)((1)Δ(g)) produced by the polymeric film doped with porphyrin can successfully inactivate C. albicans in cell suspensions and deposited on the film surface.     

BioMEMS and cellular biology: perspectives and applications

The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.