Evaluation of Acoustophoretic and Dielectrophoretic Forces for Droplet Injection in Droplet-Based Microfluidic Devices.

Acoustophoretic forces have been successfully implemented into droplet-based microfluidic devices to manipulate droplets. These acoustophoretic forces in droplet microfluidic devices are typically generated as in acoustofluidic devices through transducer actuation of a piezoelectric substrate such as lithium niobate (LiNbO3), which is inherently accompanied by the emergence of electrical fields. Understanding acoustophoretic versus dielectrophoretic forces produced by electrodes and transducers within active microfluidic devices is important for the optimization of device performance during design iterations. In this case study, we design microfluidic devices with a droplet injection module and report an experimental strategy to deduce the respective contribution of the acoustophoretic versus dielectrophoretic forces for the observed droplet injection. Our PDMS-based devices comprise a standard oil-in-water droplet-generating module connected to a T-junction injection module featuring actuating electrodes. We use two different electrode geometries produced within the same PDMS slab as the droplet production/injection channels by filling low-melting-point metal alloy into channels that template the electrode geometries. When these electrodes are constructed on LiNbO3 as the substrate, they have a dual function as a piezoelectric transducer, which we call embedded liquid metal interdigitated transducers (elmIDTs). To decipher the contribution of acoustophoretic versus dielectrophoretic forces, we build the same devices on either piezoelectric LiNbO3 or nonpiezo active glass substrates with different combinations of physical device characteristics (i.e., elmIDT geometry and alignment) and operate in a range of phase spaces (i.e., frequency, voltage, and transducer polarity). We characterize devices using techniques such as laser Doppler vibrometry (LDV) and infrared imaging, along with evaluating droplet injection for our series of device designs, constructions, and operating parameters. Although we find that LiNbO3 device designs generate acoustic fields, we demonstrate that droplet injection occurs only due to dielectrophoretic forces. We deduce that droplet injection is caused by the coupled dielectrophoretic forces arising from the operation of elmIDTs rather than by acoustophoretic forces for this specific device design. We arrive at this conclusion because equivalent droplet injection occurs without the presence of an acoustic field using the same electrode designs on nonpiezo active glass substrate devices. This work establishes a methodology to pinpoint the major contributing force of droplet manipulation in droplet-based acoustomicrofluidics.

Implementing standardized diversity, equity, and inclusion approaches within scientific communities.

Within the global scientific community, there are disparate approaches to diversity, equity, and inclusion (DEI) practices, leading to inequalities that hinder progress. Here, we frame this problem through historical perspectives in the global north and propose a DEI framework adaptable by institutions regardless of location, improving the academic environment for researchers globally.

Creating inclusive environments in cell biology by casual mentoring.

Informal mentoring affects the development of cell biologists by providing essential career, scientific, and educational guidance to mentees. In this piece, we discuss the importance of formally recognizing casual mentorship to encourage this crucial form of mentorship that contributes to the advancement of an inclusive cell biology community.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Synthesis and Assembly of Virtual Collaborations.

The interplay between academics and society within the environment of the COVID-19 pandemic has impacted on scientists across the world, prompting reevaluation of how virtual toolboxes can be used to support responsible collaborative research practices. We provide awareness of virtual resources and activities that enable scientific discovery using safe and efficient practices.

Centrifugal Generation of Droplet-Based 3D Cell Cultures.

Quickly and easily producing uniform populations of microsphere-based 3D cell cultures using droplet-based templating methods has the potential to enable widespread use of such platforms in drug discovery or cancer research. Here, we advance the design of centrifuge-based droplet generation devices, describe the use of this platform for droplet generation with controlled cell occupancy, and demonstrate weeklong culture duration. Using simple-to-construct devices and easily implemented protocols, the initial concentration of encapsulated cells is adjustable up to hundreds of cells per microsphere. This work demonstrates the first instance of using centrifugal droplet-generating devices to produce large numbers of cell-encapsulating microspheres. Applications of this versatile methodology include the rapid formation of templated 3D cell culture populations suitable for suspension culture or large batch bioreactor studies that require uniform populations.

Oil-Free Acoustofluidic Droplet Generation for Multicellular Tumor Spheroid Culture.

We present an easy-to-assemble microfluidic system for synthesizing cell-loaded dextran/alginate (DEX/ALG) hydrogel spheres using an aqueous two-phase system (ATPS) for templated fabrication of multicellular tumor spheroids (MTSs). An audio speaker driven by an amplified output of a waveform generator or smartphone provides acoustic modulation to drive the breakup of an ATPS into MTS template droplets within microcapillary fluidic devices. We apply extensions of Plateau-Rayleigh theory to help define the flow and frequency parameter space necessary for acoustofluidic ATPS droplet formation in these devices. This method provides a simple droplet microfluidic approach using off-the-shelf acoustic components for quickly initiating MTSs and subsequent 3D cell culture.

Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells.

Poly(N-isopropyl acrylamide) (pNIPAM) is a "smart" polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinning parameters were varied (needle gauge, collection time, and molecular weight of the polymer) to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.