Microfluidic devices for the rapid and automated processing of sample populations

Microfluidic devices for the rapid and automated processing of sample populations are provided. Described are multiplexer microfluidic devices configured to serially deliver a plurality of distinct sample populations to a sample processing element rapidly and automatically, without cross-contaminating the distinct sample populations. Also provided are microfluidic sample processing elements that can be used to rapidly and automatically manipulate and/or interrogate members of a sample population. The microfluidic devices can be used to improve the throughput and quality of experiments involving model organisms, such as C. elegans.Board of Regents, University of Texas Syste

An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells

Automated biosorter platforms, including recently developed microfluidic devices, enable and accelerate high-throughput and/or high-resolution bioassays on small animal models. However, time-consuming delivery of different organism populations to these systems introduces a major bottleneck to executing large-scale screens. Current population delivery strategies rely on suction from conventional well plates through tubing periodically exposed to air, leading to certain disadvantages: 1) bubble introduction to the sample, interfering with analysis in the downstream system, 2) substantial time drain from added bubble-cleaning steps, and 3) the need for complex mechanical systems to manipulate well plate position. To address these concerns, we developed a multiwell-format microfluidic platform that can deliver multiple distinct animal populations from on-chip wells using multiplexed valve control. This Population Delivery Chip could operate autonomously as part of a relatively simple setup that did not require any of the major mechanical moving parts typical of plate-handling systems to address a given well. We demonstrated automatic serial delivery of 16 distinct C. elegans worm populations to a single outlet without introducing any bubbles to the samples, causing cross-contamination, or damaging the animals. The device achieved delivery of more than 90% of the population preloaded into a given well in 4.7 seconds; an order of magnitude faster than delivery modalities in current use. This platform could potentially handle other similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. The device’s architecture and microchannel dimensions allow simple expansion for processing larger numbers of populations.The authors would like to thank the National Institutes of Health (www.nih.gov) for its generous support of this research. Specifically, the grants that made this work possible are the NIH Director's Transformative Award (NIH R01 AG041135), NIH R21 NS067340, and NIH R01 NS060129. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biomedical EngineeringElectrical and Computer EngineeringMechanical Engineerin

Automated microfluidic platforms to facilitate nerve degeneration studies with C. elegans

With its well-characterized genome, simple anatomy, and vast array of uses in molecular biology, the roundworm, Caenorhabditis elegans (C. elegans) is a well-established model organism in neurobiology. Concurrently, neurodegenerative diseases are some the most devastating and least understood ailments in modern medicine, making high-throughput approaches to understand their fundamental mechanisms imperative to developing new therapies. The worm's physical length-scales and simple genetics make it an ideal in vivo tool for high-throughput screening platforms. Concurrently, microfluidic technology has been used to make devices that manipulate these animals in a multitude of fashions to study various biological phenomena. With these considerations in mind, we have developed microfluidic platforms to facilitate optical interrogation of neurodegenerative and neuroregenerative phenomena in C. elegans for large-scale screens. First we developed a multiwell format device with 16 on-chip reservoirs to house and quickly deliver distinct worm populations to any liquid-format imaging platform. The system achieved unprecedented delivery speeds, avoided any population cross-contamination, and maintained animal viability. We then expanded this platform into a 64-well device that acted as a modular plug and play system for simple manipulation by conventional high-throughput liquid handling systems. The chip could be manipulated in the same fashion as a multiwell plate and interfaced with a novel pneumatic gasket system to achieve delivery speeds that were two-fold faster than those attained on the 16-well device. In addition, we worked to develop potential optical interrogation platform that could be fed populations of worms by the aforementioned delivery systems. This microfluidic chip consisted of an array of parallel traps to house individual worms over long durations for time-lapse studies of nerve regeneration after cuts to single axons mediated by a femtosecond pulse laser. Specifically, the platform was designed for regeneration studies in the C. elegans PQR neuron.Biomedical Engineerin

Parallelized microfluidic devices for high-throughput nerve regeneration studies in Caenorhabditis elegans

textThe nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut.Biomedical Engineerin

A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans

Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only

Automated worm population delivery sequence.

<p>A) Schematic of the device showing areas active during the sequence example as the worms are pre-staged at the first set of control valves. An image of pre-staged <i>C</i>. <i>elegans</i> worms is below the schematic (scale bar is 1 mm). B) Illustration of all steps for one full sequence cycle. Step 1: Appropriate valves open as the gasket is pressurized to send <i>Well</i> 1’s population to the main channel, where <i>Main </i><i>Channel </i><i>Flush</i> then accelerates the worms’ transport to the main exit. Step 2: Excess worms are cleared from the main channel towards the <i>Main </i><i>Outlet</i> via flow from <i>Main </i><i>Channel </i><i>Flush</i>. Step 3: Flow from <i>Exit </i><i>Flush</i> delivers the worms from the <i>Main </i><i>Outlet</i> to an off-chip location. Step 4: “Flushback”; <i>Exit </i><i>Flush</i> flow is redirected backwards to clear any remaining worms in the well channel back to <i>Well 1</i>. This step is executed on <i>Wells 1-4</i> only after finishing Steps 1-3 on each of them. C) Timings for each step.</p

Population mixing eliminated during automated delivery at 20 psi (~138 kPa).

<p>The graphs show the fraction of animals collected after delivery from a given well that are of the same strain initially loaded into the well. The actual average number of collected worms over the average number of those initially loaded is indicated above each bar. A) Four distinct strains loaded in each <b>row</b>. B) Four distinct strains loaded in each <b>column</b>. A corresponding color-coded schematic on the right of both graphs indicates into which wells the strains were loaded at the beginning of both experiments. Each color represents a single type of strain.</p

A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans.

Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner

<i>Population Delivery Chip</i> design.

<p>A) A schematic of the device indicating the flow layer (blue) and control valve layer (red). There are 16 on-chip wells arranged in a 96-well plate format for initial loading of different worm populations. Columns and wells of the array are numbered according to order of delivery. Valves <i>V1-V8</i> are multiplexer control valves and <i>V9-V12</i> control flow in the main channel. B) An image of the device with its microfluidic channels loaded with food coloring dye, showing the flow layer (green) and control valve layer (orange) (scale bar ~1mm). C) A macro-scale view of the device with the 16-well array indicated by the yellow dashed lines and a schematic of worms loaded into one of the conical wells. D) A macro-scale view of the entire chip/gasket system with pressurized input lines in the experimental setup.</p

Worm population delivery as a function of applied pressure.

<p>The fraction of worm populations loaded in 4 representative on-chip wells from 4 different columns of the <i>Population </i><i>Delivery </i><i>Chip</i> that are delivered to the outlet of the device as a function of pressure applied at the gasket and the <i>Main </i><i>Channel </i><i>Flush</i>.</p