BIOE 301D: Hands-on microfluidics laboratory

Microfluidic devices developed for biomedical research in academic labs often fall short of their intended biological and medical potential due to a persistent technology transfer gap between the engineers who design them and the biologists who could benefit most from their use. To address this, we have developed a new inquiry-based approach for graduate bioengineering education that closes this technology transfer gap by leveraging graduate student talent and creativity to meet the unmet needs of biological laboratories. Unlike many traditional fabrication laboratories in which devices created by students are discarded at the end of the course, this approach directs student efforts towards designing and fabricating devices desperately needed by collaborating bioscience laboratories. In the process, student experience first-hand the unexpected challenges inherent to cutting-edge research and learn to think creatively to solve them.

YEAR 2: WINTER 2017-2018

Teaching Team: Polly Fordyce, Siavash Ahrar, Yuan (soso) Xue, Alec Tarashansky

Project #1: A planarian guillotine

Student TEAM: Bauer LeSavage, Jack Silberstein, Jon Calles, and Suhas Rao

CollaboratoRS: Sam Bray and Alec Tarashansky in bo Wang’s lab

The planarian flatworm S. mediterranea is a unique model of development that can regenerate entire organisms from fragments as small as 1/279th of the original host. This extreme regenerative capacity is thought to result from the planarian’s high stem cell content (up to 30% of total cells). Chimeric worms created by fusing fragments from different worm species provide a powerful tool to study the collective action and coordination of these stem cells in regenerating an entire organism. This competition is best studied by bisecting worms along their longitudinal axis and fusing halves from opposite strains together; however, this requires the ability to consistently and efficiently bisect worms with a throughput and precision not possible via current methods. To address this, we developed a microfluidic guillotine to quickly and consistently bisect planaria and recover the halves for further regeneration studies. Our device consists of an 18 mm long, 175 µm tall, and 700-900 µm wide channel ending in a sharp PDMS blade (6 degree angle) such that worms are flowed down the channel, collide with the blade and are bisected into two pieces, with each half flowing out of its own outlet channel. We demonstrated that individual worms could be bisected and that bisected worms were viable and capable of future regeneration.

  Figure 1.  Schematic showing microfluidic planarian guillotines with varying channel widths after the guillotine blade.

Figure 1. Schematic showing microfluidic planarian guillotines with varying channel widths after the guillotine blade.

Video 1. High speed video showing bisection of a single planarian using the microfluidic guillotine.

Project #2: a device for long-term culture of C. elegans

Student TEAM: Prashanth Srinivasan, Pengyang Li, Spencer Cesar

Collaborator: Lauren Booth from Anne Brunet’s lab

The free-living nematode Caenorhabditis elegans is a model organism for the study of changes during aging. Recently, several studies have reported that pheromones secreted by male worms cause reduced lifespan and accelerated aging in hermaphrodite (her ) worms. However, the identity of these pheromones and their mechanisms of lifespan reduction are unknown. Current methods for studying sexual interactions in C. elegans are laborious, time-intensive, and error-prone because they require manual separation of sexes and removal of progeny. Moreover, the low throughput of manual worm separation precludes the study of context-dependent pheromone secretions. A previous attempt to maintain sex separation using a microfluidic device relied on failure-prone screw valves and a single-channel loading strategy susceptible to cross-contamination of the sexes. Here we report the design, fabrication, and initial testing of a valve-free microfluidic device capable of maintaining male and her worm populations in chemical contact but physical isolation for probing the molecular mechanisms of pheromone-mediated lifespan reduction. The final device should also satisfy following criteria: able to keep 10-20 worms for 21 days (each sex); eggs laid can be removed periodically; worms in the device can be fed with E.coli ; and transparent enough to monitor and image the worms.

  Figure 1 . Device design schematic.

Figure 1. Device design schematic.

Figure 2. Hermaphrodite and male C. elegans worms swimming in microfluidic device.

Project #3: a valved device for co-culture of macrophage and bacteria

Student Team: Peipei Lyu, Andres Aranda-Diaz, Punnag Padhy, and Thomas Lozanoski

Collaborator: Keara Lane from Markus Covert’s lab

When a macrophage becomes infected with or phagocytoses a bacterium, (e.g. E. coli or S. typhimurium), the infected cell engages in a coordinated inflammatory program to destroy the pathogen; however, the response is not always successful, and sometimes the pathogens evade the immune system, leading to severe infections. Understanding the mechanisms that govern immunosuppression will help medical scientists develop effective vaccines and treatments for problematic infections. One hypothesis for immunosuppression is that the macrophages that phagocytose the bacterium are inhibited from relaying inflammatory signals to their neighboring immune cells. Current methods for studying bacterial infection of mammalian cells with live-cell imaging do not enable the response to infection to be separated from responses due to paracrine signaling. For example, conditioned media only captures signaling at one time point and does not lend itself to studying feedback between infected and non-infected cells. To address these issues, we microfabricated a device capable of spatially and temporally separating different cell populations so that the infection process can be imaged and the effects of its downstream signaling can be measured.

Movie 1. Pneumatic valve actuation.

  Figure 1.  Fluorescence image showing macrophages (green) and  E. coli  (red) within a chamber of the microfluidic device.

Figure 1. Fluorescence image showing macrophages (green) and E. coli (red) within a chamber of the microfluidic device.

Project #4: a microfluidic droplet generator for testing polymer hydrogels

Student Team: An Ju, Loza Tadesse, Xinzi Wang

Collaborator: XINMING TONG from Fan yang’s lab

Hydrogel microspheres have been shown to enable encapsulation of cells and other biomolecules and reduce shear damage specially for injecting to an organism. Here, we investigated pre-encapsulation of mesenchymal stem cells in hydrogel microspheres using a microfluidic platform. We fabricated and tested two droplet forming designs (T-junction and flow-focusing), investigated hydrogel types including their polymerization parameter, and tested cell loading behavior of the droplets.

  Figure 1.  Microfluidic droplet generator connected to syringe pumps on microscope illumination stage.

Figure 1. Microfluidic droplet generator connected to syringe pumps on microscope illumination stage.

  Figure 2.  Droplets produced via T-junction (top) and flow focusing (bottom) devices at different flow rates.

Figure 2. Droplets produced via T-junction (top) and flow focusing (bottom) devices at different flow rates.

Year 1: Winter 2016-2017

Teaching Team: Polly Fordyce, Kara Brower, & Diego Oyarzun

PROJECT #1: a microfluidic device for studying toxoplasma gondii

Student TEAM: Sam Bray, cooper galvin, ali hemmatifar, deze kong, and tim schnabel

CollaboratorS: terence theisen and ian foe from john boothroyd and matt bogyo’s labs

Extravillous trophoblasts (EVT) grow out from the fetal placenta and invade the maternal uterus. While the establishment of EVT cells in the uterus is a vital stage in establishing pregnancy, the rarity of these cells has made them difficult to study. Furthermore, some EVTs differentiate into extremely large (>100 ɥm) and polyploid (>100 N) cells and we know little to nothing about the functions of these unique cells. To address this issue, we attempted to construct a microfluidic device with physical barriers for sorting and capture of large EVTs. We have engineered a single-layer microfluidic device to capture cells using physical separators at defined widths (90, 75, and 50 um). Using reverse flow, we selectively captured objects of defined diameters from individual chambers. This device will allow for the isolation of large EVTs for subsequent downstream analysis and functional studies (RNAseq, proteomics, motility studies).

  Figure 1 . Shear stress modeling results from COMSOL and particle image velocimetry.

Figure 1. Shear stress modeling results from COMSOL and particle image velocimetry.

  Figure 2.  Example images showing overnight growth of cells.

Figure 2. Example images showing overnight growth of cells.

TEAM #2: Placentaur: a device for isolating very large cells from a cell mixture

Student Team: Alexander Tarashansky, Terence Theisen, Shreya Deshmukh, Nelson Hall, and Yuan Xue

Collaborator: Elisa Zhang from Julie Baker’s lab

Extravillous trophoblasts (EVT) grow out from the fetal placenta and invade the maternal uterus. While the establishment of EVT cells in the uterus is a vital stage in establishing pregnancy, the rarity of these cells has made them difficult to study. Furthermore, some EVTs differentiate into extremely large (>100 ɥm) and polyploid (>100 N) cells and we know little to nothing about the functions of these unique cells. To address this issue, we attempted to construct a microfluidic device with physical barriers for sorting and capture of large EVTs. We have engineered a single-layer microfluidic device to capture cells using physical separators at defined widths (90, 75, and 50 um). Using reverse flow, we selectively captured objects of defined diameters from individual chambers. This device will allow for the isolation of large EVTs for subsequent downstream analysis and functional studies (RNAseq, proteomics, motility studies).

  FIgure 1.  Device schematic. (A) Each separation chamber has different pillar spacings to capture cells of different sizes. (B) Integrated valves allow selective retrieval of particular cell sizes.

FIgure 1. Device schematic. (A) Each separation chamber has different pillar spacings to capture cells of different sizes. (B) Integrated valves allow selective retrieval of particular cell sizes.

  Figure 2.  Image of the microfluidic device containing pillars with 50 µm gaps between them and 50 µm diameter beads successfully captured under constant pressure flow.

Figure 2. Image of the microfluidic device containing pillars with 50 µm gaps between them and 50 µm diameter beads successfully captured under constant pressure flow.

PROJECT #3: Traptasia: Devices to capture and study the cnidarian apitasia

Student Team: salil bhate, daniel hunt, louai labanieh, sarah lensch, and will van treuren

Collaborators: Cawa Tran and Heather Cartwright from John Pringle’s lab and the Carnegie Institute

Coral reefs are some of the most productive ecosystems on earth. Driving this productivity is a symbiosis between corals (phylum Cnidaria) and dinoflagellates (predominantly in the genus Symbiodinium) where the coral provides inorganic nutrients and the algae provides glucose or other fixed carbon. Coral bleaching, the phenomena where coral eject their symbionts when stressed, is a major concern for coral reefs around the globe. Unfortunately, coral reefs are currently threatened by systemic anthropogenic stressors, primarily ocean warming and acidification. Understanding the mechanics of coral bleaching is essential to responding to this threat. Here, we develop a microfluidic device that enables much more statistically rigorous quantification of coral biology in a model system, increasing the length of viable experiments by a factor of 3 and the number of replicates by a factor of 50.

  Figure 1.  Image showing array of traps with  Aiptasia  larvae within them.

Figure 1. Image showing array of traps with Aiptasia larvae within them.

Movie 1. Trapped Aiptasia larvae expelling symbiotic algae under stress.

This project was continued by some of the team members after the course finished, leading to a manuscript currently under review.

Please see the full bioRXiv post here!