The authors acknowledge the financial support from NSERC and the Canada Research Chair in Smart Medical Devices, and are grateful for the various tools supplied by CMC Microsystems and ReSMiQ. Thanks are due to Laurent Mouden, for his contributions in this work.

1. Removable PDMS-based Interconnector for Low-pressure Microfluidic Applications15-16
<ol>
	<li>Preparation 
	This section describes different steps to prepare the glass and PDMS in order to produce the interconnector.
	<ol>
		<li>Prepare the PDMS: Mix the PDMS and curing agent with (10:1) ratio.</li>
		<li>Ensure that the oven temperature is stabilized at a constant temperature of 80 &#176;C. Depending on the oven, use a measurement device to sense the temperature of the oven and then manually monitor it.</li>
		<li>Thoroughly clean the Petri dish with ethanol.</li>
		<li>Clean the surface of the microfluidic access holes where the PDMS interconnector will be placed.</li>
	</ol>
	</li>
	<li>Fabrication 
	The interconnector described in this paper is essentially a compliant gasket. This gasket is formed from PDMS and contains holes into which microtubes may be inserted. It self-adheres to the surface of a microfluidic chip, to form a leak-proof seal between the microtube and access holes to microchannels in the chip. 
	Use the following steps to fabricate the interconnector.
	<ol>
		<li>Pour a thin layer (approximately 2 mm) of the PDMS (liquid) into a Petri dish.</li>
		<li>Let the PDMS slightly polymerize at a temperature of 80 &#176;C in the oven for 12-15 min. Place the microfluidic chip to which the connection will be made in the oven at the same time, so that it will be at the same temperature.</li>
		<li>When the PDMS starts to polymerize, remove it from the curing oven.</li>
		<li>Use a&#160;biopsy punch to form holes of the desired diameter in the microfluidic chip surface.</li>
		<li>Using the blade, cut the required piece of PDMS. 
		Note: It is recommended to initially cut out a larger piece of material than that will eventually be required. To avoid using multiple connectors, it is desirable that any given PDMS interconnector be cut to a square shape, and holes punched, so as to conform to the maximum possible number of holes on the user&#39;s chip.</li>
		<li>Keep the holes slightly smaller than the outside diameter of the microtube.</li>
		<li>Quickly remove the PDMS from the Petri dishes and place it onto the microfluidic chip.</li>
		<li>Ensure that there is no dust on either the PDMS or chip and ensure that the access holes on the microfluidic chip are aligned with those of PDMS.</li>
		<li>Using a micropositioner, insert tubes (e.g. Teflon tubes) through the PDMS interconnector. The tubes should be 110% of microfluidic access hole diameter to prevent liquid leakage. The same tubes can be connected to the liquid injection system depending on the user&#39;s application. 
		Note: Always place the tubes using a micropositioner, the PDMS layer is not very thick and can be detached if the tubes are twisted.</li>
		<li>If necessary, add epoxy between the tube and the PDMS for better adherence. 
		An example of the completed interconnector is shown in the figures and in Ghallab and Badawy2.</li>
	</ol>
	</li>
</ol>
2. Interconnecting Microtubes in Microfluidic Applications with Epoxy17&#160;
When fabricating an interconnector, ensure the edge of the microtube is correctly cut. Use a new sharp blade to cut the edge of the microtube straight and to ensure that the contact between microtubes and access hole is uniform.
<ol>
	<li>Using the micropositioner, carefully place the microtube in the access hole of the microfluidic chip.</li>
	<li>Apply a small pressure on the epoxy dispenser to deposit epoxy on the access holes or manually place it.</li>
	<li>Allow the epoxy to cure using the following steps:
	<ol>
		<li>UV curable Epoxy: Expose the UV epoxy to a UV source for 3-10 min, depending on the quantity of the epoxy used. For more details, refer to the epoxy specifications.</li>
		<li>Standard epoxy: If the microfluidic substrate is to be used only at RT, keep epoxy curing during 24 hr&#160;at RT. If the substrate is required to withstand high temperatures, rapidly solidify the epoxy by exposing it to 100 &#176;C using an oven or a heater, depending on the application. For more details, refer to the epoxy specifications.</li>
	</ol>
	</li>
</ol>
3. Assembly Technique for Reusable Microfluidic Chips with Electrical Interface17
<ol>
	<li>Assembling the Components 
	Figure 2 shows the links between the electrical connectors on the PCB and the electrical pads on the microfluidic chip. To assemble the PCB/microfluidic chip/Plexiglas components, follow these steps: 
	CAUTION: Avoid excessive compression pressure, which can break the microfluidic chip.
	<ol>
		<li>Solder the surface-mount multipin electrical connectors to the PCB.</li>
		<li>If there is surface contamination on the microfluidic chip&#39;s electrical pads, clean with plasma oxygen; otherwise clean the pads with ethanol.</li>
		<li>Use a sharp blade to cut the conductive tape into small pieces with the same dimensions as the microfluidic chip&#39;s electrical pads, as shown in Figures&#160;1 and 2.</li>
		<li>Using forceps, place the conductive tape on the PCB electrical pads.</li>
		<li>Align and then place the microfluidic chip onto the PCB. 
		Note: For placement precision, use an alignment machine.</li>
		<li>Place an insulation layer (such as paper) on the PCB to avoid short circuits before installing the metal brackets, depending on the location of the PCB traces.</li>
		<li>Attach the metal bracket using machine screws and nuts.</li>
	</ol>
	</li>
	<li>Disassembling
	<ol>
		<li>Remove the machine screws, nuts and the metal brackets.</li>
		<li>Gently separate the microfluidic chip and the PCB.</li>
		<li>Use ethanol to remove the conductive tape adhesive from the electrical pads on the PCB and microfluidic chip prior to reassembly.</li>
	</ol>
	</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoretic+chip+with+multilayer+electrodes+and+micro-cavity+array+for+trapping+and+programmably+releasing+single+cells.">Dielectrophoretic chip with multilayer electrodes and micro-cavity array for trapping and programmably releasing single cells.</a> Biomed. Microdev. 14, 271-278 (2012).</li><li>Xie, L., Premachandran, C., Chew, M., Chong, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Disposable+Bio-Microfluidic+Package+With+Reagents+Self-Contained+Reservoirs+and+Micro-Valves+for+a+DNA+Lab-on-a-Chip+(LOC)+Application.">Development of a Disposable Bio-Microfluidic Package With Reagents Self-Contained Reservoirs and Micro-Valves for a DNA Lab-on-a-Chip (LOC) Application.</a> IEEE Trans. Adv. 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S., Dalton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cost+effective,+re-configurable+electrokinetic+microfluidic+chip+platform.">A cost effective, re-configurable electrokinetic microfluidic chip platform.</a> Sens. Actuators B Chem. 123, 628-635 (2007).</li><li>Miled, M. 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A., Sawan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removable+PDMS-based+Interconnector+for+Low-pressure+Microfluidic+Applications.">Removable PDMS-based Interconnector for Low-pressure Microfluidic Applications.</a> CMC application note. , (2012).</li><li>Miled, M. 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There is nothing to disclose.

The PDMS interconnector is intended for low-pressure applications, liquid leakage into the chip access hole when the PDMS interconnector is subsequently placed on the access hole must be prevented. Therefore, the recommended thickness of PDMS is less than 2 mm. A greater thickness can induce a weak adhesion of the PDMS to the microfluidic chip surface and is not recommended. An unsuccessful trial was made using a thickness of 4 mm. This work focuses on the adherence of the PDMS interconnector to the glass while syringe p...

Recent progresses in microfabrication have led to more complex microfluidic devices including a large number of electrodes and different types of control algorithms. However, these advances highlighted new challenges in packaging. Furthermore, Lab-on-Chip (LoC) is a multidisciplinary concept where microfluidic devices, microelectromechanical systems (MEMS), microelectronic chips among other parts are connected together. Consequently it is impossible to develop a packaging process without considering different constraints of LoC components such as microelectronics circuitry sensitivity and protection, tubing, and mechanical aspects. In addition, packaging the components of a given LoC is closely related to the application1,2. As an example, in optical applications, the transparence of the device is crucial, especially for testing purposes. Thus, it is important to monitor system behavior by keeping the device observable. In applications related to cell or particle manipulations, using low-voltage techniques is more attractive, with the emergence of new microfabrication technologies which facilitate reduced electrode dimensions (from few a micrometers to submicron scale3-5) but considerably increase the number of electrodes to 100 or more.
On the other hand, LoCs dedicated for chemical and biological analysis require long-term observation. Thus, the system packaging and test bench must avoid biological contamination as well as any failure in the electronic devices of LoC in addition to keeping the system observable.
In recently published papers, microfluidic and MEMS are the main parts of a LoC, but other modules may be used for the monitoring of the system. Consequently, testing and validating microfluidic architectures became a challenging issue with the new microfabrication approaches and the high complexity and throughput architectures. Up to now there are no standard testing platforms or supports for microfluidic system, as it is the case for microelectronics dies.
Researchers proposed different packaging techniques for specific uses, such as Xie et al.6&#160;who designed a bio-microfluidic packaging including a microelectronics chip to control DNA separation in microfluidic channels. Beebe et al.7&#160;designed a microfluidic channel with integrated fluid reservoirs and stimuli-responsive hydrogel valves to manipulate fluid samples. Other systems propose the use of electrolytic bubble-based flow sensors to detect pressures through an electrical field to generate air bubbles and measure corresponding pressure8.&#160;The sensing system measures the impedance of the bubble and then determines the pressure, which requires special packaging designed to avoid introducing any interference into the measurements. In another work, a room temperature microfluidic packaging method was introduced based on sequential plasma activation process9. Despite the fact that this process could lead to a strongly assembled device, it has limitations when it is used for prototyping purpose or when the device is used several times. The latter may imply frequent replacement of some parts of the system. Moreover, Lee et al. introduced a new packaging technique, called Biolab-on-IC, based on using magnetic fields for individual magnetic bead manipulation, where the microfluidic architecture is designed on the top of the integrated circuit10. In addition, the reliability of packaging and integration of microfluidic systems, considering the system size, optical aspects, and connection parts, are critical issues as detailed by Han and Frazier11. This latter work reflects a typical approach for LoC systems, where several parameters must be precisely fixed and measured during the packaging.
Consequently, microfluidic packaging is a challenging issue for new a generation of microfluidic devices. Packaging includes heterogeneous parts such as tubing, electrical connections and microfluidic support. For tubing, the main issue is the very limited space available to connect the tubes to access the microfluidic holes. The diameter of the current commercially available connectors is in the range of 6 mm; however, the distance between the two access holes is becoming smaller with the reduction of overall system dimensions12-13. Therefore, we have proposed a tubing technique based on PDMS. This tubing process is specially elaborated for the prototyping phase. In fact, owing to the chemical constraint, changing the connection tube after each usage is mandatory to avoid and minimize liquid leakage inside the microstructure and to clean the microchannels. For these purposes, the described process in this paper is particularly applicable for a fast prototyping design.
On the other hand, electrical connections are critical for microfluidic devices that utilize direct or indirect electrical manipulation or measurement. Due to the wide range of microfluidic applications, device dimensions, shape, and number of electrical contacts may vary considerably. Therefore, a versatile method is required, which can be used for different applications. Kaler&#160;et al.&#160;reported results from their microfluidic rapid prototyping platform, which is based on zero insertion force (ZIF) connectors14. Instead of these later connectors, the presented methods use an anisotropic adhesive conductive film, which can be easily adapted to any shape.
Many researchers do not take into consideration the testing constraints when designing their microfluidic device. In microelectronics, there is many supports used for prototyping purpose and testing, which are called IC holders. These are mainly designed to reduce prototyping and testing times, and offer researchers and engineers an easy way to replace devices in case of failure. Similar to microelectronics, we are also proposing a new testing package for microfluidic devices limited to a 45 mm x 90 mm device size.
Our proposed techniques are targeting to cover a large number of microfluidic devices and applications. These methods can be adapted, extended and upgraded to other applications. We aim to provide one of the first versatile packaging methods for microfluidic devices that are optimized for microfluidic manipulations. For example, one end application for devices packaged with these techniques is separation of polystyrene and carboxyl modified microspheres with artificial cerebrospinal fluid. Nevertheless, they can be used in other applications with different solutions.
In the remainder of this article, three different tubing techniques, 2 electrical connection methods and one microfluidic packaging method are presented and compared.

<table border="0" cellpadding="0" cellspacing="0" width="643">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Epoxy 731</td>
			<td style="width:191px;">Epotek</td>
			<td align="right" style="width:157px;">731</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDMS</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#160;184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive conductive film</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Epoxy</td>
			<td>Epotek</td>
			<td>OG159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropump</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCB</td>
			<td>Advanced Circuits</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plexiglass</td>
			<td colspan="2">Ecole Polytechnique</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive conductive film</td>
			<td>3M&#160;</td>
			<td align="right">9703</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput microfluidic rapid and low cost prototyping packaging methods

本研究では、3種類のパッケージングとアセンブリ技術が提示されています。一回限りの使用および再使用可能なパッケージング技術：彼らは、2つのカテゴリに分類することができる。 1回使用パッケージング技術は、電気的接続のためのUVベースおよび温度の穴にアクセスするためにマイクロチューブを接続するためにエポキシ樹脂を硬化、集積回路接続用のワイヤボンディング、及び銀エポキシを使用する。この方法は、1 PSIに近い比較的高い圧力をサポートすることができ、マイクロ流体アーキテクチャを強化する任意のサポートを必要としない堅牢な組立技術に基づいています。 再利用可能なパッケージング技術は、PDMSベースのマイクロチューブのインターコネクタとの電気的接続のための異方性接着フィルムで構成されています。これらのデバイスは、より敏感かつ脆弱である。従って、プレキシガラス支持体は、異方性接着フィルムが使用されるときに電気的接触を改善するために、また、strengtheするマイクロ流体構造に追加されるNマイクロ流体アーキテクチャ。また、マイクロマニピュレータは、アクセスホールに接続すること薄いPDMS層を使用しながら、チューブを維持するために必要とされる。 0.45〜3ミリメートルの範囲の異なるPDMS層の厚さは、注入速度に対する最良の付着を比較するために試験される。応用注入速度は、それぞれ0.45〜3ミリメートルのPDMS層のために50〜300μL/時まで変化させている。これらの技術は、低圧の用途に主に適用可能である。しかしながら、それらは永久にガラス基板にPDMSを密封するために酸素プラズマ処理を介して高圧もののために拡張することができる。この技術の主な利点は、それが再使用可能であるという事実に加えて、マイクロチャネルの長さ（3mm以上低いの範囲）が非常に短い場合には観察可能な装置を保つからなる。

Figure 3 illustrates a detailed schematic of the proposed packaging technique. The packaging solution was tested with several PDMS thicknesses and sizes to characterize a transparent and efficient packaging process for hybrid microelectronics/microfluidic microsystems as shown in Figures 4 and 5. The dimensions of the designed PDMS interconnector layer are 6 mm x 6 mm and it is used to connect two access holes. However, with major commercially available connectors, it is...

Watch this Scientific Journal Video about High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods at JoVE.com

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

ハイスループットマイクロ流体の迅速かつ低コストのプロトタイピング包装方法

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

high-throughput-microfluidic-rapid-low-cost-prototyping-packaging

Research

JoVE Journal

Bioengineering

7.3K ビュー.  Polytechnique Montreal. In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

ビデオ: High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

相関ライト/電子顕微鏡のための低コストクライオ光学顕微鏡のステージの作製

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

生物工学

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

マイクロ流体プラットフォームを使用した高スループットのタンパク質発現ジェネレータ

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

ハイスループットマイクロカプセル化シングルセルと複数セル

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

液体射出成形用の3Dプリント金型を用いた医療機器を迅速かつ低コストプロトタイピング

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

ハイスループットの使用<em&gt;インビトロ</emオーラル多種バイオフィルムを開発するために&gt;マイクロ流体システム

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

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.

ハイスループットシングルセルの単離および培養のためのマイクロ流体プラットフォーム

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 ...

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.

マイクロ流体プローブとのライブの細胞層の急速なサブトラクティブパターニング

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

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

オープンソース、低コストのバイオインク、食品溶融物押出3Dプリンタの設計

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...