Hossein Tavana

Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells

Microscale biopatterning allows regulation of cell-material interactions1,2 and cell shape3, and enables multiplexed high throughput studies4,5,6,7,8 in a cell and reagent efficient manner. The majority of available techniques rely on physical contact of a stamp3, pin8, or mask9,10 with mainly a dry surface. Inkjet and piezoelectric printing11 is performed in a non-contact manner but still requires a substantially dry substrate to ensure fidelity of printed patterns. These existing methods, therefore, are limited for patterning onto delicate surfaces of living cells because physical contact or substantially dry conditions are damaging to them. Microfluidic patterning with laminar streams12,13 does allow non-contact patterning in fully aqueous environments but with limited throughput and reagent diffusion across interfacial flows. Here, we describe a polymeric aqueous two-phase system (ATPS) that enables patterning nanoliters of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate media formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. Utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.

Integrated elastomeric components for autonomous regulation of sequential and oscillatory flow switching in microfluidic devices

A critical need for enhancing usability and capabilities of microfluidic technologies is the development of standardized, scalable, and versatile control systems1,2. Electronically controlled valves and pumps typically used for dynamic flow regulation, although useful, can limit convenience, scalability, and robustness3–5. This shortcoming has motivated development of device-embedded non-electrical flow-control systems. Existing approaches to regulate operation timing on-chip, however, still require external signals such as timed generation of fluid flow, bubbles, liquid plugs or droplets, or an alteration of chemical compositions or temperature6–16. Here, we describe a strategy to provide device-embedded flow switching and clocking functions. Physical gaps and cavities interconnected by holes are fabricated into a three-layer elastomer structure to form networks of fluidic gates that can spontaneously generate cascading and oscillatory flow output using only a constant flow of Newtonian fluids as the device input. The resulting microfluidic substrate architecture is simple, scalable, and should be applicable to various materials. This flow-powered fluidic gating scheme brings the autonomous signal processing ability of microelectronic circuits to microfluidics where there is the added diversity in current information of having distinct chemical or particulate species and richness in current operation of having chemical reactions and physical interactions.

Polymeric Aqueous Biphasic Systems for Non‐Contact Cell Printing on Cells: Engineering Heterocellular Embryonic Stem Cell Niches

The ability to generate heterocellular niches through direct spatial patterning of one type of cells onto a living layer of another cell type would benefit a wide range of cell biological studies including stem cell research. Most cell patterning approaches, however, rely on patterned material adhesiveness to indirectly guide cell patterning,[1–5] and cannot position cells on an already existing cell monolayer. Cells can be embedded directly into soft supported membranes and gels using solid pins,[6, 7] but the physical contact involved may damage delicate substrates such as living cells. Individual cells or cell sheets have been gently placed onto a cell monolayer, but with limitations in control of positioning and resolution.[8–10] Thermal or piezoelectric inkjet printers utilize thermal energy or applied electric voltage to eject the cell suspension from a nozzle and direct it toward the substrate.[11–13] Although this approach is non-contact and can potentially accommodate several cell types, concentrated buffer solutions[11] and special additives[14] are required and still often induce chemical, mechanical, or thermal stresses during cell suspension preparation and printing stages. Furthermore, dedicated cartridges are needed for each cell type, and pattern fidelity is an issue because cells have to be printed on a gel immersed in aqueous media.[14] Pressure-mediated extrusion of cell-seeded hydrogels through needles also creates continuous pattern of cells,[15] though with impaired direct cell-cell contact due to gel encapsulation. Therefore, existing approaches face limitations in terms of contact-free printing with and onto live cells for engineering heterocellular stem cell niches.

Epithelium damage and protection during reopening of occluded airways in a physiologic microfluidic pulmonary airway model

Airways of the peripheral lung are prone to closure at low lung volumes. Deficiency or dysfunction of pulmonary surfactant during various lung diseases compounds this event by destabilizing the liquid lining of small airways and giving rise to occluding liquid plugs in airways. Propagation of liquid plugs in airways during inflation of the lung exerts large mechanical forces on airway cells. We describe a microfluidic model of small airways of the lung that mimics airway architecture, recreates physiologic levels of pulmonary pressures, and allows studying cellular response to repeated liquid plug propagation events. Substantial cellular injury happens due to the propagation of liquid plugs devoid of surfactant. We show that addition of a physiologic concentration of a clinical surfactant, Survanta, to propagating liquid plugs protects the epithelium and significantly reduces cell death. Although the protective role of surfactants has been demonstrated in models of a propagating air finger in liquid-filled airways, this is the first time to study the protective role of surfactants in liquid plugs where fluid mechanical stresses are expected to be higher than in air fingers. Our parallel computational simulations revealed a significant decrease in mechanical forces in the presence of surfactant, confirming the experimental observations. The results support the practice of providing exogenous surfactant to patients in certain clinical settings as a protective mechanism against pathologic flows. More importantly, this platform provides a useful model to investigate various surface tension-mediated lung diseases at the cellular level.

Microprinted feeder cells guide embryonic stem cell fate.

We introduce a non-contact approach to microprint multiple types of feeder cells in a microarray format using immiscible aqueous solutions of two biopolymers. Droplets of cell suspension in the denser aqueous phase are printed on a substrate residing within a bath of the immersion aqueous phase. Due to their affinity to the denser phase, cells remain localized within the drops and adhere to regions of the substrate underneath the drops. We show the utility of this technology for creating duplex heterocellular stem cell niches by printing two different support cell types on a gel surface and overlaying them with mouse embryonic stem cells (mESCs). As desired, the type of printed support cell spatially direct the fate of overlaid mESCs. Interestingly, we found that interspaced mESCs colonies on differentiation-inducing feeder cells show enhanced neuronal differentiation and give rise to dense networks of neurons. This cell printing technology provides unprecedented capabilities to efficiently identify the role of various feeder cells in guiding the fate of stem cells.

Ultralow interfacial tensions of aqueous two-phase systems measured using drop shape.

Aqueous solutions of different polymers can separate and form aqueous two-phase systems (ATPS). ATPS provide an aqueous, biocompatible, and mild environment for separation and fractionation of biomolecules. The interfacial tension between the two aqueous phases plays a major role in ATPS-mediated partition of biomolecules. Because of the structure of the two aqueous phases, the interfacial tensions between the phases can be 3-4 orders of magnitude smaller than conventional fluid-liquid systems: ∼1-100 μJ/m(2) for ATPS compared to ∼72 mJ/m(2) for the water-vapor interface. This poses a major challenge for the experimental measurements of reproducible interfacial tension data for these systems. We address the need for precise determination of ultralow interfacial tensions by systematically studying a series of polymeric ATPS comprising of polyethylene glycol (PEG) and dextran (DEX) as the phase-forming polymers. Sessile and pendant drops of the denser DEX phase are formed within the immersion PEG phase. An axisymmetric drop shape analysis (ADSA) is used to determine interfacial tensions of eight different ATPS. Specific criteria are used to reproducibly determine ultralow interfacial tensions of the ATPS from pendant and sessile drops. Importantly, for a given ATPS, pendant drop and sessile drop experiments return values within 0.001 mJ/m(2) indicating reliability of our measurements. Then, the pendant drop technique is used to measure interfacial tensions of all eight ATPS. Our measured values range from 0.012 ± 0.001 mJ/m(2) to 0.381 ± 0.006 mJ/m(2) and vary with the concentration of polymers in equilibrated phases of ATPS. Measurements of ultralow interfacial tensions with such reproducibility will broadly benefit studies involving partition of different biomolecules in ATPS and elucidate the critical effect of interfacial tension.

Dynamics of liquid plugs of buffer and surfactant solutions in a micro-engineered pulmonary airway model.

We describe a bioinspired microfluidic system that resembles pulmonary airways and enables on-chip generation of airway occluding liquid plugs from a stratified air-liquid two-phase flow. User-defined changes in the air stream pressure facilitated by mechanical components and tuning the wettability of the microchannels enable generation of well-defined liquid plugs. Significant differences are observed in liquid plug generation and propagation when surfactant is added to the buffer. The plug flow patterns suggest a protective role of surfactant for airway epithelial cells against pathological flow-induced mechanical stresses. We discuss the implications of the findings for clinical settings. This approach and the described platform will enable systematic investigation of the effect of different degrees of fluid mechanical stresses on lung injury at the cellular level and administration of exogenous therapeutic surfactants.

Uniform cell seeding and generation of overlapping gradient profiles in a multiplexed microchamber device with normally-closed valves

Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.

Interfacial Tension Effect on Cell Partition in Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPS) provide a mild environment for the partition and separation of cells. We report a combined experimental and theoretical study on the effect of interfacial tension of polymeric ATPS on the partitioning of cells between two phases and their interface. Two-phase systems are generated using polyethylene glycol and dextran of specific properties as phase-forming polymers and culture media as the solvent component. Ultralow interfacial tensions of the solutions are precisely measured using an axisymmetric drop shape analysis method. Partition experiments show that two-phase systems with an interfacial tension of 30 μJ/m(2) result in distribution of majority of cells to the bottom dextran phase. An increase in the interfacial tension results in a distribution of cells toward the interface. An independent cancer cell spheroid formation assay confirms these observations: a drop of the dextran phase containing cancer cells is dispensed into the immersion polyethylene glycol phase to form a cell-containing drop. Only at very small interfacial tensions do cells remain within the drop to aggregate into a spheroid. We perform a thermodynamic modeling of cell partition to determine variations of free energy associated with displacement of cells in ATPS with respect to the ultralow interfacial tensions. This modeling corroborates with the experimental results and demonstrates that at the smallest interfacial tension of 30 μJ/m(2), the free energy is a minimum with cells in the bottom phase. Increasing the interfacial tension shifts the minimum energy and partition of cells toward the interfacial region of the two aqueous phases. Examining differences in the partition behavior and minimum free energy modeling of A431.H9 cancer cells and mouse embryonic stem cells shows that the surface properties of cells further modulate partition in ATPS. This combined approach provides a fundamental understanding of interfacial tension role on cell partition in ATPS and a framework for future studies.

A Robust Polynomial Fitting Approach for Contact Angle Measurements

Polynomial fitting to drop profile offers an alternative to well-established drop shape techniques for contact angle measurements from sessile drops without a need for liquid physical properties. Here, we evaluate the accuracy of contact angles resulting from fitting polynomials of various orders to drop profiles in a Cartesian coordinate system, over a wide range of contact angles. We develop a differentiator mask to automatically find a range of required number of pixels from a drop profile over which a stable contact angle is obtained. The polynomial order that results in the longest stable regime and returns the lowest standard error and the highest correlation coefficient is selected to determine drop contact angles. We find that, unlike previous reports, a single polynomial order cannot be used to accurately estimate a wide range of contact angles and that a larger order polynomial is needed for drops with larger contact angles. Our method returns contact angles with an accuracy of <0.4° for solid-liquid systems with θ < ~60°. This compares well with the axisymmetric drop shape analysis-profile (ADSA-P) methodology results. Above about 60°, we observe significant deviations from ADSA-P results, most likely because a polynomial cannot trace the profile of drops with close-to-vertical and vertical segments. To overcome this limitation, we implement a new polynomial fitting scheme by transforming drop profiles into polar coordinate system. This eliminates the well-known problem with high curvature drops and enables estimating contact angles in a wide range with a fourth-order polynomial. We show that this approach returns dynamic contact angles with less than 0.7° error as compared to ADSA-P, for the solid-liquid systems tested. This new approach is a powerful alternative to drop shape techniques for estimating contact angles of drops regardless of drop symmetry and without a need for liquid properties.