Biochemical Gradients Research Articles

Cells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment

The transition to increasingly sophisticated microfluidic systems has led to the emergence of "organ-on-chip" technology that can faithfully recapitulate organ-level function. Given the rapid progress at the interface between microfluidics and cell biology, there is need to provide a focused evaluation of the state-of-the-art in microfluidic systems for cancer research to advance development, accelerate discovery of novel insights, and facilitate cooperation between engineers, biologists and oncologists in the clinic. Here, we provide a focused review of microfluidics technology from cells- and tissues- to organs-on-chips with application toward studying the tumor microenvironment. Key aspects of the tumor microenvironment including angiogenesis, hypoxia, biochemical gradients, tumor-stromal interactions, and the extracellular matrix are summarized for both solid tumors and non-solid hematologic malignancies. An overview of microfluidic systems designed specifically to answer questions related to different aspects of the tumor microenvironment is provided, followed by an examination of how these systems offer new opportunities to study outstanding challenges related to the major cancer hallmarks. Challenges also remain for microfluidics engineers, but it is hoped that cooperation between engineers and biologists at the intersection of their respective fields will lead to significant impact on the utility of organs-on-chips in cancer research.

Regulation of cell behavior and tissue patterning by bioelectrical signals: challenges and opportunities for biomedical engineering.

Achieving control over cell behavior and pattern formation requires molecular-level understanding of regulatory mechanisms. Alongside transcriptional networks and biochemical gradients, there functions an important system of cellular communication and control: transmembrane voltage gradients (V(mem)). Bioelectrical signals encoded in spatiotemporal changes of V(mem) control cell proliferation, migration, and differentiation. Moreover, endogenous bioelectrical gradients serve as instructive cues mediating anatomical polarity and other organ-level aspects of morphogenesis. In the past decade, significant advances in molecular physiology have enabled the development of new genetic and biophysical tools for the investigation and functional manipulation of bioelectric cues. Recent data implicate V(mem) as a crucial epigenetic regulator of patterning events in embryogenesis, regeneration, and cancer. We review new conceptual and methodological developments in this fascinating field. Bioelectricity offers a novel way of quantitatively understanding regulation of growth and form in vivo, and it reveals tractable, powerful control points that will enable truly transformative applications in bioengineering, regenerative medicine, and synthetic biology.

Future of lab on a chip

Biological and medical applications of lab a on a chip is a timely topic that is active area of research worldwide. Biomedical Engineering Letters is well positioned to contribute to reporting important results in this area. The field is continually evolving as lab on chip technologies and associated fabrication methods mature and the focus of research turns more and more towards the applications. A recent example is the application of lab on a chip to tackle the problem of the capture and analysis of circulating tumor cells (CTCs). CTCs are rare cells (approximately 1 in a billion) that have the potential to improve patient diagnosis, monitoring and response to treatment. Another example is the application of lab on a chip the measurement of immune cell response which may lead to improved asthma diagnosis as well as improving our understanding of the mechanisms that regulated directed cell migration. The development of lab on a chip for global health and point of care diagnostics are other emerging application areas. Encouragingly, the focus of much current work has moved beyond technology development and demonstration and into clinical use. These are examples that provide appropriate matches between the problem and the technology. The ability of lab on a chip to control the microenvironment (e.g. create stable biochemical gradients) opens up new avenues of biological inquiry and the potential for more personalized diagnosis and treatment. Lab on a chip can enable “more with less” which will enable the use of small numbers of primary cells from biopsies for companion diagnostics. The forces dominant at the microscale provide unique capabilities that are now being applied in practical ways to address challenges in medicine and biology such as single cell analysis and biomolecular sensing. Such advances allow new lines of inquiry and discovery as well as higher resolution measurements. The future is bright at the interface between lab on a chip and biology/medicine. This issue contains many examples of progress towards increased understanding of biology and improved patient care. I look forward to BMEL’s continued leadership in bringing the latest updates in lab a chip to a broad audience. In closing, I am pleased to serve on the editorial board of Biomedical Engineering Letters and to have the opportunity to contribute this editorial to this issue that is focused on lab on a chip.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7±0.1 to 0.9±0.2 GPa) and cementum (0.6±0.1 to 0.8±0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.

Golgi apparatus: Homotypic fusion maintains biochemical gradients within the Golgi and improves the accuracy of protein maturation

Eukaryotic cells are compartmentalized into organelles which, although constantly exchanging proteins and lipids with their environment, maintain a relatively well-defined biochemical identity. How can such large heterogeneities of chemical composition between (and within) organelles be maintained if different organelles are in constant contact through mass transport? Generic nonlinearities in the transport processes, as would result from specific molecular interactions, can cause the spontaneous chemical differentiation of interacting organelles and compartments within organelles. For the Golgi apparatus, the role of which is to process an incoming flux of lipids and proteins, this spontaneous differentiation decreases inter-cisternal exchange and increases the protein transit time under conditions of high incoming flux, This mechanism enables the Golgi apparatus to spontaneously adjust the protein transit time to the amount of protein requiring processing, thereby improving the processing accuracy of even a limited amount of maturation enzymes.

Investigation of Lipid Distribution by Coherent Anti-Stokes Raman Scattering

The chemical identity of individual lipids in cellular systems is believed to modulate the function of organelles and macro molecular assemblies. It has been shown that lipid composition can alter the rate of membrane protein conformational changes. In particular, variations in saturated and unsaturated lipids are believed to modulate the mechanical properties of the bilayer matrix supporting membrane proteins. Thus far, it has been challenging to identify the specific lipids that interact to modulate intact membranes. To study these phenomena, we have developed a multiplex coherent anti-Stokes microscope to spectroscopically distinguish the chemical composition of lipids in model and cellular systems. CARS imaging is a valuable tool for studying lipids in biological systems. We seek to gain spectroscopic information to discern biochemical gradients and interactions. The microscope utilizes a super continuum stokes beam and a spectrally narrow picosecond probe to measure a selection of vibrational modes simultaneously providing a chemical fingerprint for each pixel. Using this microscope we have detected vibrational bands that can be attributed to specific lipid components, such as the choline head group and are exploring its use in intact cellular systems.

Microfluidic chip for spatially and temporally controlled biochemical gradient generation in standard cell-culture Petri dishes

This paper reports the development, modeling, and testing of an original microfluidic chip capable of generating both time-evolving and spatially varying gradients in standard Petri dishes. It consists of three sets of five independently controlled parallel channels, and its architecture allows the generation of complex gradient profiles that can be flexibly positioned and dynamically altered in an open cell-chamber environment. A detailed fabrication protocol for the production of these chips using multilayer soft lithography is reported. A comprehensive computational model is also presented based on COMSOL Multiphysics software that includes both diffusion and advection of the fluid as it exits the microchannels. The results of the simulation are successfully applied to model single-channel experiments. The chip is then tested in multi-channel mode, and its ability to produce complex spatially varied concentration profiles is demonstrated. The achievement of steady state of the gradient profile in less than 5 min also allows for the dynamic variation of the profile. Finally, we apply the present chip architecture to investigate the migration of mouse neutrophils in an Interleukin-8 gradient. We report quantitatively on cell migration driven by Interleukin-8 gradient and provide migration speed distribution.

A modular cell culture device for generating arrays of gradients using stacked microfluidic flows

Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.

A new micro-fluidic channel that provides stable biochemical gradients for cell migration and chemotaxis

Cell migration and chemotaxis play an important role in many physiological and pathological processes. Traditional methods such as agarose plate culture and the transwell chamber model can only detect cell migration induced by a single factor. Microfluidic chips overcome this limitation and further provide multi-parameter conditions and real-time observation. However, microchips are mostly constructed using photolithography, a typically expensive procedure. We introduce an accessible platform made of polydimethylsiloxane, which provides the structure of the microchannel with microwires. These microwires were elongated to form a cross-channel structure with a 500-μm depth. The micro-fluid channel demonstrated a long-lasting concentration gradient that was maintained for up to 2 h, and a pressure gradient that was maintained for up to 3 d. The chemotactic responses of human endothelial cells within the cross- channel were further studied, and the feasibility and applicability of producing this technology were verified.

Microfluidic chip with temporal and spatial concentration generation capabilities for biological applications

This paper details the control and operation of a new microfluidic chip whose architecture of three sets of five fluidic channels is capable of generating both spatially and temporally varying concentration gradients. It is housed in a Petri dish where the cells are first seeded and preconditioned and then exposed to the biochemical gradients produced by the chip. The flow in the fluidic channels is regulated by crisscrossing control channels with the contact interfaces acting as valves. The pneumatic control and operation of the chip have been automated with LABVIEW and thoroughly tested. The results for the single channel case have also been validated by a comprehensive computational model whose formulation is described.

Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone

Substrates with mechanical property gradients and various extracellular matrix ligand loadings were evaluated for their ability to direct bone marrow stromal cell differentiation along osteogenic and tenogenic lineages. After verifying reproducible mechanical compliance characteristics of commercial hydrogel gradient substrates, substrates were functionalized with whole length fibronectin or collagen, both of which are found in skeletal structures and are relevant to cell-matrix signalling. Bone marrow stromal cells were seeded onto the substrates in growth media and cultured first to examine cell attachment and morphology, indicating higher levels of attachment on collagen substrates after 1 h, and increased spreading and organization trends after 24 h. Differentiation studies showed an increase in osteoblast differentiation on fibronectin substrates while collagen substrates lacked osteogenic differentiation. Osteogenic differentiation decreased on substrates of lower stiffness and lower ligand density. Molecular investigations revealed an increase in relevant signalling molecules for osteoblasts that were consistent with differentiation studies, but detected the presence of tenoblast markers on collagen substrates within a narrow range of stiffness. Our results indicate that mechanovariant substrates do hold promise as a culture platform for directed differentiation to tendon and bone by altering gene level expression of relevant signalling molecules. This study aids in understanding the molecular mechanisms that drive differentiation from substrate based cues, and could aid the design of therapeutic biomaterials at the transition from tendon to bone.

The contribution of deep‐sea macrohabitat heterogeneity to global nematode diversity

AbstractThe great variety of geological and hydrological conditions in the deep sea generates many different habitats. Some are only recently explored, although their true extent and geographical coverage are still not fully established. Both continental margins and mid‐oceanic seafloors are much more complex ecologically, geologically, chemically and hydrodynamically than originally thought. As a result, fundamental patterns of species distribution first observed and explained in the context of relatively monotonous slopes and abyssal plains must now be re‐evaluated in the light of this newly recognized habitat heterogeneity. Based on a global database of nematode genus composition, collected as part of the Census of Marine Life, we show that macrohabitat heterogeneity contributes significantly to total deep‐sea nematode diversity on a global scale. Different deep‐sea settings harbour specific nematode assemblages. Some of them, like coral rubble zones or nodule areas, are very diverse habitats. Factors such as increased substrate complexity in the case of nodules and corals seem to facilitate the co‐existence of a large number of genera with different modes of life, ranging from sediment dwelling to epifaunal. Furthermore, strong biochemical gradients in the case of vents or seeps are responsible for the success of particular genera, which are not prominent in more typical soft sediments. Many nematode deep‐sea genera are cosmopolitan, inhabiting a variety of deep‐sea habitats and oceans, whereas only 21% of all deep‐sea genera recorded are restricted to a single habitat. In addition to habitat heterogeneity, regional differences are important in structuring nematode assemblages. For instance, seeps from different regions yield different genera that thrive on the sulphidic sediments. This study also shows that many areas and habitats remain highly under‐sampled, affecting our ability to understand fully the contribution of habitat heterogeneity versus regional differences to global nematode diversity.

The roles of habitat heterogeneity in generating and maintaining biodiversity on continental margins: an introduction

(introduction to Special Issue: The roles of habitat heterogeneity in generating and maintaining biodiversity on continental margins A Contribution to the Census of Marine Life)

Microfluidic Platforms for Studies of Angiogenesis, Cell Migration, and Cell–Cell Interactions

Recent advances in microfluidic technologies have opened the door for creating more realistic in vitro cell culture methods that replicate many aspects of the true in vivo microenvironment. These new designs (i) provide enormous flexibility in controlling the critical biochemical and biomechanical factors that influence cell behavior, (ii) allow for the introduction of multiple cell types in a single system, (iii) provide for the establishment of biochemical gradients in two- or three-dimensional geometries, and (iv) allow for high quality, time-lapse imaging. Here, some of the recent developments are reviewed, with a focus on studies from our own laboratory in three separate areas: angiogenesis, cell migration in the context of tumor cell-endothelial interactions, and liver tissue engineering.

Fundamentals of cataract development

AbstractThe lens is a highly specialized tissue in the vertebrate eye, whose growth characteristics and metabolic turn‐over are designed for longevity in respect to its optical function. It grows throughout life and does neither shed any of its cells, nor excrete any degraded proteins. The lens manages to maintain an optical and a biochemical gradient based on the high protein content (crystallins) and slow metabolic turn‐over in the superficial fibre cells. It is well equipped with anti‐oxidative defence mechanisms, can seal off groups of damaged fibre cells and changes its transmission properties during ageing. The 3 main ageing characteristics in the lens are a tissue layer specific increase in light scattering, a yellowish discoloration of the proteins and a loss of accommodative capacity. Cataract development can be triggered by a wide selection of factors including genetic predisposition, certain diseases, optical and ionizing radiation, chemical compounds, drugs, nutritional and environmental factors, as well as trauma. Patho‐physiological mechanisms involved are DNA damage in epithelial cells, protein glycosylation and aggregation and molecular cross linking in lens fibre cells, metabolic breakdown of selected groups of fibre cells or of a whole layer, protein degradation and finally globular degeneration and liquefaction. Ageing in this context is the time line which allows an increasing number of noxious factors to interact in the lens at decreasing metabolic capacities. While the sensitivity of the lens to oxidative damage increases during ageing, its sensitivity to low dose ionizing radiation decreases. The function of molecular receptors for acetylcholine, steroids, the sigma receptor and other receptors found in the lens is still unknown, but might explain the specific reaction to certain drugs.

Cell migration into scaffolds under co-culture conditions in a microfluidic platform

Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

The Effects of Hydration on Growth of the House Cricket,Acheta domesticus

Maintenance of biochemical gradients, membrane fluidity, and sustained periods of activity are key physiological and behavioral functions of water for animals living in desiccating environments. Water stress may reduce the organism's ability to maintain these functions and as such, may reduce an organism's growth. However, few studies have examined this potential effect. The effects of altered hydration state of the house cricket, Acheta domesticus L. (Orthoptera: Gryllidae) on individual growth were studied under laboratory conditions. Crickets were permitted access to water for three different durations each day, resulting in significant differences in hydration state (32% greater hydration for maximum than minimum duration of water availability). Growth was 59% and 72% greater in dry mass and length, respectively, between the lowest and highest hydration state treatments. These findings may be representative for a variety of animal species and environments and could have important ecological implications.

Three-Dimensional Biochemical Patterning of Click-Based Composite Hydrogels via Thiolene Photopolymerization

Hydrogels formed from (meth)acrylated poly(ethylene glycol) precursors are commonly used in a variety of biomedical applications ranging from tissue engineering to biosensors. While this approach has proven quite diverse, a major limitation to this approach is the heterogeneities and nonidealities that arise in the gels from the chain polymerization process, which increases the difficulty in relating the network structure to the final physical properties of the gel. Here we have exploited the specificity and fidelity of the [3+2] cycloaddition reaction to synthesize hydrogels with controlled architectures and improved mechanical properties. Moreover, we demonstrate a general approach toward the integration of multifunctional photoreactive polypeptide sequences into the network structure that provides a facile way to independently tune the 3D chemical and physical properties of the gel. Standard photolithographic techniques were used to generate a variety of two- and three-dimensional patterns as well as controlled biochemical gradients within existing preformed hydrogels.

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.

Aortic glutathione-related antioxidant defences in rabbits subjected to suprarenal aortic coarctation hypertension

In seven rabbits subjected to suprarenal aortic coarctation hypertension, the segments above and below the coarctation were tested for the antioxidant defences (i.e. acid-soluble thiol compounds, selenium-dependent and selenium-indepdent glutathione peroxidase, glutathione reductase, glutathione transferase) and thiobarbituric acid-reactive substances. Seven sham-operated rabbits served as controls. Systolic blood pressure proximal to the ligature increased significantly with respect to pre-operative values after 16 days (117 ± 8.3 vs 71.7 ± 5.2 mmHg, P < 0.05), while pressure distal to the ligature remained normotensive. Higher values of acid-soluble thiol compounds. thiobarbituric acid-reactive substances and increased activities of selenium-dependent glutathione peroxidase. glutathione reductase and glutathione transferase were assayed in the suprarenal with respect to the subrenal segment in both groups. However, the values of the upper segments were more elevated in the experimental group than in controls, but no differences were observed in the lower segments. Glutathione peroxidase activity assayed with cumene hydroperoxide was higher than the activity assaysed with hydrogen peroxide in the hypertensive segments, but no differences were detected in the substenotic and control segments. Furthermore, an isoenzymatic form of glutathione transferase, analogous to rat 8-8 glutathione transferase isoenzyme, was detected by immunodiffusion in the hypertensive aorta. The following conclusions may be drawn: (1) a biochemical gradient in glutathione-related enzymes, acid-soluble thiol compounds and thiobarbituric acid-reactive substances between the proximal and distal aorta seems to exist in control rabbits; (2) suprarenal aortic coarctation induces a significant increase in glutathione-related antioxidant defences and thiobarbituric acid-reactive substances of the hypertensive aortic wall.