Piezoelectric ink jet processing of materials for medical and biological applications

Many advanced medical and biological devices require microscale patterning of cells, proteins, and other biological materials. This article describes the use of piezoelectric ink jet processing in the fabrication of biosensors, cell-based assays, and other microscale medical devices. A microelectromechanical system-based piezoelectric transducer was used to develop uniform fluid flow through nozzles and to prepare well-defined microscale patterns of proteins, monofunctional acrylate ester, sinapinic acid, deoxyribonucleic acid (DNA), and DNA scaffolds on relevant substrates. Our results demonstrate that piezoelectric ink jet deposition is a powerful non-contact, non-destructive additive process for developing biosensors, cell culture systems, and other devices for medical and biological applications.     

Versatile and inexpensive Hall-Effect force sensor for mechanical characterization of soft biological materials

Mismatch of hierarchical structure and mechanical properties between tissue-engineered implants and native tissue may result in signal cues that negatively impact repair and remodeling. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve necessary macroscale properties in the final implant. However, characterizing microscale mechanical properties is challenging, and current methods do not provide the versatility and sensitivity required to measure these fragile, soft biological materials. Here, we developed a novel, highly sensitive Hall-Effect based force sensor that is capable of measuring mechanical properties of biological materials over wide force ranges (μN to N), allowing its use at all steps in layer-by-layer fabrication of engineered tissues. The force sensor design can be easily customized to measure specific force ranges, while remaining easy to fabricate using inexpensive, commercial materials. Although we used the force sensor to characterize mechanics of single-layer cell sheets and silk fibers, the design can be easily adapted for different applications spanning larger force ranges (>N). This platform is thus a novel, versatile, and practical tool for mechanically characterizing biological and biomimetic materials.     

Transfer of macroscale tissue strain to microscale cell regions in the deformed meniscus

Cells within fibrocartilaginous tissues, including chondrocytes and fibroblasts of the meniscus, ligament, and tendon, regulate cell biosynthesis in response to local mechanical stimuli. The processes by which an applied mechanical load is transferred through the extracellular matrix to the environment of a cell are not fully understood. To better understand the role of mechanics in controlling cell phenotype and biosynthetic activity, this study was conducted to measure strain at different length scales in tissue of the fibrocartilaginous meniscus of the knee joint, and to define a quantitative parameter that describes the strain transferred from the far-field tissue to a microenvironment surrounding a cell. Experiments were performed to apply a controlled uniaxial tensile deformation to explants of porcine meniscus containing live cells. Using texture correlation analyses of confocal microscopy images, two-dimensional Lagrangian and principal strains were measured at length scales representative of the tissue (macroscale) and microenvironment in the region of a cell (microscale) to yield a strain transfer ratio as a measure of median microscale to macroscale strain. The data demonstrate that principal strains at the microscale are coupled to and amplified from macroscale principal strains for a majority of cell microenvironments located across diverse microstructural regions, with average strain transfer ratios of 1.6 and 2.9 for the maximum and minimum principal strains, respectively. Lagrangian strain components calculated along the experimental axes of applied deformations exhibited considerable spatial heterogeneity and intersample variability, and suggest the existence of both strain amplification and attenuation. This feature is consistent with an in-plane rotation of the principal strain axes relative to the experimental axes at the microscale that may result from fiber sliding, fiber twisting, and fiber-matrix interactions that are believed to be important for regulating deformation in other fibrocartilaginous tissues. The findings for consistent amplification of macroscale to microscale principal strains suggest a coordinated pattern of strain transfer from applied deformation to the microscale environment of a cell that is largely independent of these microstructural features in the fibrocartilaginous meniscus.     

Micro- and nanotechnology in cell separation

This review describes recent work in cell separation using micro- and nanoscale technologies. These devices offer several advantages over conventional, macroscale separation systems in terms of sample volumes, low cost, portability, and potential for integration with other analytical techniques. More importantly, and in the context of modern medicine, these technologies provide tools for point-of-care diagnostics, drug discovery, and chemical or biological agent detection. This review describes work in five broad categories of cell separation based on (1) size, (2) magnetic attraction, (3) fluorescence, (4) adhesion to surfaces, and (5) new emerging technologies. The examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field.     

Tools for studying and modulating (cardiac muscle) cell mechanics and mechanosensing across the scales

Cardiomyocytes generate force for the contraction of the heart to pump blood into the lungs and body. At the same time, they are exquisitely tuned to the mechanical environment and react to e.g. changes in cell and extracellular matrix stiffness or altered stretching due to reduced ejection fraction in heart disease, by adapting their cytoskeleton, force generation and cell mechanics. Both mechanical sensing and cell mechanical adaptations are multiscale processes. Receptor interactions with the extracellular matrix at the nanoscale will lead to clustering of receptors and modification of the cytoskeleton. This in turn alters mechanosensing, force generation, cell and nuclear stiffness and viscoelasticity at the microscale. Further, this affects cell shape, orientation, maturation and tissue integration at the microscale to macroscale. A variety of tools have been developed and adapted to measure cardiomyocyte receptor-ligand interactions and forces or mechanics at the different ranges, resulting in a wealth of new information about cardiomyocyte mechanobiology. Here, we take stock at the different tools for exploring cardiomyocyte mechanosensing and cell mechanics at the different scales from the nanoscale to microscale and macroscale.     

Explicit Separation of Growth and Motility in a New Tumor Cord Model

We investigate a new model of tumor growth in which cell motility is considered an explicitly separate process from growth. Bulk tumor expansion is modeled by individual cell motility in a density-dependent diffusion process. This model is implemented in the context of an in vivo system, the tumor cord. We investigate numerically microscale density distributions of different cell classes and macroscale whole tumor growth rates as functions of the strength of transitions between classes. Our results indicate that the total tumor growth follows a classical von Bertalanffy growth profile, as many in vivo tumors are observed to do. This provides a quick validation for the model hypotheses. The microscale and macroscale properties are both sensitive to fluctuations in the transition parameters, and grossly adopt one of two phenotypic profiles based on their parameter regime. We analyze these profiles and use the observations to classify parameter regimes by their phenotypes. This classification yields a novel hypothesis for the early evolutionary selection of the metastatic phenotype by selecting against less motile cells which grow to higher densities and may therefore induce local collapse of the vascular network.     

Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Molecularly imprinted polymers in pseudoimmunoassay

Immunoassays are a class of analytical techniques based on the selective affinity of a biological antibody for its antigen. Competitive binding assays, of which the radioimmunoassay (RIA) was the first example, are based on the competition between analyte and a labelled probe for a limited number of binding sites. Molecularly imprinted polymers (MIPs) have been shown to be suitable replacements for biological antibodies in such techniques. Molecularly imprinted sorbent assays (MIAs) similar to RIA have been developed for a range of analytes of clinical and environmental interest. Limits of detection and selectivities of such assays are often similar to those using biological antibodies. Some assays have been used for measurements directly in biological fluids. The field is reviewed and it is shown that some perceived disadvantages of MIPs do not hinder their application in competitive binding assays: many MIAs have been demonstrated in aqueous solvents, and it has been shown that the quantity of template required to prepare imprinted polymers can be drastically reduced, and that binding site heterogeneity is not a problem as long as the sites which bind the probe most strongly are selective. Finally, recent developments including assays in microtitre plates, the use of enzyme-labelled probes, flow-injection assays and a scintillation proximity MIA are discussed.     

Self-adhesive microculture system for extended live cell imaging

Abstract

Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.     

Stress and strain localization in stretched collagenous tissues via a multiscale modelling approach

Mechanobiology of cells in soft collagenous tissues is highly affected by both tissue response at the macroscale and stress/strain localization mechanisms due to features at lower scales. In this paper, the macroscale mechanical behaviour of soft collagenous tissues is modelled by a three-level multiscale approach, based on a multi-step homogenisation technique from nanoscale up to the macroscale. Nanoscale effects, related to both intermolecular cross-links and collagen mechanics, are accounted for, together with geometric nonlinearities at the microscale. Moreover, an effective submodelling procedure is conceived in order to evaluate the local stress and strain fields at the microscale, which is around and within cells. Numerical results, obtained by using an incremental finite element formulation and addressing stretched tendinous tissues, prove consistency and accuracy of the model at both macroscale and microscale, confirming also the effectiveness of the multiscale modelling concept for successfully analysing physiopathological processes in biological tissues.     

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Status and developments of animal cell technology using suspension culture techniques

In this review the state of the art in animal cell technology, using suspension culture techniques is updated as far as the end of 1980. We have tried to discuss, on a broad basis, the current status and potential developments of both, the purely biological and the biochemical engineering aspects which may be important to improve the performance and design of animal cell technologies. The process economics could be considerably improved by the use of transformed animal cell substrates, the use of cheaper cultivation media and by methodological and engineering means. Most of these aspects are in the state of realization. Nevertheless, for a great variety of biological active substances which do not require so – or posttranslational processing, recombinant DNA-techniques (e.g. genetic engineering) are a promising alternative for the production of animal cell derived substances.     

Macroscale versus microscale methods for physiological analysis of biofilms formed in 96-well microtiter plates

Microtiter plates with 96 wells have become one of the preferred platforms for biofilm studies mainly because they enable high-throughput assays. In this work, macroscale and microscale methods were used to study the impact of hydrodynamic conditions on the physiology and location of Escherichia coli JM109(DE3) biofilms formed in microtiter plates. Biofilms were formed in shaking and static conditions, and two macroscale parameters were assayed: the total amount of biofilm was measured by the crystal violet assay and the metabolic activity was determined by the resazurin assay. From the macroscale point of view, there were no statistically significant differences between the biofilms formed in static and shaking conditions. However, at a microscale level, the differences between both conditions were revealed using scanning electron microscopy (SEM). It was observed that biofilm morphology and spatial distribution along the wall were different in these conditions. Simulation of the hydrodynamic conditions inside the wells at a microscale was performed by computational fluid dynamics (CFD). These simulations showed that the shear strain rate was unevenly distributed on the walls during shaking conditions and that regions of higher shear strain rate were obtained closer to the air/liquid interface. Additionally, it was shown that wall regions subjected to higher shear strain rates were associated with the formation of biofilms containing cells of smaller size. Conversely, regions with lower shear strain rate were prone to have a more uniform spatial distribution of adhered cells of larger size. The results presented on this work highlight the wealth of information that may be gathered by complementing macroscale approaches with a microscale analysis of the experiments.     

Nuclear equivalence, nuclear transfer, and the cell cycle

The last 20 years have seen the development of techniques for the production of mammals by nuclear transfer. Originally limited to the swapping of pronuclei and the use of early cleavage-stage embryos as nuclear donors, nuclear transfer came of age in 1995 with the birth of 2 Welsh Mountain lambs, Megan and Morag, that were produced using cultured differentiated cells as donors of genetic material. In 1996, Dolly was the first animal to be produced using the genetic material from an adult-derived somatic cell. The techniques used in the production of these animals have now been reproduced in both sheep and cattle, and as predicted, successful development has been obtained using donor cells taken directly ex vivo. This article reviews the current status of mammalian nuclear transfer and the biological background to these successes.     

The living cell as an ecosystem: Hierarchical analog and symmetry

Biology has benefited from a number of analogies and metaphors, in its attempt to rationalize the hierarchical structure of life's manifestations. Oft-cited is the comparison of a living organism to a society. Phenomenological similitude between the two extremes of the biological spectrum - the microscale of the cell and the macroscale of socioecosystems - has become strikingly evident, with the recent accumulation of empirical information on the metabolic infrastructure of the living cell. From the universal perspective of 'physiological determinism' reciprocal (or symmetric) relations are apparent at both levels of complexity. Unification of the biological hierarchy may follow a similar epistemological course to that in contemporary physics.     

Coupling microscale vegetation–soil water and macroscale vegetation–precipitation feedbacks in semiarid ecosystems

At macroscale, land–atmosphere exchange of energy and water in semiarid zones such as the Sahel constitutes a strong positive feedback between vegetation density and precipitation. At microscale, however, additional positive feedbacks between hydrology and vegetation such as increase of infiltration due to increase of vegetation, have been reported and have a large impact on vegetation distribution and spatial pattern formation. If both macroscale and microscale positive feedbacks are present in the same region, it is reasonable to assume that these feedback mechanisms are connected. In this study, we develop and analyse a soil‐vegetation‐atmosphere model coupling large‐scale evapotranspiration–precipitation feedback with a model of microscale vegetation–hydrology feedback to study the integration of these nonlinearities at disparate scales. From our results, two important conclusions can be drawn: (1) it is important to account for spatially explicit vegetation dynamics at the microscale in climate models (the strength of the precipitation feedback increased up to 35% by accounting for these microscale dynamics); (2) studies on resilience of ecosystems to climate change should always be cast within a framework of possible large‐scale atmospheric feedback mechanism (substantial changes in vegetation resilience resulted from incorporating macroscale precipitation feedback). Analysis of full‐coupled modelling shows that both type of feedbacks markedly influence each other and that they should both be accounted for in climate change models.     

Separation technologies for stem cell bioprocessing

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell‐derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato‐oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost‐effective. Important examples are the need for high‐resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity‐based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical‐based methods requiring no cell labeling and integrated with microscale technologies. Biotechnol. Bioeng. 2012; 109: 2699–2709. © 2012 Wiley Periodicals, Inc.     

Differences in estimates of cisplatin-induced cell kill in vitro between colorimetric and cell count/colony assays

Summary The aim of this study was to evaluate some bioassays that are different in principle: cell counting, colony forming assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB), crystal violet, and alamarBlue, with respect to their ability to measure cisplatin-induced cell death of in vitro-cultivated squamous cell carcinoma of the head and neck (SCCHN). Cisplatin was applied in concentrations of 1.0, 5.0, 10.0, 50.0, and 100 μM. The cells were incubated for 1 h, and the cell survival was measured 5 d after treatment. We found the colorimetric assays and cell counting to be comparable. The colony forming assay indicated a higher degree of cell kill compared with the other techniques. Measurement of cell survival after treatment with cisplatin can be done by use of any of the above tested assays. However, the majority of SCCHN cell lines available do not form colonies easily, or at all. Therefore, comparing the chemosensitivity between such cell lines is limited to alternative assays. In this respect, any of the tested colorimetric assays can be used. However, they seem to underestimate cell kill. Cell counting is also an alternative. This technique, however, is time consuming and operator dependent, as in the case of manual counting, or relatively expensive when counting is performed electronically, compared with the colorimetric assays.     

Metallic nanoparticle radiosensitisation of ion radiotherapy: A review

The use of gold nanoparticle (GNP) and other metal nanoparticle (MNP) radiosensitisers to enhance radiotherapy offers the potential of improved treatment outcomes. Originally intended for use with X-ray therapy, the possibility of enhanced hadron therapy is desirable due to the superior sparing of healthy tissue in hadron therapy compared to conventional X-ray therapy. While MNPs were not expected to be effective radiosensitisers for hadron therapy due to the limited Z dependence of interactions, recent experimental measurements have contradicted this expectation. Key experimental measurements and Monte Carlo simulations of MNP radiosensitisation for hadron irradiation are reviewed in the current work. Numerous experimental measurements have found a large radiosensitisation effect due to MNPs for proton and carbon ion irradiation. Experiments have also indicated that the radiosensitisation is due in large part to enhanced reactive oxygen species (ROS) production. Simulations have found a large radial dose and ROS enhancement on the nanoscale around a single MNP. However, the short range of the dose enhancement is insufficient for a large macroscale dose enhancement or enhanced biological effect in a cell model considering dose to the nucleus from GNPs in the cytoplasm (a distribution observed in most experiments).