Micropillar sequence designs for fundamental inertial flow transformations

The ability to control the shape of a flow in a passive microfluidic device enables potential applications in chemical reaction control, particle separation, and complex material fabrication. Recent work has demonstrated the concept of sculpting fluid streams in a microchannel using a set of pillars or other structures that individually deform a flow in a predictable pre-computed manner. These individual pillars are then placed in a defined sequence within the channel to yield the composition of the individual flow deformations – and ultimately complex user-defined flow shapes. In this way, an elegant mathematical operation can yield the final flow shape for a sequence without an experiment or additional numerical simulation. Although these approaches allow for programming complex flow shapes without understanding the detailed fluid mechanics, the design of an arbitrary flow shape of interest remains difficult, requiring significant design iteration. The development of intuitive basic operations (i.e. higher-level functions that consist of combinations of obstacles) that act on the flow field to create a basis for more complex transformations would be useful in systematically achieving a desired flow shape. Here, we show eight transformations that could serve as a partial basis for more complex transformations. We initially used in-house, freely available custom software (uFlow), which allowed us to arrive at these transformations that include making a fluid stream concave and convex, tilting, stretching, splitting, adding a vertex, shifting, and encapsulating another flow stream. The pillar sequences corresponding to these transformations were subsequently fabricated and optically analyzed using confocal imaging – yielding close agreement with uFlow-predicted shapes. We performed topological analysis on each transformation, characterizing potential sequences leading to these outputs and trends associated with changing diameter and placement of the pillars. We classify operations into four sets of sequence-building concatenations: stacking, recursion, mirroring, and shaping. The developed basis should help in the design of microfluidic systems that have a phenomenal variety of applications, such as optofluidic lensing, enhanced heat transfer, or new polymer fiber design

Shaped 3D microcarriers for adherent cell culture and analysis

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting, which modulates protein expression and eliminates intercellular connections. To allow adherent culture and processing in flow, we present 3D-shaped hydrogel cell microcarriers, which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth, and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow, important for processing in flow and imaging flow cytometry. We designed a method to rapidly design, using the genetic algorithm, and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach. The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling, and is poised to enable adherent cell culture, in-flow analysis, and sorting in a single format

Inertial microfluidic programming of microparticle-laden flows for solution transfer around cells and particles

Control of particles/cells and the surrounding fluid is enabling toward the purification of complex cellular samples, which still remains a bottleneck for point-of-care diagnostic devices. We explore a newly developed approach to engineer fluid stream motion while simultaneously controlling particles using inertial lift force. We use inertial flow deformations induced by sequences of simple pillar microstructures to control the fluid stream. Instead of iterative experimental procedures to identify optimal sequences of structures, we use software that numerically predicts the total deformation function for any pillar sequence. Using this program, we engineer the cross-stream translation of a fluid stream to achieve solution exchange around particles, where both the particles and fluid stream remain focused and can be extracted at high purity. An extraction device, called a pillar separation device, is then designed and validated with suspensions of rigid particles to identify optimal operating parameters. At a flow rate of 250 A mu L/min, up to 96 % beads and 70.5 % of an initial buffer stream inputted into the system can be collected downstream in separate outlets, respectively, with 10.9 % buffer and 0.3 % bead contamination. This device was further applied to a functionalized bead bioassay, achieving high-yield and continuous separation of 98 % of biotin-coated beads from 72.2 % of extra FITC-biotin. In a last study, we performed the extraction of 80 % of leukocytes from lysed blood, which validates our platform can be applied on living cells and used for various functions of cellular sample preparation

Modulating motility of intracellular vesicles in cortical neurons with nanomagnetic forces on-chip

Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat cortical neurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6-126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients

Shaped 3D microcarriers for adherent cell culture and analysis.

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting, which modulates protein expression and eliminates intercellular connections. To allow adherent culture and processing in flow, we present 3D-shaped hydrogel cell microcarriers, which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth, and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow, important for processing in flow and imaging flow cytometry. We designed a method to rapidly design, using the genetic algorithm, and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach. The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling, and is poised to enable adherent cell culture, in-flow analysis, and sorting in a single format

Micropillar sequence designs for fundamental inertial flow transformations

The ability to control the shape of a flow in a passive microfluidic device enables potential applications in chemical reaction control, particle separation, and complex material fabrication. Recent work has demonstrated the concept of sculpting fluid streams in a microchannel using a set of pillars or other structures that individually deform a flow in a predictable pre-computed manner. These individual pillars are then placed in a defined sequence within the channel to yield the composition of the individual flow deformations – and ultimately complex user-defined flow shapes. In this way, an elegant mathematical operation can yield the final flow shape for a sequence without an experiment or additional numerical simulation. Although these approaches allow for programming complex flow shapes without understanding the detailed fluid mechanics, the design of an arbitrary flow shape of interest remains difficult, requiring significant design iteration. The development of intuitive basic operations (i.e. higher-level functions that consist of combinations of obstacles) that act on the flow field to create a basis for more complex transformations would be useful in systematically achieving a desired flow shape. Here, we show eight transformations that could serve as a partial basis for more complex transformations. We initially used in-house, freely available custom software (uFlow), which allowed us to arrive at these transformations that include making a fluid stream concave and convex, tilting, stretching, splitting, adding a vertex, shifting, and encapsulating another flow stream. The pillar sequences corresponding to these transformations were subsequently fabricated and optically analyzed using confocal imaging – yielding close agreement with uFlow-predicted shapes. We performed topological analysis on each transformation, characterizing potential sequences leading to these outputs and trends associated with changing diameter and placement of the pillars. We classify operations into four sets of sequence-building concatenations: stacking, recursion, mirroring, and shaping. The developed basis should help in the design of microfluidic systems that have a phenomenal variety of applications, such as optofluidic lensing, enhanced heat transfer, or new polymer fiber design.This article is published as Stoecklein, Daniel, Chueh-Yu Wu, Keegan Owsley, Yu Xie, Dino Di Carlo, and Baskar Ganapathysubramanian. "Micropillar sequence designs for fundamental inertial flow transformations." Lab on a Chip 14, no. 21 (2014): 4197-4204. DOI:10.1039/C4LC00653D. Posted with permission.</p

Shaped 3D microcarriers for adherent cell culture and analysis

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting, which modulates protein expression and eliminates intercellular connections. To allow adherent culture and processing in flow, we present 3D-shaped hydrogel cell microcarriers, which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth, and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow, important for processing in flow and imaging flow cytometry. We designed a method to rapidly design, using the genetic algorithm, and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach. The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling, and is poised to enable adherent cell culture, in-flow analysis, and sorting in a single format.This article is published as Wu, Chueh-Yu, Daniel Stoecklein, Aditya Kommajosula, Jonathan Lin, Keegan Owsley, Baskar Ganapathysubramanian, and Dino Di Carlo. "Shaped 3D microcarriers for adherent cell culture and analysis." Microsystems & Nanoengineering 4, no. 1 (2018): 21. DOI: 10.1038/s41378-018-0020-7. Posted with permission.</p

Promoter Element Arising from the Fusion of Standard BioBrick Parts

We characterize the appearance of a constitutive promoter element in the commonly used cI repressor-encoding BioBrick BBa_C0051. We have termed this promoter element pKAT. Full pKAT activity is created by the ordered assembly of sequences in BBa_C0051 downstream of the <i>cI</i> gene encoding the 11 amino acid LVA proteolytic degradation tag, a BioBrick standard double-TAA stop codon, a genetic barcode, and part of the RFC10 SpeI-XbaI BioBrick scar. Placing BBa_C0051 or other pKAT containing parts upstream of other functional RNA coding elements in a polycistronic context may therefore lead to the unintended transcription of the downstream elements. The frequent reuse of pKAT or pKAT-like containing basic parts in the Registry of Biological Parts has resulted in approximately 5% of registry parts encoding at least one instance of a predicted pKAT promoter located directly upstream of a ribosome binding site and ATG start codon. This example highlights that even seemingly simple modifications of a part’s sequence (in this case addition of degradation tags and barcodes) may be sufficient to unexpectedly change the contextual behavior of a part and reaffirms the inherent challenge in carefully characterizing the behavior of standardized biological parts across a broad range of reasonable use scenarios