Wang, B. L. ; Ghaderi, A. ; Zhou, H. ; Agresti, J. ; Weitz, D. A. ; Fink, G. R. ; Stephanopoulos, G. Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nat Biotech 2014, 32, 473 - 478. Publisher's VersionAbstract

Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 104 cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. We also enriched L-lactate-producing Escherichia coli clones 5,800[times] from a population containing one L-lactate producer per 104D-lactate producers. Our approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals and pharmaceuticals.

Pessi, J. ; Santos, H. A. ; Miroshnyk, I. ; JoukoYliruusi, ; Weitz, D. A. ; Mirza, S. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery. International Journal of Pharmaceutics 2014, 472, 82 - 87. Publisher's VersionAbstract

Abstract In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water–oil–water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23–47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of \{BSA\} in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

Guo, M. ; Ehrlicher, A.  J. ; Jensen, M.  H. ; Renz, M. ; Moore, J.  R. ; Goldman, R.  D. ; Lippincott-Schwartz, J. ; Mackintosh, F.  C. ; Weitz, D.  A. Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy. Cell 2014, 158, 822 - 832. Publisher's Version 2014_cell_guo.pdf

doi: 10.1016/j.cell.2014.06.051

Datta, S. S. ; Abbaspourrad, A. ; Weitz, D. A. Expansion and rupture of charged microcapsules. Mater. Horiz. 2014, 1 92-95. Publisher's VersionAbstract

We study the deformations of pH-responsive spherical microcapsules - micrometer-scale liquid drops surrounded by thin{,} solid shells - under the influence of electrostatic forces. When exposed to a large concentration of NaOH{,} the microcapsules become highly charged{,} and expand isotropically. We find that the extent of this expansion can be understood by coupling electrostatics with shell theory; moreover{,} the expansion dynamics is well described by Darcy{'}s law for fluid flow through the microcapsule shell. Unexpectedly{,} however{,} below a threshold NaOH concentration{,} the microcapsules begin to disintegrate{,} and eventually rupture; they then expand non-uniformly{,} ultimately forming large{,} jellyfish-like structures. Our results highlight the fascinating range of behaviors exhibited by pH-responsive microcapsules{,} driven by the interplay between electrostatic and mechanical forces.

Carroll, N. J. ; Jensen, K. H. ; Parsa, S. ; Holbrook, M. N. ; Weitz, D. A. Measurement of Flow Velocity and Inference of Liquid Viscosity in a Microfluidic Channel by Fluorescence Photobleaching. Langmuir 2014, 30, 4868-4874. Publisher's Version [PDF]
Datta, S. S. ; Dupin, J. - B. ; Weitz, D. A. Fluid breakup during simultaneous two-phase flow through a three-dimensional porous medium. Physics of Fluids 2014, 26, n/a–n/a. Publisher's Version [PDF]
Chen, L. ; Li, Y. ; Fan, J. ; Bisoyi, H. K. ; Weitz, D. A. ; Li, Q. Photoresponsive Monodisperse Cholesteric Liquid Crystalline Microshells for Tunable Omnidirectional Lasing Enabled by a Visible Light-Driven Chiral Molecular Switch. Advanced Optical Materials 2014, n/a–n/a. Publisher's Version [PDF]
Arriaga, L. R. ; Datta, S. S. ; Kim, S. - H. ; Amstad, E. ; Kodger, T. E. ; Monroy, F. ; Weitz, D. A. Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation. Small 2014, 10, 950–956. Publisher's VersionAbstract

A microfluidic approach is reported for the high-throughput, continuous production of giant unilamellar vesicles (GUVs) using water-in-oil-in-water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.

Cohen, S. I. A. ; Rajah, L. ; Yoon, C. H. ; Buell, A. K. ; White, D. A. ; Sperling, R. A. ; Vendruscolo, M. ; Terentjev, E. M. ; Dobson, C. M. ; Weitz, D. A. ; et al. Spatial Propagation of Protein Polymerization. Physical Review Letters 2014, 112, 098101. Publisher's Version [PDF]
Jensen*, M.  H. ; Morris*, E.  J. ; Gallant, C.  M. ; Morgan, K.  G. ; Weitz, D.  A. ; Moore, J.  R. Mechanism of Calponin Stabilization of Cross-Linked Actin Networks. Biophysical Journal 2014, 106, 793 - 800. Publisher's Version [PDF]
[*Equal contribution]
Datta, S. S. ; Ramakrishnan, T. S. ; Weitz, D. A. Mobilization of a trapped non-wetting fluid from a three-dimensional porous medium. Physics of Fluids 2014, 26, 022002. Publisher's Version [PDF]
Comunian, T. A. *; Abbaspourrad*, A. ; Favaro-Trindade, C. S. ; Weitz, D. A. Fabrication of solid lipid microcapsules containing ascorbic acid using a microfluidic technique. Food Chemistry 2014, 152, 271 - 275. Publisher's Version [PDF]
[*Equal contribution]
Datta, S. S. *; Abbaspourrad*, A. ; Amstad, E. ; Fan, J. ; Kim, S. - H. ; Romanowsky, M. ; Shum, H. C. ; Sun, B. J. ; Utada, A. S. ; Windbergs, M. ; et al. Double Emulsion Templated Solid Microcapsules: Mechanics And Controlled Release. Advanced Materials 2014, n/a–n/a. Publisher's VersionAbstract
We describe how droplet microfluidics can be used to fabricate solid-shelled microcapsules having precisely controlled release behavior. Glass capillary devices enable the production of monodisperse double emulsion drops, which can then be used as templates for microcapsule formation. The exquisite control afforded by microfluidics can be used to tune the compositions and geometrical characteristics of the microcapsules with exceptional precision. We review the use of this approach to fabricate microcapsules that only release their contents when exposed to a specific stimulus – such as a change in temperature, exposure to light, a change in the chemical environment, or an external stress – only after a prescribed time delay, and at a prescribed rate.
[*Equal contribution]
Röding, M. ; Guo, M. ; Weitz, D. A. ; Rudemo, M. ; Särkkä, A. Identifying directional persistence in intracellular particle motion using Hidden Markov Models. Mathematical Biosciences 2014, 248, 140 - 145. Publisher's Version [PDF]
Herranz-Blanco, B. ; Arriaga, L. R. ; Makila, E. ; Correia, A. ; Shrestha, N. ; Mirza, S. ; Weitz, D. A. ; Salonen, J. ; Hirvonen, J. ; Santos, H. A. Microfluidic assembly of multistage porous silicon-lipid vesicles for controlled drug release. Lab on a Chip 2014, 14, 1083 - 1086. Publisher's VersionAbstract

A reliable microfluidic platform for the generation of stable and monodisperse multistage drug delivery systems is reported. A glass-capillary flow-focusing droplet generation device was used to encapsulate thermally hydrocarbonized porous silicon (PSi) microparticles into the aqueous cores of double emulsion drops, yielding the formation of a multistage PSi-lipid vesicle. This composite system enables a large loading capacity for hydrophobic drugs.

Zhang, W. ; Seminara, A. ; Suaris, M. ; Brenner, M. P. ; Weitz, D. A. ; Angelini, T. E. Nutrient depletion in Bacillus subtilis biofilms triggers matrix production. New Journal of Physics 2014, 16, 015028. Publisher's VersionAbstract

Many types of bacteria form colonies that grow into physically robust and strongly adhesiveaggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation.

Campas, O. ; Mammoto, T. ; Hasso, S. ; Sperling, R. A. ; O'Connell, D. ; Bischof, A. G. ; Maas, R. ; Weitz, D. A. ; Mahadevan, L. ; Ingber, D. E. Quantifying cell-generated mechanical forces within living embryonic tissues. Nat Meth 2014, 11, 183 - 189. Publisher's VersionAbstract

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN [mu]m-2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

Lee, M. ; Collins, J. W. ; Aubrecht, D. M. ; Sperling, R. A. ; Solomon, L. ; Ha, J. - W. ; Yi, G. - R. ; Weitz, D. A. ; Manoharan, V. N. Synchronized reinjection and coalescence of droplets in microfluidics. Lab on a Chip 2014, 14, 509-513.Abstract

Coalescence of two kinds of pre-processed droplets is necessary to perform chemical and biological assays in droplet-based microfluidics. However, a robust technique to accomplish this does not exist. Here we present a microfluidic device to synchronize the reinjection of two different kinds of droplets and coalesce them, using hydrostatic pressure in conjunction with a conventional syringe pump. We use a device consisting of two opposing T-junctions for reinjecting two kinds of droplets and control the flows of the droplets by applying gravity-driven hydrostatic pressure. The hydrostatic-pressure operation facilitates balancing the droplet reinjection rates and allows us to synchronize the reinjection. Furthermore, we present a simple but robust module to coalesce two droplets that sequentially come into the module, regardless of their arrival times. These re-injection and coalescence techniques might be used in lab-on-chip applications requiring droplets with controlled numbers of solid materials, which can be made by coalescing two pre-processed droplets that are formed and sorted in devices.

Amstad, E. ; Datta, S. S. ; Weitz, D. A. The microfluidic post-array device: High throughput production of single emulsion drops. Lab on a Chip 2014, 14, 705-709.Abstract

We present a microfluidic device that enables high throughput production of relatively monodisperse emulsion drops while controlling the average size. The device consists of a two-dimensional array of regularly-spaced posts. Large drops of a highly polydisperse crude emulsion are input into the device and are successively split by the posts, ultimately yielding a finer emulsion consisting of smaller, and much more monodisperse drops. The size distribution of the resultant emulsion depends only weakly on the viscosities of the input fluids and allows fluids of very high viscosities to be used. The average size and polydispersity of the drops depend strongly on the device geometry enabling both control and optimization. We use this device to produce drops of a highly viscous monomer solution and subsequently solidify them into polymeric microparticles. The production rate of these devices is similar to that achieved by membrane emulsification techniques, yet the control over the drop size is superior; thus these post-array microfluidic devices are potentially useful for industrial applications.

The role of vimentin intermediate filaments in cortical and cytoplasmic mechanics
Guo, M. ; Ehrlicher, A. J. ; Mahammad, S. ; Fabich, H. ; Jensen, M. H. ; Moore, J. R. ; Fredberg, J. J. ; Goldman, R. D. ; Weitz, D. A. The role of vimentin intermediate filaments in cortical and cytoplasmic mechanics. Biophysical journal 2013, 105, 1562–1568. [PDF]