Nanoliter scale microbioreactor array for quantitative cell biology

A nanoliter scale microbioreactor array was designed for multiplexed quantitative cell biology. An addressable 8 x 8 array of three nanoliter chambers was demonstrated for observing the serum response of HeLa human cancer cells in 64 parallel cultures. The individual culture unit was designed with a "C" shaped ring that effectively decoupled the central cell growth regions from the outer fluid transport channels. The chamber layout mimics physiological tissue conditions by implementing an outer channel for convective "blood" flow that feeds cells through diffusion into the low shear "interstitial" space. The 2 microm opening at the base of the "C" ring established a differential fluidic resistance up to 3 orders of magnitude greater than the fluid transport channel within a single mold microfluidic device. Three-dimensional (3D) finite element simulation were used to predict fluid transport properties based on chamber dimensions and verified experimentally. The microbioreactor array provided a continuous flow culture environment with a Peclet number (0.02) and shear stress (0.01 Pa) that approximated in vivo tissue conditions without limiting mass transport (10 s nutrient turnover). This microfluidic design overcomes the major problems encountered in multiplexing nanoliter culture environments by enabling uniform cell loading, eliminating shear, and pressure stresses on cultured cells, providing stable control of fluidic addressing, and permitting continuous on-chip optical monitoring.     

Evaluation of different culture techniques of osteoblasts on 3D scaffolds

Bones adjust their structure to withstand the mechanical demands they experience. It is suggested that flow-derived shear stress may be the most significant and primary mediator of mechanical stimulation. In this study, we designed and fabricated a fluid flow cell culture system that can load shear stress onto cells cultured on 3D scaffolds. We evaluated the effect of different culture techniques, namely, (1) continuous perfusion fluid flow, (2) intermittent perfusion fluid flow, and (3) static condition, on the proliferation of osteoblasts seeded on partially deproteinized bones. The flow rate was set at 1 ml/min for all the cells cultured using flow perfusion and the experiment was conducted for 12 days. Scanning electron microscopy analysis indicated an increase in cell proliferation for scaffolds subjected to fluid shear stress. In addition, the long axes of these cells lengthened along the flowing fluid direction. Continuous perfusion significantly enhanced cell proliferation compared to either intermittent perfusion or static condition. All the results demonstrated that fluid shear stress is able to enhance the proliferation of cells and change the form of cells.     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.     

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.     

Computational simulation of hemodynamic-driven growth and remodeling of embryonic atrioventricular valves

Embryonic heart valves develop under continuous and demanding hemodynamic loading. The particular contributions of fluid pressure and shear tractions in valve morphogenesis are difficult to decouple experimentally. To better understand how fluid loads could direct valve formation, we developed a computational model of avian embryonic atrioventricular (AV) valve (cushion) growth and remodeling using experimentally derived parameters for the blood flow and the cushion stiffness. Through an iterative scheme, we first solved the fluid loads on the axisymmetric AV canal and cushion model geometry. We then applied the fluid loads to the cushion and integrated the evolution equations to determine the growth and remodeling. After a set time of growth, we updated the fluid domain to reflect the change in cushion geometry and resolved for the fluid forces. The rate of growth and remodeling was assumed to be a function of the difference between the current stress and an isotropic homeostatic stress state. The magnitude of the homeostatic stress modulated the rate of volume addition during the evolution. We found that the pressure distribution on the AV cushion was sufficient to generate leaflet-like elongation in the direction of flow, through inducing tissue resorption on the inflow side of cushion and expansion on the outflow side. Conversely, shear tractions minimally altered tissue volume, but regulated the remodeling of tissue near the cushion surface, particular at the leading edge. Significant shear and circumferential residual stresses developed as the cushion evolved. This model offers insight into how natural and perturbed mechanical environments may direct AV valvulogenesis and provides an initial framework on which to incorporate more mechano-biological details.     

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

Three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression

Here, we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen gels. Epithelial cells are seeded into the cavities and covered with a second layer of collagen. The cells reorganize into hollow tissues corresponding to the geometry of the cavities. Patterned tissue arrays can be produced in 3-4 h and will undergo morphogenesis over the following 1-3 d. The protocol can easily be adapted to study a variety of tissues and aspects of normal and neoplastic development.     

Perfusion flow bioreactor for 3D in situ imaging: investigating cell/biomaterials interactions

The capability to image real time cell/material interactions in a three-dimensional (3D) culture environment will aid in the advancement of tissue engineering. This paper describes a perfusion flow bioreactor designed to hold tissue engineering scaffolds and allow for in situ imaging using an upright microscope. The bioreactor can hold a scaffold of desirable thickness for implantation (>2 mm). Coupling 3D culture and perfusion flow leads to the creation of a more biomimetic environment. We examined the ability of the bioreactor to maintain cell viability outside of an incubator environment (temperature and pH stability), investigated the flow features of the system (flow induced shear stress), and determined the image quality in order to perform time-lapsed imaging of two-dimensional (2D) and 3D cell culture. In situ imaging was performed on 2D and 3D, culture samples and cell viability was measured under perfusion flow (2.5 mL/min, 0.016 Pa). The visualization of cell response to their environment, in real time, will help to further elucidate the influences of biomaterial surface features, scaffold architectures, and the influence of flow induced shear on cell response and growth of new tissue.     

A fiber matrix model for interstitial fluid flow and permeability in ligaments and tendons

Collagen fibrils in ligaments and tendons are highly organized into parallel arrays which influence interstitial fluid transport. Finite element (FE) models were developed analogous to the fibrillar arrays in ligaments and tendons to investigate interstitial fluid flow and tissue permeability as a function of interfibrillar spacing and fluid properties. Collagen fibrils were assumed to be a periodic square array of impermeable cylinders. A two-dimensional FE model was used to study transverse fluid flow and a three-dimensional model was used to study flow parallel to the collagen fibrils. Parametric FE analysis provided data to formulate empirical expressions for permeability (kappa) as a function of porosity (phi). Results show that longitudinal permeability (kappa = 1.1.10(-15)phi 2.5[1 - phi]-0.333) can be up to 50 times higher than transverse permeability (kappa = 1.2.10(-15)phi 0.5[phi - phi min]2.5) in a compact array. Maximum fluid shear stresses occur at the narrowest zones of adjacent fibrils (1.21 Pa or 12.1 dyn/cm2 at 10 microns/s of average transverse influx). If interstitial fluid is highly non-Newtonian, the permeability should be considered as flow (shear)-dependent. The computational results suggest that tissue permeability in ligaments and tendons is highly anisotropic, porosity-dependent, and can be estimated by analytic expressions.     

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Mechanotransduction in leukocyte activation: a review

We review recent evidence which suggests that leukocytes in the circulation and in the tissue may readily respond to physiological levels of fluid shear stress in the range between about 1 and 10 dyn/cm 2, a range that is below the level to achieve a significant passive, viscoelastic response. The response of activated neutrophilic leukocytes to fluid shear consists of a rapid retraction of lamellipodia with membrane detachment from integrin binding sites. In contrast, a subgroup of non-activated neutrophils may project pseudopods after exposure to fluid shear stress. The evidence suggests that G-protein coupled receptor downregulation by fluid shear with concomitant downregulation of Rac-related small GTPases and depolymerization of F-actin serves to retract the lamellipodia in conjunction with proteolytic cleavage of beta 2 integrin to facilitate membrane detachment. Furthermore, there exists a mechanism to up- and down-regulate the fluid shear-response, which involves nitric oxide and the second messenger cyclic guanosine monophosphate (cGMP). Many physiological activities of circulating leukocytes are under the influence of fluid shear stress, including transendothelial migration of lymphocytes. We describe a disease model with chronic hypertension that suffers from an attenuated fluid shear-response with far reaching implications for microvascular blood flow.     

A numerical study on the effect of side hole number and arrangement in venous cannulae

Insertion of cannulae into vessels may apply non-physiological load and stress on blood cells, such that adenosine diphosphate may increase and result in hemolysis. Authors used the computational method to simulate the blood flow inside of the cannula. We limited the research to within the drainage cannulae. Nine different cannulae categorized by the number of side holes of 4, 12, and 20, and also categorized by the array type as staggered array, in-line array, and alternative in-line array were studied and compared to the cannulae with no side holes by using computational fluid dynamics. We evaluated the flow rate, the wall shear stress, and the shear rate, and compared them with one another to estimate the effect of the side holes. The flow rate is not proportional to the number of the side holes. However, larger number of side holes can reduce the mean shear rate.     

A driving force for change: interstitial flow as a morphoregulator

Dynamic stresses that are present in all living tissues drive small fluid flows, called interstitial flows, through the extracellular matrix. Interstitial flow not only helps to transport nutrients throughout the tissue, but also has important roles in tissue maintenance and pathobiology that have been, until recently, largely overlooked. Here, we present evidence for the various effects of interstitial flow on cell biology, including its roles in embryonic development, tissue morphogenesis and remodeling, inflammation and lymphedema, tumor biology and immune cell trafficking. We also discuss possible mechanisms by which interstitial flow can induce morphoregulation, including direct shear stress, matrix-cell transduction (as has been proposed in the endothelial glycocalyx) and the newly emerging concept of autologous gradient formation.     

Effect of serum concentration on hybridoma viable cell density and production of monoclonal antibodies in CSTRs and on shear sensitivity in air-lift loop reactors

The death rate of hybridoma cells, grown in a continuous culture, has been studied in a small air-lift loop reactor as a function of reactor height and injected gas flow rate. The first-order death-rate constant was found to be proportional to the reciprocal height and to the gas flow rate, in accordance with the hypothetical killing volume model for insect cells in bubble columns. Furthermore, the effect of the serum concentration on viable cell concentration and cell productivity has been investigated in a continuous culture. A serum component became growth limiting when the serum concentration was decreased from 2% to 1%. No effect of the serum concentration on specific cell productivity could be measured. Samples from this culture were also studied in the air-lift loop reactor to determine the effect of serum concentration on the shear sensitivity. The cells' shear sensitivity increased with decreasing serum concentration. The protective effect of serum was found to be physical as well as physiological.     

Influence of hydrodynamic imposed shear stress on the activation of factor X in the presence of tissue factor-factor VIIa complex in a continuous flow reactor

Summary The effect of shear stress on the ability of tissue factor-factor VIIa complex to activate factor X in a continuous flow reactor was studied. Tissue factor immobilized in a phospholipid bilayer on the inner surface of a capillary tube was exposed to a perfusate containing factors VIIa and X flowing at flow rates of 12.7, and 204 l/min, corresponding to wall shear rates of 100, and 1760 sec^-1. The maximum flux (moles formed per unit surface area per unit time) of factor Xa (activated form of factor X) produced at the wall decreased as the shear stress at the wall was increased from 1 to 3 dynes/cm² (3-fold) at a constant shear rate of 100 sec^-1. In contrast, at higher shear rate (1760 sec^-1), increasing shear stress from 16 to 48 dynes/cm² had no significant influence on factor Xa production. The decreased production of factor Xa at higher shear stress (low shear rate 100 sec^-1) probably reflects the transport limitation of factor X to the wall. Apparently shear stress can directly influence the activation of factor X at low shear rates.     

Modeling of flow‐induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering

Novel tissue‐culture bioreactors employ flow‐induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three‐dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear‐stress values within the physiological range of those naturally sensed by vascular cells (1–10 dyne/cm²), and will thereby provide suitable conditions for vascular tissue‐engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell‐layer thicknesses of 0, 50, 75, 100, and 125 µm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear‐stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell‐layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in‐depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. Biotechnol. Bioeng. 2010; 105: 645–654. © 2009 Wiley Periodicals, Inc.     

Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

Fractal dimension of antibody‐PEG precipitate: Light microscopy for the reconstruction of 3D precipitate structures

Protein and in particular antibody precipitation by PEG is a cost‐effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo‐TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.1–0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro‐ to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.     

The mouse anterior chamber angle and trabecular meshwork develop without cell death

Background

The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice.

Results

The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation.

Conclusions

These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.