Name: a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level
Uploaded: 2017-01-10T15:00:00
Description: Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation Bubblesâ€“Cell Interaction and the Resultant Bioeffects at the Single-cell Lev

We would like to acknowledge the use of the clean room facility SMIF at Duke University. We also want to thank Hao Qiang for his assistance in measuring the jet velocity. The authors thank Todd Rumbaugh of Hadland Imaging for providing the Shimadzu HPV-X camera used in this study.The work was funded in part by NIH through grants 5R03EB017886-02 and 4R37DK052985-20.

1. Microfabrication
NOTE: All the microfabrication procedures are performed in a cleanroom. A chrome mask is designed prior to the microfabrication, see Figure 2.
<img alt="Figure 2" src="/files/ftp_upload/55106/55106fig2.jpg" /> 
Figure 2: Schematic of the channel design in the microfluidic chip and the dimensions of the working units. a) Mask design of the aligned PDMS microchannels (green) and ...

<ol>
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Single-cell analysis, in combination with live-cell imaging, has greatly enhanced our understanding of the dynamic and often variable processes in individual cells, such as phenotype development and immune response23. In contrast to the conventional cell culture in dishes or flasks, microfluidic systems enable precise control of the microenvironment, down to the single-cell level, in real time. Consequently, advances in microfluidic technology and techniques have largely improved the throughput and reproducibi...

There is a growing recognition that cellular heterogeneity, arising from the stochastic expression of genes, proteins, and metabolites, exists within a large cell population and serves as a fundamental principle in biology to allow for cell adaptation and evolution1. Therefore, it is often inaccurate and unreliable to use population-based bulk measurements to understand the function of individual cells and their interactions. Developing new technologies for single-cell analysis is therefore of high interest in biological and pharmacological research, and can be used, for example, to better understand the key signaling pathways and processes in stem cell biology and cancer therapy2-4. In recent years, the emergence of microfluidic platforms has greatly facilitated single-cell analysis, where the positioning, treatment, and observation of the response from individual cells have been performed with novel analytical strategies5.
Cavitation plays an important role in a diverse range of biomedical applications, including the treatment of cancers by high-intensity focused ultrasound (HIFU)6, the non-invasive fragmentation of kidney stones by shock wave lithotripsy (SWL)7, drug or gene delivery by sonoporation8, and the recently reported destruction of cells or tissues by hydrodynamic bubble cavitation9,10. Despite this, the dynamic processes of cavitation bubble(s) interactions with biological tissue and cells have not been well understood. This is due to the randomness in cavitation initiation and bubble dynamics produced by ultrasound, shock waves, and local hydraulic pressure; furthermore, there is a lack of enabling techniques to resolve the inherently complex and fast responses of biological cells, especially at the single-cell level.
Because of these challenges, it is not surprising that very few studies have been reported to investigate bubble-cell interactions under well-controlled experimental conditions. For example, membrane poration of individual cells trapped in suspension11 and the impulsive large deformation of human red blood cells12 have been demonstrated using laser-generated single bubbles in microfluidic channels. The latter technique, however, can only produce very small deformation in eukaryotic cells because of the presence of the nucleus13. Moreover, it is difficult to monitor downstream bioeffects when treating cells in suspension. In other studies, ultrasound excitation of a cell-bound microbubble (or ultrasound contrast agent) for producing membrane poration and/or intracellular calcium responses in single adherent cells has been reported8. Membrane poration of single adherent cells can also be produced by using laser-generated tandem bubbles in a thin liquid layer containing light-absorbing Trypan blue solution14, or by an oscillating gas bubble generated by microsecond laser pulses irradiating through an optically absorbing substrate in microchambers15. When compared, the optically absorbing substrate has an advantage over the laser-absorbing Trypan blue solution because the latter is toxic to cells. More importantly, laser-generated bubbles are more controllable in terms of bubble size and location than acoustically excited bubbles. Nevertheless, in all these previous studies, the cell shape, orientation, and adhesion conditions were not controlled, which may substantially influence the cell response and bioeffects produced by mechanical stresses16.
To overcome these drawbacks in previous studies, we have recently developed an experimental system for bubble generation, cell patterning, bubble-bubble-cell interactions, and real-time bioassays of cell response in a microfluidic chip constructed by using a unique combination of microfabrication techniques. Three main features that distinguish our experimental system from others in the field are: 1) the patterning of micron-sized gold dots on the glass substrate to enable localized laser absorption for bubble generation17; 2) the patterning of micron-sized islands of extracellular matrix (ECM) for cell adhesion on the same substrate to control both the location and geometry of individual cells; and 3) the compression of the dimension of the bubble-bubble-cell interaction domain from 3D to a quasi-2D space to facilitate in-plane visualization of bubble-bubble interactions, jetting flow fields, cell deformation, and bioeffects, all captured in one streamlined imaging sequence (Figure 1d).
<img alt="Figure 1" src="/files/ftp_upload/55106/55106fig1.jpg" /> 
Figure 1: The microfluidic chip and schematics of different assays. a) An assembled microfluidic chip with channels filled with blue ink for visualization. b) A region inside the microfluidic chip with patterned cells and gold dots (the distance between the two gold dots in proximity is 40 &#181;m). Many pairs of working units can be arranged in a channel. c) Close-up image of a single working unit consisting of a pair of gold dots and a HeLa cell adhered to the cell-patterning region. d) Schematic of the device operation. A single cell adheres to and spreads on the &#34;H&#34;-shaped island coated with fibronectin. A pair of cavitation bubbles (tandem bubble) with anti-phase oscillation are produced by illuminating pulsed laser beams on the gold dots (see Figure 4a), leading to the generation of a fast and localized jet moving towards the target cell nearby. The cell may be deformed, porated for macromolecular uptake, and/or stimulated with a calcium response, depending upon the standoff distance (Sd) of the cell to the tandem bubble. <a href="http://cloudflare.jove.com/files/ftp_upload/55106/55106fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This platform can be further combined with fluorescence assays and functionalized beads attached to the cell surface for cavitation-induced bioeffects. In particular, this platform opens the way for reliable and quantifiable assays at the single-cell level. Up to now, we have used the device for the analysis of tandem bubble-induced cell membrane deformation, cell poration and intracellular uptake, viability, apoptosis, and intracellular calcium response. In the following protocol, we describe the process of chip fabrication and the procedure for analyzing the various bioeffects mentioned above. Moreover, the operations of the chip are also described.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reagent/Materials</td>
			<td style="width:171px;"></td>
			<td style="width:155px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">75x38mm Plain Microscope Slides</td>
			<td style="width:171px;">Corning</td>
			<td style="width:155px;">2947-75X38</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Acetone</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320110</td>
			<td style="width:179px;">ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Isopropyl alcohol</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">W292907</td>
			<td style="width:179px;">&#8805;99.7%, FCC, FG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Sulfuric acid</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320501</td>
			<td style="width:179px;">ACS reagent, 95.0-98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Hydrogen peroxide</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">216763</td>
			<td style="width:179px;">30 wt.% in H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Primer P-20</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">MCC Primer 80/20</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">NFR photoresist</td>
			<td style="width:171px;">JSR</td>
			<td style="width:155px;">NFR016D2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Photomask</td>
			<td style="width:171px;">Photoplotstore</td>
			<td style="width:155px;">N/A</td>
			<td style="width:179px;">4x4 Direct write mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">MF-319 Developer</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">Microposit MF-319</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">1165 Photoresist Remover</td>
			<td style="width:171px;">Dow Chemical, Co.</td>
			<td style="width:155px;">DEM-10018073</td>
			<td style="width:179px;">1-methyl-2-pyrrolidinone based</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">S1813 photoresist</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">S1813</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PLL-g-PEG</td>
			<td style="width:171px;">SuSoS</td>
			<td style="width:155px;">PLL(20)-g[3.5]- PEG(2)</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HEPES</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">15630080</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Paraffin film</td>
			<td style="width:171px;">HACH</td>
			<td style="width:155px;">251764</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SU-2025 photoresist</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">SU-2025</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">PDMS</td>
			<td style="width:171px;">Dow Corning</td>
			<td style="width:155px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microbore Tubing</td>
			<td style="width:171px;">Saint-Gobain PPL Corp.</td>
			<td style="width:155px;">S-54-HL</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Metal pins</td>
			<td style="width:171px;">New England Small Tube</td>
			<td style="width:155px;">NE-1300-01</td>
			<td style="width:179px;">&#160;Cut Tube (straight), 0.025&#8221; OD x 0.017&#8221; ID x 0.50&#8221; Long</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">HeLa cells</td>
			<td style="width:171px;">Duke Cell Culture Facility</td>
			<td style="width:155px;">(307-CCL-2) HeLa, p.148</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DPBS(1X) buffer</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">14190144</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMEM culture medium</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11995065</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Fibronectin Bovine Protein, Plasma</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">33010018</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">25200056</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Propidium Iodide</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">P21493</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 1.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">08226-15</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 2.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">18327-10</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">22980</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Sulfo-NHS</td>
			<td style="width:171px;">Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td style="width:155px;">24510</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Peptite-2000</td>
			<td style="width:171px;">Advanced BioMatrix</td>
			<td style="width:155px;">5020-5MG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">FITC Annexin V</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">A13199</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Fura-2, AM</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">F1221</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMSO</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">D2650</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">F-127</td>
			<td style="width:171px;">invitrogen</td>
			<td style="width:155px;">P6866</td>
			<td style="width:179px;">0.2 &#181;m filtered (10% Solution in Water)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reduced serum media&#160;</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11058021</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Plasma asher</td>
			<td style="width:171px;">Emitech</td>
			<td style="width:155px;">K-1050X</td>
			<td style="width:179px;">O2 / Ar plasma ashing of photoresist and other organic materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Mask aligner</td>
			<td style="width:171px;">SUSS MicroTec&#160;</td>
			<td style="width:155px;">Karl Suss MA6/BA6</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">E-beam evaporator</td>
			<td style="width:171px;">CHA Industries</td>
			<td style="width:155px;">CHA Industries Solution E-Beam</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">RIE</td>
			<td style="width:171px;">Trion Technology&#160;</td>
			<td style="width:155px;">Trion Technology Phantom II</td>
			<td style="width:179px;">(oxide/ nitride/ polymer) etching</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Stereoscope</td>
			<td style="width:171px;">AmScope</td>
			<td style="width:155px;">American Scope SM-4TZ-FRL</td>
			<td style="width:179px;">Stereo Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Syringe pump</td>
			<td style="width:171px;">Chemyx Inc</td>
			<td style="width:155px;">NanoJet</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:175px;">Cell culture incubator</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">AutoFlow NU-8500 Water Jacket CO2 Incubator</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Biological Safety Cabinets</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">NU-425-400</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Water bath</td>
			<td style="width:171px;">VWR</td>
			<td style="width:155px;">1122s</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Centrifuge</td>
			<td style="width:171px;">IEC</td>
			<td style="width:155px;">Centra CL2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microscope</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">Axio Observer Z1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 1)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Tempest</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 2)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Orion</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Delay generator</td>
			<td style="width:171px;">Berkeley Nucleonics&#160;</td>
			<td style="width:155px;">BNC 565-8c</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Flash lamp</td>
			<td style="width:171px;">Dyna-Lite</td>
			<td style="width:155px;">ML1000 fiber-coupled flashtube</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">DRS Hadland</td>
			<td style="width:155px;">&#160;Imacon 200</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed&#160; camera</td>
			<td style="width:171px;">Shimadzu</td>
			<td style="width:155px;">HPV-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">Vision Research</td>
			<td style="width:155px;">Phantom V7.3</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PIV software</td>
			<td style="width:171px;">LaVision</td>
			<td style="width:155px;">DaVis 7.2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">camera</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioCam MRc 5</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">software</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioVision</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PTI system</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">S/N: 1705 RAM-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">EasyRatio software</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">Easy Ratio Pro 2</td>
			<td style="width:179px;">version 2.3.125.86</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">63&#215; objective</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">LD Plan Neofluar</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

The microfluidic platform described in this work can be used to investigate bubble-bubble interactions and to analyze a variety of cavitation-induced bioeffects at the single-cell level. Here, we present several examples to demonstrate a variety of experimental studies and bioassays that can be performed in our experimental system. We will first illustrate the transient interactions of tandem bubbles with the jet formation, the visualization of the resultant flow field, and the calculatio...

Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation Bubblesâ€“Cell Interaction and the Resultant Bioeffects at the Single-cell Lev

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&amp;#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

The overall goal of this experimental procedure is to investigate cavitation induced bioeffects in microfluidic confinement using surface patterning to precisely control the generation of tandem bubbles and the location and shape of the individual target cells. This microfluidic system enables experimentation of this transit cavitation bubbles and cells that is relevant to therapeutic and sound publication and sound operation. The main advantage of this technique comes from the improve the precision from surface patterning.It allows us to study the bioeffects of individual cells and is a high stream loading from reliable bubble-bubble interaction. Visual demonstration of the procedures is critical as the surface patterning and cell preparation in the chip steps are complex involving various techniques and tips. Perform all the microfabrication procedures in a clean room while wearing a clean room suit.Design the area of each gold dot within 25 to 30 square microns, so that it is large enough to absorb laser energy for bubble generation, but small enough to avoid individual cells adhering to it. Clean the glass slide in a chemical hood according to the text protocol, then proceed to the spin coating hood. Program the spin coder to accelerate to 1000 RPM over two seconds and to maintain that speed for five seconds then have it ramp to 3000 RPM over three seconds and maintain that speed for 30 seconds.Next, secure the glass slide to the spin coder using the vacuum. Then, cover the slide with P20 and start the spin cycle. Next, apply NFR negative photo resist using the same cycle.Now, bake the slide on a hot plate at 95 degrees celsius for 60 seconds. Once cooled to room temperature perform the photolithography. Mount the chrome mask onto the mask aligner and make sure that the pattern side is facing down towards the slide.Then, set the photolithography recipe to hard exposure mode with nine seconds of UV exposure and align the glass substrate with the mask. After the UV exposure, bake the slide at 95 degrees celsius for one minute, and then let it cool as before. To develop the pattern on the slide, place it in developer solution for 60 seconds, then wash the slide with distilled water and dry it with nitrogen gas.After confirming the pattern by microscopic inspection bake the slide at 120 degrees celsius for five minutes and let it cool to room temperature. Now, clean the slide with plasma in the reactive ion etching machine for 90 seconds at 500 tor and 100 watts. Using an E-beam evaporator, tape the sample to the plate holder.Program the machine to deposit a 5 nanometer layer of titanium, followed by a 15 nanometer layer of gold. Once that position is complete, ventilate the machine. Next, soak the slide overnight in a beaker of photoresist remover solvent to remove the gold resting on top of the NFR resist.The next day, wash the slide with acetone, followed by IPA and then dry it with nitrogen. Rinse the slide with DI water and dry it again with nitrogen. Now, heat the slide at 115 degrees celsius for five minutes.Then, clean the gold dot pattern slide using an oxygen plasma asher at 100 watts for 90 seconds. Set the area of each fibronectin encoded island to be within 700 to 900 square microns to facilitate adequate hela cell spreading in a square region while minimizing the chances of multiple cells aggregating on the island. Fabrication is much like that of the gold pattern.Spin code the slide as before using S18 positive photoresist and bake on the coding at 115 degrees celsius. Then, perform photolithography, aligning the marks on the mask with those on the substrate. Check the pattern on the central portion to confirm the correct angle and adjust the standoff distance from the gold dots to the H pattern.Run the UV exposure for nine seconds with no post bake. The next difference is that the development step only takes 45 seconds. For the reactive ion etching, set the parimeters to 500 tor 100 watts, and 90 seconds.Then, apply a drop of PLLG peg passivating solution onto a piece of paraffin film and sandwich the solution to the pattern side of the slide. Avoid trapping bubbles. After 45 minutes, remove the slide from the film.Rinse the slide with DI water and then dry it with nitrogen. Next, soak the slide consecutively in photoresist remover 1165. Then 50 percent 1165 in DI water.Then just DI water. During each bath, agitate the solution in an ultrasound bath for 90 seconds. Now, dry the slide on a hot plate, then seal it in a desiccator and store it at 4 degrees celsius.Assemble the slides into a microchannel chip as described in the text protocol. Pay careful attention to shield the patterned area of the glass substrate with the small PDMS slab before using reactive ion etching to remove the PLLG peg from the peripheral area. Retrieve the patterned glass from the RIE machine and remove the small PDMS slab.After treating the microchannel PDMS with a reduced dose of oxygen plasma, align it to the patterned glass substrate under a stereoscope. Bring the micro channel PDMS and the patterned glass substrate in conformal contact. Now, proceed to use the chip.First, prime the chip by flowing PBS down the microchannel for 30 minutes at one microliter per minute. Then infuse the chip with fibronectin solution for 45 minutes at the same flow rate. While waiting, prepare the cells at five million cells per milliliter.Healthy status and high confluencey is critical to this procedure. Later, replace the solution flowing through the chip with PBS and increase the flow rate to ten microliters per minute. Let the PBS flow for five minutes.Next, inject the prepared cells into the chip with a syringe. When one drop has flowed out of the tubing stop the injection and clamp the outlet. Then, place the chip in an incubator for 30 minutes.Later, release the outlet and flush the chip with cell culture medium at ten microliters per minute for five minutes. After five minutes, drop the flow to 0.75 microliters per minute and transfer the chip and pump to an incubator for two hours. After two hours, proceed with tandem bubble generation viewing the chip under an inverted microscope with two pulsed NDI lasers on timing controls directed at the gold dot pattern and with a high speed camera ready to capture the results.For 50 micron bubbles, set the delay between the two lasers to 2.5 microseconds and set their power to ten microjoules. Then, synchronize the high speed camera recording with the lasers and make records at two million frames per second with 200 nanosecond exposures. Thus, the dynamics of bubble expansion, collapse bubble-bubble interaction, and jet formation are recorded.Bubble-bubble interactions and a variety of cavitation induced bioeffects were studied at the single cell level using the described technique, such as the transient interactions of tandem bubbles with the jet formation the visualization of the resultant flow field and the calculation of the jet speed. Directional jetting flow around the tandem bubble is around ten meters per second, is about ten microns wide and is thus capable of producing an impulsive and localized sheer stress and stress gradient onto nearby target cells. Tandem bubble induced cell membrane deformation was also studied.Membrane deformation and recovery are highlighted by the displacement of functionalized beads attached to the leading edge of the cell membrane. From the coordinates of a triad of adjacent beads, the local area strain was calculated. This schematic shows the maximum area change at different locations on the cell surface.The leading edge is primarily stretched, while the trailing edge or lateral sides of the cell are compressed indicating heterogeneity and cell deformation produced by the tandem bubble induced jetting flow. The temporal variation of the area strain at the cell leading edge is comprised of a few rapid oscillations followed by a large and sustained stretch for about 100 microseconds, and a subsequent gradual recovery on a timescale of several milliseconds. After watching this video, you should have a good understanding of how to make a controllable bubble-bubble interaction to study the bioeffects of single cells with controlled shape and location from surface patterning.Following this procedure, other measures like cytoskeleton thinning and the conjugal imaging can be further performed to study cell cytoskeleton rearrangement of the tandem bubble treatment. After its development, this technique will pave the way for researchers in the field of subpretic ultrasound to explore captation induced bioeffects on the single cell level. While attempting this procedure, it is important to remember to maintain a good cell confluency and condition for the success during the cell patterning.Do not forget that working with clean room chemicals and lasers can be extremely hazardous and precautions such as wearing gloves and laser goggles should always be taken while performing this procedure.

a-microfluidic-system-with-surface-patterning-for-investigating

Research

JoVE Journal

Bioengineering

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...