Automated hydrodynamic trap for single cell analysis in free solution

Johnson Chavarria, Eric

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https://hdl.handle.net/2142/49482 Copy

Description

Title

Automated hydrodynamic trap for single cell analysis in free solution

Author(s)

Johnson Chavarria, Eric

Issue Date

2014-05-30T16:46:25Z

Director of Research (if dissertation) or Advisor (if thesis)

Schroeder, Charles M.

Doctoral Committee Chair(s)

Schroeder, Charles M.

Committee Member(s)

• Myong, Su-A
• Kuhlman, Thomas E.
• Tajkhorshid, Emad
• Selvin, Paul R.

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Computnl Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• hydrodynamic trap
• single cell
• microfluidics
• planar extensional flow
• cell sorting

Abstract

Over the last several years, microfluidic-based techniques have been developed to study inducible gene expression at the single cell level, albeit without the ability to control external stimuli with precise methods. Most are limited by long diffusive timescales to alternate environmental concentrations. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. Recent studies have reported the ability of biological systems to implement lowpass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals. This cellular adaptation is critical for survival in fluctuating environmental conditions, yet we still lack a complete understanding of this phenomenon. The hydrodynamic trap was developed as a step forward in elucidate single cells in changing environmental conditions. To become the single cell microbioreactor (SCM), our trapping technique went through four generations. Our first generation was directed at sequencing organisms that were uncultivated. Rumen fauna from cattle, sheep, and reindeer consist of a heterogeneous cell mixture including Oscillospira spp. These bacterium have distinct septation patterns and form spores. In particular, O. guilliermondii has been observed in these rumen sample for almost a century yet we are still unable to cultivate them within the lab. Our hydrodynamic trapping technique was able to isolate and sort up to 50 O. guilliermondii cells from a heterogeneous environmental rumen sample. Our proof-of-principle demonstration produced ≻80 % sample purity. Our second generation was aimed at live cell growth for defining our trapping technique as a non perturbative method. Many other methods of confinement produce perturbations through optical, magnetic, acoustic, or electrokinetic fields. Our technique utilizes the sole action of fluid flow for confining target cells. We observed Escherichia coli cells run and tumble within our trap. In addition, we observed increasing growth rates over 5 generations. Our results demonstrate that cells beneficially adapt to the trap environment. This non-perturbative nature of our trap is useful for observing cells over extended periods of time. Finally, our third and final generation was aimed at elucidating gene expression under oscillating nutrient conditions. We observed gene expression under periodic concentrations of a lactose mimic. We also, performed diffusion experiments of TetR:EYFP on a chromosomal binding array by rapidly switching to concentrations of aTc. Using the SCM, we are able to observe the rapid release and subsequent intracellular diffusion of the Tet repressor within the nucleoid region of Gram-negative bacteria. Overall, this microfluidic bioreactor provides a direct method for sustaining periodic environmental conditions, measuring growth rates, detecting gene expression, and observing intracellular diffusion within single cells suspended at a stagnation point. Over the last several years, microfluidic-based techniques have been developed to study inducible gene expression at the single cell level, albeit without the ability to control external stimuli with precise methods. Most are limited by long diffusive timescales to alternate environmental concentrations. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. Recent studies have reported the ability of biological systems to implement lowpass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals. This cellular adaptation is critical for survival in fluctuating environmental conditions, yet we still lack a complete understanding of this phenomenon. The hydrodynamic trap was developed as a step forward in elucidate single cells in changing environmental conditions. To become the single cell microbioreactor (SCM), our trapping technique went through four generations. Our first generation was directed at sequencing organisms that were uncultivated. Rumen fauna from cattle, sheep, and reindeer consist of a heterogeneous cell mixture including Oscillospira spp. These bacterium have distinct septation patterns and form spores. In particular, O. guilliermondii has been observed in these rumen sample for almost a century yet we are still unable to cultivate them within the lab. Our hydrodynamic trapping technique was able to isolate and sort up to 50 O. guilliermondii cells from a heterogeneous environmental rumen sample. Our proof-of-principle demonstration produced ≻80 % sample purity. Our second generation was aimed at live cell growth for defining our trapping technique as a non perturbative method. Many other methods of confinement produce perturbations through optical, magnetic, acoustic, or electrokinetic fields. Our technique utilizes the sole action of fluid flow for confining target cells. We observed Escherichia coli cells run and tumble within our trap. In addition, we observed increasing growth rates over 5 generations. Our results demonstrate that cells beneficially adapt to the trap environment. This non-perturbative nature of our trap is useful for observing cells over extended periods of time. Finally, our third and final generation was aimed at elucidating gene expression under oscillating nutrient conditions. We observed gene expression under periodic concentrations of a lactose mimic. We also, performed diffusion experiments of TetR:EYFP on a chromosomal binding array by rapidly switching to concentrations of aTc. Using the SCM, we are able to observe the rapid release and subsequent intracellular diffusion of the Tet repressor within the nucleoid region of Gram-negative bacteria. Overall, this microfluidic bioreactor provides a direct method for sustaining periodic environmental conditions, measuring growth rates, detecting gene expression, and observing intracellular diffusion within single cells suspended at a stagnation point.

Graduation Semester

2014-05

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http://hdl.handle.net/2142/49482

Copyright and License Information

Copyright 2014 Eric Johnson Chavarria. Reprint permission from Applied Physics Letters.

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