Open access to novel dual flow chamber technology for in vitro cell mechanotransduction, toxicity and pharamacokinetic studies

<p>Abstract</p> <p>Background</p> <p>A major stumbling block for researchers developing experimental models of mechanotransduction is the control of experimental variables, in particular the transmission of the mechanical forces at the cellular level. A previous evaluation of state of the art commercial perfusion chambers showed that flow regimes, applied to impart a defined mechanical stimulus to cells, are poorly controlled and that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable.</p> <p>Methods</p> <p>This study provides a novel chamber design to provide both physiologically-based flow regimes, improvements in control of experimental variables, as well as ease of use compared to commercial chambers. This novel design achieves controlled stresses through five gasket designs and both single- and dual-flow regimes.</p> <p>Results</p> <p>The imparted shear stress within the gasket geometry is well controlled. Fifty percent of the entire area of the 10 × 21 mm universal gasket (Gasket I, designed to impart constant magnitude shear stresses in the center of the chamber where outcome measures are taken), is exposed to target stresses. In the 8 mm diameter circular area at the center of the chamber (where outcome measures are made), over 92% of the area is exposed to the target stress (± 2.5%). In addition, other gasket geometries provide specific gradients of stress that vary with distance from the chamber inlet. Bench-top testing of the novel chamber prototype shows improvements, in the ease of use as well as in performance, compared to the other commercial chambers. The design of the chamber eliminates flow deviations due to leakage and bubbles and allows actual flow profiles to better conform with those predicted in computational models.</p> <p>Conclusion</p> <p>The novel flow chamber design provides predictable and well defined mechanical forces at the surface of a cell monolayer, showing improvement over previously tested commercial chambers. The predictability of the imparted stress improves both experiment repeatability as well as the accuracy of inter-study comparisons. Carefully controlling the stresses on cells is critical in effectively mimicking <it>in vivo </it>situations. Overall, the improved perfusion flow chamber provides the needed resolution, standardization and <it>in vitro </it>model analogous to <it>in vivo </it>conditions to make the step towards greater use in research and the opportunity to enter the diagnostic and therapeutic market.</p

Prospective Design, Rapid Prototyping, and Testing of Smart Dressings, Drug Delivery Patches, and Replacement Body Parts Using Microscopy Aided Design and ManufacturE (MADAME)

Natural materials exhibit smart properties including gradients in biophysical properties that engender higher order functions, as well as stimuli-responsive properties which integrate sensor and/or actuator capacities. Elucidation of mechanisms underpinning such smart material properties (i), and translation of that understanding (ii), represent two of the biggest challenges in emulating natural design paradigms for design and manufacture of disruptive materials, parts, and products. Microscopy Aided Design And ManufacturE (MADAME) stands for a computer-aided additive manufacturing platform that incorporates multidimensional (multi-D) printing and computer-controlled weaving. MADAME enables the creation of composite design motifs emulating e.g., patterns of woven protein fibers as well as gradients in different caliber porosities, mechanical, and molecular properties, found in natural tissues, from the skin on bones (periosteum) to tree bark. Insodoing, MADAME provides a means to manufacture a new genre of smart materials, products and replacement body parts that exhibit advantageous properties both under the influence of as well as harnessing dynamic mechanical loads to activate material properties (mechanoactive properties). This Technical Report introduces the MADAME technology platform and its associated machine-based workflow (pipeline), provides basic technical background of the novel technology and its applications, and discusses advantages and disadvantages of the approach in context of current 3 and 4D printing platforms

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

BACKGROUND: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. METHODS: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm(2 )for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (μPIV) experiments. RESULTS: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within ±10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. CONCLUSION: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms

Effect of lacunocanalicular architecture on hydraulic conductance in bone tissue: implications for bone health and evolution. The Anatomical Record

on hydraulic conductance in bone tissue: Implications for bone health and evolution. Th

A spike in circulating cytokines TNF-α and TGF-β alters barrier function between vascular and musculoskeletal tissues

Abstract Molecular transport between the circulatory and musculoskeletal systems regulates articular joint physiology in health and disease. Osteoarthritis (OA) is a degenerative joint disease linked to systemic and local inflammation. Inflammatory events involve cytokines, which are secreted by cells of the immune system and modulate molecular transport across tissue interfaces (referred to as tight junction [TJ] barrier function). In a previous study from our group, OA knee joint tissues were shown to exhibit size separation of different sized molecules delivered as a single bolus to the heart (Ngo et al. in Sci. Rep. 8:10254, 2018). Here, in a follow up study of parallel design, we test the hypothesis that two common cytokines, with multifaceted roles in the etiology of osteoarthritis as well as immune state in general, modulate the barrier function properties of joint tissue interfaces. Specifically, we probe the effect of an acute cytokine increase (spike) on molecular transport within tissues and across tissue interfaces of the circulatory and musculoskeletal systems. A single bolus of fluorescent-tagged 70 kDa dextran, was delivered intracardially, either alone, or with either the pro-inflammatory cytokine TNF-α or the anti-inflammatory cytokine TGF-β, to skeletally mature (11 to 13-month-old) guinea pigs (Dunkin-Hartley, a spontaneous OA animal model). After five minutes' circulation, whole knee joints were serial sectioned and fluorescent block face cryo-imaged at near-single-cell resolution. The 70 kDa fluorescent-tagged tracer is analogous in size to albumin, the most prevalent blood transporter protein, and quantification of tracer fluorescence intensity gave a measure of tracer concentration. Within five minutes, a spike (acute doubling) in circulating cytokines TNF-α or TGF-β significantly disrupted barrier function between the circulatory and musculoskeletal systems, with barrier function essentially abrogated in the TNF-α group. In the entire volume of the joint (including all tissue compartments and the bounding musculature), tracer concentration was significantly decreased in the TGF-β- and TNF-α- compared to the control-group. These studies implicate inflammatory cytokines as gatekeepers for molecular passage within and between tissue compartments of our joints and may open new means to delay the onset and mitigate the progression of degenerative joint diseases such as OA, using pharmaceutical and/or physical measures

Multiscale Modeling of Emergent Stem Cell Structure and Function Underlying Emergent Lineage Commitment

International audienc