A MICROFLUIDICS PLATFORM FOR WOUNDING AND REGENERATION STUDIES OF SINGLE CELLS L.C. Gerber1, M. Slabodnick2, W.F. Marshall2, and S.K.Y. Tang1*

1Department of Mechanical Engineering, Stanford University, CA 94305,USA and 2Biochemistry & Biophysics, UC San Francisco, CA 94143, USA.

ABSTRACT

In this study, we explore the use of microfluidic devices for reproducible wounding and dissection of Stentor coeruleus, a canonical single-cell regeneration model organism. Stentor coeruleus is a large unicellular ciliate protozoan with a size of about 500 µm. It possesses the striking ability to heal wounds and to regenerate into multiple fully operational organisms after being dissected into small fragments – as small as 1/100th of the initial cell size – within 24 hours (Figure 1).1 KEYWORDS: Stentor coeruleus, dissection, wounding, healing, regeneration

INTRODUCTION

The overall aim of this study is to understand how cells heal wounds, how robust the healing process is, and how the wounds affect the cell’s metabolism. Wound healing is a fundamental biological process for maintaining homeostasis and, ultimately, survival.2 While wound healing at the tissue level is well understood,3 the mechanism by which individual cells can heal wounds within themselves remains relatively unknown.4 Many cells, such as cardiac myocytes or skeletal muscle cells, are regularly wounded under normal physiological conditions, such as during stretching and contracting cycles. As these cells cannot be replaced easily, self-healing is the only approach to restore normal functions.5 The inability of a wound to heal can lead to serious diseases, such as muscular dystrophies, respiratory, and heart failures.6

Figure 1: Historic Stentor drawing. A single-celled Stentor is cut into three parts, which will then re-store themselves and gradually become complete Stentor over time. Adapted from “The problem of age, growth, and death,” C.S Minot, Pop. Sci. Mon., 71 (1907), 364.

THEORY

Conventional single cell wounding experiments using Stentor Coeruleus as a model are performed manually using glass needles under a stereoscope. This process requires high dexterity and is time-consuming. Here, we show that the use of microfluidic techniques allows wounding a large number of cells reproducibly. By using a droplet-based system, we were also able to encapsulate individual cells and cell fragments into microliter-droplets. This ability allows us to control the cellular microenvironment and to track the individual regeneration processes.

978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 706 18th International Conference on MiniaturizedSystems for Chemistry and Life Sciences

October 26-30, 2014, San Antonio, Texas, USA

EXPERIMENTAL Figure 2 shows a microfluidic channel possessing a Y-junction passive splitter fabricated in

polydimethylsiloxane (PDMS) with a height of 150 µm and an upstream width of 300 µm. Stentor cells suspended in growth media were introduced into this channel. At the Y-junction, the cells split into two fragments, and were subsequently separated into two narrow downstream channels. Current work in progress is to quantify the effect of shear on the quality of the dissection and to investigate on the ability of the cells to heal and regenerate.

Figure 2: a) Schematic drawing of a static knife. b) Device molded in PDMS with a single inlet tube and two outlet tubes. c) Stentor cell flowing from left to right, just before hitting the Y-splitter. d) The cell is pressed through the constriction and forced to split into two individual parts. e) Dissected Stentor cell after 20 minutes and f) fully regenerated after 22 hours.

RESULTS AND DISCUSSION

Figure 3 shows that it is possible to encapsulate a cell into a micro-droplet using a flow-focusing device. Cells survived for at least four days after encapsulation in drops. Current work is in progress to demonstrate the dissection of cell within the droplet, which also contains metabolic indicators to measure the metabolism of the cell after dissection. The capability to generate and investigate a large number of drops in a high throughput manner will enable novel metabolism measurements and statistical analyses at the single-cell level not possible with existing tools.7 This work will be an important step towards understanding wound healing and regeneration processes at the single cell level.

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Figure 3: a) Encapsulation of Stentor in drops. The dispersed phase (flow rate: 1 ml/h) is stand-ard Stentor growth medium; the continuous phase (flow rate: 1 ml/h) is HFE with 2% RAS (a bi-ocompatible surfactant). b) The drops are stable for several days and the Stentor remain alive and are able to regenerate and divide inside a single droplet.

CONCLUSION

Microfluidic techniques allow to bisect and wound Stentor cells in a high-throughput manner. PDMS is an optimal material, as it is non-toxic to cells and proteins, transparent, compatible with water and most polar organic solvents, and permeable to oxygen and CO2.

ACKNOWLEDGEMENTS

We acknowledge support from the Stanford Center for Integrated Systems, the 3M Non-tenured Fac-ulty Award, and the Swiss National Science Foundation. REFERENCES [1] F. E. Lillie, "On the Smallest Parts of Stentor Capable of Regeneration: A Contribution on the

Limits of Divisibility of Living Matter", 1896. [2] K. J. Sonnemann, and W. M. Bement, "Wound repair: toward understanding and integration of

single-cell and multicellular wound responses", Annual review of cell and developmental biology, 27, 2011.

[3] S. Guo, and L. A. DiPietro, "Factors affecting wound healing", Journal of dental research, 89, 3, 2010.

[4] A. Puliafito, L. Hufnagel, P. Neveu, S. Streichan, A. Sigal, D. K. Fygenson, and B. I. Shraiman, "Collective and single cell behavior in epithelial contact inhibition", Proceedings of the National Academy of Sciences, 109, 3, 2012.

[5] D. L. MacIntyre, W. D. Reid, and D. C. McKenzie, "Delayed muscle soreness", Sports Medicine, 20, 1, 1995.

[6] G. C. Gurtner, S. Werner, Y. Barrandon, and M. T. Longaker, "Wound repair and regeneration", Nature, 453, 7193, 2008.

[7] Y. Chen, A. W. Gani, and S. K. Tang, "Characterization of sensitivity and specificity in leaky droplet-based assays", Lab on a Chip, 12, 23, 2012.

CONTACT * S.K.Y. Tang; phone: +1 650-723-5385; sindy@stanford.edu

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