The authors thank William Nance (University of Michigan) for help in formulating the biofilm growth protocols and John Battista (Fluxion, San Francisco, CA) for advice concerning technological issues relating to the Bioflux system. This work was supported by the National Institutes of Health (NIH: R21DE018820 to A. H. R.) and University of Michigan start-up funds to A. H. R.

The saliva collection protocol described herein was reviewed by the University of Michigan Institutional Review Board for Human Subject Research. 
NOTE: With regard to institutional reviews for human subject work of this type, prior arrangements and permissions should be garnered from the host institution. In particular, depending on the institution, IRB or ethics approval might need to be sought and approved before saliva collection from human volunteers can proceed. As an aid to preparing an application, a useful NIH algorithm/chart can be found here: <a href="http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDecisionChart.pdf">http://grants1.nih.gov/grants/policy/hs/PrivateInfoOrBioSpecimensDecisionChart.pdf</a>
1. Preparation of Pooled Saliva for Use as an Environmentally Germane Growth Medium
<ol>
	<li>Recruit &#62;5 individuals for saliva donation. Do not take any identifying information, ensure no individuals will donate saliva if they are ill, or have taken oral antibiotics in the past 3 months, or have consumed food or liquid, with the exception of water, in the previous 2 hr before donation.</li>
	<li>Collect saliva in 50 ml plastic tubes. Pool the collected saliva in a plastic beaker, keeping it on ice. Do not use glass as polymers in the saliva will adhere to the internal glass surfaces.</li>
	<li>Add Dithiothreitol (DTT) to a final concentration of 2.5 mM from a 100x stock. (Stock is frozen in single-use aliquots at &#8211;20 &#176;C). Stir for 10 min in a plastic beaker on ice.</li>
	<li>In order to remove particulate matter, centrifuge the pooled saliva for 30 min at 17,500 x g.</li>
	<li>Dilute the saliva with 3 volumes of dH2O to give one-fourth concentrated saliva.</li>
	<li>Filter-sterilize saliva using 0.22 &#181;m polyethersulfone (PES), low protein binding filter. Use filter with large surface area, or use several small area filters. Keep saliva in a plastic container on ice while filtering.</li>
	<li>Freeze the pooled saliva at &#8211;80&#160;&#176;C in 50 ml plastic tubes until needed to grow bacteria. Each plastic tube is for one use only and should contain no more than 35 ml as each microfluidic well holds a maximum of 1.2 ml (1.2&#160;ml x 24 wells = 28.8 ml) and space is needed in each tube as saliva expands during freezing.</li>
	<li>For use, thaw the pooled saliva at room temperature. Once thawed, filter-sterilize once more (0.22 &#181;m polyethersulfone, low protein binding filter to remove any precipitates).</li>
</ol>
2. Preparation of Pooled Saliva for Use as an Inoculum
<ol>
	<li>Recruit &#62;5 individuals for saliva donation. Do not take any identifying information, ensure no individuals will donate saliva if they are ill, have taken oral antibiotics in the past 3 months, or have consumed food or liquid, with the exception of water, in the previous 2 hr before donation. Collect samples over 30 min.</li>
	<li>Collect saliva in 50 ml plastic tubes at room temperature and pool the collected saliva in a plastic beaker at room temperature.</li>
	<li>Dilute pooled saliva with autoclaved general reagent grade glycerol to yield a stock with a final ratio of 25% glycerol, 75% cell-containing saliva [CCS].</li>
	<li>Freeze saliva at &#8211;80&#160;&#176;C in 3 ml single-use aliquots until needed.</li>
	<li>When required to inoculate the microfluidic system, aliquots are thawed at room temperature and agitated gently on a vortexer for 5 sec before being pipetted into the microfluidic system as described in step 3 of the protocol, below.</li>
</ol>
3. Growth of Oral Multi-species Biofilms
<ol>
	<li>CFS pre-treatment
	<ol>
		<li>First coat the Bioflux microfluidic channels with CFS. Add 100 &#181;l of CFS to each outlet well. Using the Bioflux control software, select &#8220;manual&#8221; and set flow from channels &#8220;B1-B24&#8221; that are to be used.</li>
		<li>Next, set shear to 1.0 dyne/cm&#178; and start flow for 2 min at room temperature to ensure homogenous distribution of the CFS throughout the channel. Ensure there is fluid in each inlet channel to verify that CFS flowed through all channels evenly.</li>
		<li>Incubate plate at room temperature for 20 min.</li>
		<li>Remove the CFS/pretreat solution that remains in the outlet wells and transfer to the inlet wells. This total volume of 100 &#181;l will serve to balance against the pressure being applied to the inoculum from the outlet well.</li>
	</ol>
	</li>
	<li>Inoculation
	<ol>
		<li>To each outlet well, add 100 &#181;l of CCS inoculum. Place the microfluidic plate on the heat plate (temperature set at 37 &#176;C), select manual within the control software, and set flow from outlet wells to the inlet wells (i.e., reverse) at 1.0 dyne/cm&#178; for exactly 6 sec.</li>
		<li>Incubate the microfluidic plate at 37 &#176;C for 40 min to allow for initial adherence and growth of the bacteria in the inoculum.</li>
	</ol>
	</li>
	<li>Overnight Growth
	<ol>
		<li>For the inoculated channels being used, aspirate the waste inoculum from each of the outlet wells. Add up to 1 ml total volume of CFS into each of the inlet wells (can be done on top of existing CFS).</li>
		<li>Incubate the microfluidic plate at 37 &#176;C, select manual and set the program to run at 0.2 dyne/cm&#178; for 20 hr.</li>
	</ol>
	</li>
	<li>Stain prewash
	<ol>
		<li>Aspirate all fluid from the inlet and outlet wells and add 100 &#181;l of PBS (pH 7.4) to each of the inlet wells. Flow for 20 min at 0.2 dyne/cm&#178;.</li>
	</ol>
	</li>
	<li>Stain mixture addition
	<ol>
		<li>For cell viability staining make 100 &#181;l of stain mixture for each channel to be stained. Specifically, add 3 &#181;l of SYTO 9 and 3 &#181;l of propidium iodide per ml of PBS using commercial cell viability staining kit such as LIVE/DEAD. This generates a staining mixture containing 10 &#181;M SYTO 9 and 60 &#181;M propidium iodide.</li>
		<li>Aspirate the remaining PBS from the inlet wells and then add 100 &#181;l of the cell viability stain mixture to each inlet well. Set to flow at 0.2 dyne/cm2 and run the solution from inlet to outlet for 45 min at room temperature.</li>
	</ol>
	</li>
	<li>Post-staining wash
	<ol>
		<li>Aspirate the remaining stain in each of the inlet wells and add 100 &#181;l of PBS to each inlet well. Set to flow at 0.2 dyne/cm&#178; and run the PBS solution from inlet to outlet for 20 min at room temperature to remove any excess stain.</li>
	</ol>
	</li>
</ol>
4. Image Collection, 3D Rendering, and Image Analysis
<ol>
	<li>Use an inverted confocal laser scanning microscope (CLSM) to collect biofilm image data from the microfluidic system. Make sure that the CLSM is able to withstand the weight of the Bioflux plate, which can reach 150 g. 
	NOTE: Given the dimensions of the microfluidic channels, the need for sensitivity to discern biofilm structure, and the requirements to detect fluorescence signal of varying intensity, fit the CLSM with a 40X 1.25 NA objective lens or one with similar optical quality, magnification, and numerical aperture.</li>
	<li>Standardize the emission capture gates with gain and offset measurements being kept constant. Ensure that the laser power does not exceed 25% as this can result in photo-bleaching.</li>
	<li>Convert CLSM files from the Leica Image File type (LIF) to OME (Open Microscopy Environment) file type. This allows for greater ease of access and compatibility between software programs. Use commercially available software such as, IMARIS to convert files to this type.</li>
</ol>
3D Rendering for Images/Figures
<ol start="4">
	<li>Once converted to OME format, use commercially available software such as IMARIS to render the images in 3D. Perform this using a combination of &#8220;Easy3D&#8221; and &#8220;Surpass&#8221; options. 
	NOTE: Attention to background signal and thresholding should be made. The histogram function in IMARIS can be used to obtain the collection range and this should be kept constant between images being analyzed.</li>
	<li>Save images using the &#8220;snapshot&#8221; function and make careful consideration to image resolution before saving file types.</li>
	<li>Assemble images into figures using image editing software such as CorelDraw or Adobe Illustrator.</li>
</ol>
3D Image Analysis for Graphs and Tables
<ol start="7">
	<li>Use freely available IMAGEJ 23 and COMSTAT/COMSTAT2 38 for image analysis. Download the packages from http://rsb.info.nih.gov/ij/ and http://comstat.dk/, respectively. Have the most recent Java update installed. 
	NOTE: The software platform JAVA will need to be installed prior to the use of the image analysis software.
	<ol>
		<li>Use the IMAGEJ software with COMSTAT2 plugin to import the OME files for each biofilm image in &#8220;hyperstack&#8221; mode and view with &#8220;split channels&#8221;.</li>
		<li>Individually analyze the two channels (red/propidium-iodide/dead being channel 0, green/SYTO-9/LIVE being channel 1) using the &#8220;histogram&#8221; function of IMAGEJ. This function lists the total number of pixels of 8-bit OME files (shown as &#8220;count&#8221;) at each color intensity from 0 to 255 (shown as &#8220;value&#8221;), with 0 being pixels with no signal (background) and 255 being pixels with complete signal saturation.</li>
		<li>Export the data into a spreadsheet program and standardize all signal values by weighting each pixel by the corresponding signal intensity. This is performed by multiplying the total count at a given signal intensity by the numerical 8-bit value (0-255) of that signal intensity.</li>
		<li>Sum all weighted values, 0-255, for both channels for each biofilm image captured. Take the sum of the weighted values for both channels to determine the percent total signal from either channel. Do this to determine the relative ratio or percent red and percent green signal (i.e. the percent &#8220;DEAD&#8221; and percent &#8220;LIVE&#8221; for each treatment).</li>
		<li>Perform statistical analyses, for example using two-tailed Student&#39;s T-test modified for unequal variance. Values of p &#60;0.05 are considered significant and those values of p &#60;0.01 are considered highly significant.</li>
	</ol>
	</li>
</ol>
5. Harvesting Biofilm Cells for Culture-independent Analysis
<ol start="1">
	<li>Remove all spent and unused saliva from inlet and outlet wells. Wash the wells with 1 ml sterile distilled water three times.</li>
	<li>Add 100 &#181;l of sterile distilled water to the inlet wells and, using the software control interface, pass the sterile distilled water forward through the channels at &#62;8.0 dyne/cm2 (flow rate &#62;745 &#181;l/hr, shear of 800 sec-1) for at least 5 min. Repeat in the reverse direction for at least 5 min and then repeat the forward and reverse washing step process.</li>
	<li>Check by light or by epifluorescence microscopy (if cells were stained/labeled) for thorough biofilm removal (Figure 2). Collect the 100 &#181;l cell-suspension and store at -80&#160;&#176;C for culture-independent analysis. A cost-effective analysis of community composition can be achieved through using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) using the approach described in Nance et al. 20</li>
</ol>

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Total microscopic and viable counts and counts of specific organisms.</a> Archives of oral biology. 8, 275-280 (1963).</li><li>Heydorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (10), 2395-2407 (2000).</li><li>Nobbs, A. H., Lamont, R. J., Jenkinson, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+adherence+and+colonization.+Microbiology+and+molecular+biology+reviews).">Streptococcus adherence and colonization. Microbiology and molecular biology reviews).</a> MMBR. 73 (3), 407-450 (2009).</li><li>Hope, C. K., Clements, D., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+spatial+distribution+of+viable+and+nonviable+bacteria+in+hydrated+microcosm+dental+plaques+by+viability+profiling.">Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling.</a> Journal of applied microbiology. 93 (3), 448-455 (2002).</li><li>Adams, H., et al. <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+laboratory+model+to+assess+the+removal+of+biofilm+from+interproximal+spaces+by+powered+tooth+brushing.">Development of a laboratory model to assess the removal of biofilm from interproximal spaces by powered tooth brushing.</a> American journal of dentistry Spec No 12B-17B. 15, 12-17 (2002).</li><li>Ledder, R. G., McBain, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+comparison+of+dentifrice+formulations+in+three+distinct+oral+microbiotas.">An in vitro comparison of dentifrice formulations in three distinct oral microbiotas.</a> Archives of oral biology. 57 (2), 139-147 (2012).</li></ol>

A. H. R. and N.S.J. have received research awards from a variety of sources such as the National Institutes of Health (NIDCR), Colgate-Palmolive (Piscataway, NJ) and the Society for Applied Microbiology to fund research studies in their lab over the past five years. All other authors: none to declare.

This methods paper highlights the basic steps required to setup and run a microfluidic system in a manner to allow for the development of oral multi-species biofilms derived from pooled human saliva and grown in filter-sterilized 25% pooled human saliva. Approaches to characterize the biofilm are given but it should be remembered that these described approaches are modifiable and additional technologies such as, for example, stains or labels can be introduced. As a matter of example, one could conceivably use labeled ant...

Biofilms are architecturally complex communities of bacteria that are aggregated on surfaces 1. These communities typically contain numerous species that interact with one another within the biofilm 2. Oral biofilms, the most visually conspicuous being dental plaque, are a persistent problem in humans and their uncontrolled development results in the generation of taxonomically diverse multi-species communities 3. The component bacteria of these diverse communities can be up to 1,000 times more resistant to antimicrobials than their free-floating (planktonic) counterparts 4-6. Failure to treat these oral biofilm communities, which can cause dental caries and periodontal disease, has resulted in a significant public health burden: over 500 million visits to the dentist office per annum in the US, and an approximately $108 billion to treat or prevent periodontal disease and dental caries 7.
&#8220;While many microbiologists advocate studying microbial behavior under natural conditions, few of them do so. This is because their morale for overcoming the difficulties is constantly sapped by the attractive ease of working with laboratory cultures.&#8221; &#8212;Smith 8.
At present, oral biofilm research is conducted using a variety of in vivo and in vitro approaches, each with their own advantages and disadvantages 9,10. In vitro approaches often use model biofilm systems that are relatively easy to set up but may lack clinical/real-world relevance 10,11. In vivo approaches typically rely upon animal model systems that may reproduce certain aspects of the human oral environment, but again suffer from limitations due to differences in anatomy, physiology, microbiology and immunology between animals and humans 12,13. It should be noted that oral biofilms can also be developed on enamel surfaces held in a stent within the mouths of human volunteers, but this approach is currently relatively costly and labor-intensive 14,15. Ultimately, novel agents or technologies to improve oral healthcare are tested in humans under controlled clinical trial conditions 11. At present, an often-used modus operandi for identifying and evaluating new oral healthcare agents is to first perform laboratory studies to discern potential efficacy, and then perform animal studies and &#8220;field trials&#8221; that employ clinicians to evaluate the success of the technology 9,16,17. Unfortunately, laboratory studies tend to rely on model systems that occupy a large footprint, are technologically challenging to use, and often contain simplified communities of one or at most a few species to derive potential real-world meaning 10,18. Given that dental plaque biofilms contain multiple species and form in a complex flowing salivary milieu, developing biofilms that contain one or a few species in artificial media is unlikely to generate communities that behave in a similar manner to those in a real-world scenario 10,19. To address the time, cost, training requirements, and the poor representative nature of laboratory model biofilm systems compared to the real-world environment, we recently developed a high throughput and environmentally germane biofilm system 20 (Figure 1). The system benefits from the use of cell-free pooled human saliva (CFS) as medium and untreated pooled human bacterial cell-containing saliva (CCS) as an inoculum. Uniquely, the system also combines microfluidic technology, a confocal laser scanning microscope, and culture-independent bacterial diversity analysis technology. Thus, the model system is environmentally germane (using saliva as an inoculum to grow multi-species biofilms at 37 &#176;C in filter-sterilized flowing saliva) and the oral biofilms contain species (including Streptococcus, Neisseria, Veillonella, and Porphyromonas species) in abundances representative of those found in early supragingival plaque 20.
When considering that this work describes the use of the newly developed model system, particular attention must be given to the amalgamation of a confocal laser scanning microscope (CLSM) microfluidics, and culture-independent diversity analysis technologies. The union of these technologies by our research group was intentional and not only adds a high-throughput capability to the newly developed model system, but also allows questions to be asked that could not be easily addressed before with other systems. Firstly, CLSM has distinct advantages over traditional microscopy as it allows for the three-dimensional analysis of biofilms. Often unappreciated, this is extremely important as biofilms are heterogeneous with respect to species composition and spatial position as well as the physiological conditions being imposed at different spatial locations within the biofilm 6,21. In concert with three-dimensional rendering software and image analysis software, the biofilm architecture, spatial relationships between component species, and extent of antimicrobial killing can be analyzed 22-24. Such abilities are not possible using standard transmitted light or epifluorescence microscopy. Next, microfluidics has garnered particular attention in the field of microbiology as it enables the study of biofilms under carefully controlled conditions (flow, temperature, pH, etc.) and only requires small volumes of liquid 25-27. As a point of comparison, growing an oral biofilm in human saliva within a flow cell model system (a system that is arguably considered the mainstay model for many oral biofilm studies) for 20 hr at a similar flow-rate and shear as that achieved in a microfluidic system requires at least 200 ml, as opposed to 800 &#181;l in the microfluidic device 28-31. Thus, a microfluidic model biofilm system enables the study of quantity-limited material under defined conditions. Finally, pyrosequencing technology has been optimized in the last decade to require only small amounts of material to perform a community analysis and is sufficiently versatile to control depth of sequencing to obtain the identity of even rare biofilm species. The use of this technology, such as bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), has allowed for pertinent questions concerning the ecology of biofilms to be addressed 32,33. Such questions imbued difficulties in the past when pyrosequencing was not available because of the time and costs required to create plasmid libraries and the complex technological and analytical steps required to derive data 33,34. Of course, a great advantage with culture-independent approaches, such as pyrosequencing, is that the bacterial species that cannot be grown in isolation within conventional laboratory media (i.e. viable but non-cultivable species) can be grown and identified within the model system and their relative abundance in the community quantified 35,36. To add perspective, as early as 1963, the late Sigmund Socransky estimated that approximately 50% of the bacteria in material isolated from the human oral gingival crevice could not be cultured using laboratory growth conditions 37.
The objective of this methods paper is to describe the approach to develop oral multi-species biofilms in a commercially available microfluidic (Bioflux) system under: (i) conditions representative of the human oral cavity and (ii) with a species composition and abundance that is comparable to supragingival plaque. Furthermore, using both freeware and commercial software, we highlight how basic biofilm architecture measures can be derived from CLSM data, with a focus on approaches to quantify biofilm biomass, roughness, and viability (based upon Live/Dead staining). Finally, the steps required to harvest biofilm material for diversity analysis by bTEFAP are described.

<table border="0" cellpadding="0" cellspacing="0" width="951">
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			<td height="20" style="height:20px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
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			<td height="20" style="height:20px;">Falcon 15mL Conical Centrifuge Tubes</td>
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			<td height="20" style="height:20px;">Dithiothreitol (White Crystals or Powder/Electrophoresis), Fisher BioReagents</td>
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			<td>BP172-5</td>
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			<td height="20" style="height:20px;">Sorval ultracentrifuge&#160; (SS-34 compatible)</td>
			<td>Thermoscientific</td>
			<td>Unit-dependent</td>
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			<td height="20" style="height:20px;">Thermo Scientific SS-34 Rotor&#160;</td>
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			<td height="20" style="height:20px;">BioFlux microfluidic system</td>
			<td>Fluxion</td>
			<td>Bioflux 200 system</td>
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			<td height="20" style="height:20px;">Bioflux 24-channel plate</td>
			<td>Fluxion</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Confocal Laser Scanning Microscope</td>
			<td>Lecia</td>
			<td>SPE or eqivalent system</td>
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			<td height="20" style="height:20px;">Epifluorescence Microscope</td>
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			<td height="20" style="height:20px;">&#160;Ultra Low Temperature Freezer -80&#176;C</td>
			<td>Multiple choices</td>
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			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SOFTWARE</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bioflux dedicated software</td>
			<td>Bioflux</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica SPE</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ</td>
			<td>Freeware (http://imagej.nih.gov/ij/)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">COMSTAT/COMSTAT 2</td>
			<td colspan="2">Freeware (http://www.comstat.dk/)</td>
		</tr>
	</tbody>
</table>

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.

3D Rendering of Biofilms
Representative results are shown in Figure 3. A useful tool in IMARIS software is the option to examine each slice of the collected biofilm stack and to combine them to create three-dimensional reconstructions. In addition, artificial shadowing effects can be added to help visually interpret three-dimensional structures. The rendered biofilms can be orientated in any direction with different magnifications to explore biofilm o...

Watch this Scientific Journal Video about Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms at JoVE.com

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.

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 ...

use-high-throughput-vitro-microfluidic-system-to-develop-oral-multi

Research

JoVE Journal

Bioengineering

13.4K visualizações.  The University of Michigan.  O objectivo deste trabalho é métodos para descrever o uso de um sistema de microfluidos para o desenvolvimento de biofilmes multi-espécies que contêm espécies normalmente identificados na placa dental supragengival humano. Métodos para descrever arquitetura biofilme, a viabilidade de biofilme, e uma abordagem para a colheita de biofilme para análises dependentes de cultura ou independentes de cultivo são realçados. 

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo microfluídicos para Recriando um microambiente do tumor<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengenharia

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.

Alto rendimento Gerador Protein Expression Usando uma plataforma microfluídica

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

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

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.

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

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Análise sistemática de<em&gt; In Vitro</em&gt; Célula de rolamento usando um sistema microfluídicos Multi-bem placa

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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.

Uma plataforma microfluídica para a isolação de alta taxa de transferência de célula única e Cultura

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 ...

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Otimizando o uso de um robô de manipulação líquidos para conduzir a uma alta taxa de transferência para a frente química genética tela de Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Uso do elevado-Throughput Microbioreactor sistema automatizado para produção de IgG1 modelo em células CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Identificação de alta duração da resistência a Pseudomonas syringae pv. Tomate no Tomate usando Ensaio de Inundação de Mudas

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Geração de Condições Ambientais Dinâmicas usando um dispositivo microfluido de alto rendimento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...