This work was funded in part by grants from the South Texas Technology Management (POCrr 2009.041), the Institute for Integration of Medicine and Science from the National Center for Research Resources (UL 1RR025767), and from the National Institute of Dental &amp; Craniofacial Research (5R21DE017294).

1. Preparation of Functionalized Slides
<ol>
 <li>Place the microscope slides in a removable slide rack, and wash twice by immersing in a staining jar containing 99% ethanol (histological grade). Wipe the slides clean using paper towels (ensuring not to generate paper dust), and dry using a jet of compressed nitrogen gas.</li>
</ol>
NOTE: Do not use Kim-Wipes to wipe the slides as it would generate fine paper dust.
<ol start="2">
 <li>Immerse the slide rack containing the slides in a staining jar filled with concentrated sulphuric acid and incubate at room temperature overnight.</li>
</ol>
NOTE: To avoid contact with skin, use chemical-resistant gloves and safety goggles while working with concentrated acids and toxic and corrosive chemicals.
<ol start="3">
 <li>Sonicate the slides for 30 min and wash with Milli-Q (18 M&Omega;) water for 30 min, follow with another wash in acetone for 5 min. This treatment exposes the silanol groups on the glass surface.</li>
 <li>Coat the clean slides with 2.5% (vol/vol in water) of 3-aminopropyltriethoxysilane (APTES) solution, by immersing the slide rack in APTES for 30 min, washing 3 times in Milli-Q water for 15 min each wash, and baking the slides in a furnace at 110 &deg;C for 15 min. Baking allows cross-linking of the APTES, resulting in -NH2-functionalization of the surface.</li>
</ol>
NOTE: Make the APTES solution in a plastic container since APTES deposits preferentially on the walls of glass container.
<ol start="5">
 <li>Using a spin coater, coat the slides with 1% (wt/vol in toluene) Polystyrene-Co-Maleic Anhydride (PS-MA) (Sigma) to achieve a mono-layer of hydrophobic coating 6. Add 2.0 mL of PS-MA onto a clean glass slide mounted on a spin coater and coat at 3,000 rpm for 30 s. These conditions may vary based on spin coating parameters such as coating solution, substrate, thickness of coating and governed by the following equation 7</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/3845/3845eq1.jpg" /> 
where h is thickness of film coating, e is rate of evaporation, &eta; , C and &rho; are viscosity, concentration and density of the coating solution, respectively, and &omega; is angular velocity.
NOTE: Toluene is harmful when inhaled for a long time and use of a fume hood is recommended.
<ol start="6">
 <li>The slides can be stored in a slide rack for up to one month in dry, dust-free conditions at 2- 8 &deg;C.</li>
</ol>
2. Preparation of Yeast Inocula and Collagen Encapsulation
<ol>
 <li>Prepare an overnight culture of C. albicans strain SC5314, in Yeast Peptone Dextrose [YPD; 10 g/L yeast extract, 20 g/L peptone and 20 g/L dextrose] liquid medium by inoculating a single colony of C. albicans into 10 - 20 mL of YPD8. Incubate culture in an orbital shaker (about 150 - 200 rpm) at 30 &deg;C overnight. Harvest 1 mL Candida albicans yeast cells from overnight YPD cultures (by centrifugation at 5,000 rpm for 5-10 minutes) and wash twice for 10 min in 1 mL of sterile phosphate buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4) (Sigma) per wash. Harvest washed cells also by centrifugation at 1900 rpm for 10 minutes. Resuspend the washed cells in 1 mL reconstruction buffer (0.2 N NaOH solution with 2.2% (wt/vol) sodium bicarbonate and 4.8% (wt/vol) HEPES, pH 7.2).</li>
</ol>
NOTE: C. albicans is a Risk Group 1/BSL1 microorganism. Always remember to use good aseptic/sterile techniques for work with this microorganism and follow institutional procedures for proper disposal of biohazardous materials.
<ol start="2">
 <li>Count cells using a hemocytometer on a bright field microscope and adjust to a cell density of 5&times;107 cells/mL.</li>
 <li>Further dilute the suspension ten times by addition of 10&times; RPMI-1640 supplemented with L-glutamine and buffered with morpholinepropanesulfonic (MOPS) acid (pH 7.2).</li>
 <li>Encapsulate the yeast cells in collagen by mixing the cell suspension in RPMI-1640 with collagen (1.8 mg/mL) (Type 1 from rat tail, BD Biosciences, Bedford, MA), to obtain a final concentration of 4&times; 106 cells/mL. Keep the collagen-cell suspension on ice to prevent the gelation of collagen before printing.</li>
</ol>
3. Preparation of CaBChip
<ol>
 <li>Clean and disinfect all surfaces of the microarrayer, including the source plate station, wash and vacuum station, vacuum slide platter and printing chamber, by wiping with 70% isopropanol.</li>
 <li>Place and hold by vacuum the desired number of PS-MA-coated glass slides on the slide deck of the microarrayer.</li>
 <li>Vortex the cell suspension in collagen vigorously and aspirate 100 &mu;L of the well-mixed suspension into a well of a 96-well plate, just before printing. Place this source plate in the loading station.</li>
 <li>Switch on the humidifier to maintain a relative humidity of 100% in the microarray chamber throughout the printing process.</li>
 <li>Print 50 nL of cell suspension in an array of 48 rows and 16 columns with spots spaced 1.2 mm apart using a microarray spotter (Omnigrid Micro, Digilab Inc., Holliston, MA) by non-contact deposition using conically tapered 190 &mu;m orifice ceramic tips (Digilab).</li>
 <li>Prime, rinse and vacuum-dry the tips twice after each round of printing.</li>
 <li>Immediately after printing, place the slide in an air-tight, humidified chamber (Hybridization cassette, ArrayIt Corporation, Sunnyvale, CA) and incubate at 37 &deg;C for 24 hr to allow for biofilm formation.</li>
</ol>
4. Susceptibility Testing of Preformed Biofilms in CaBChip Against Antifungal Agents
<ol>
 <li>From stock solutions or powder of antifungal drugs, prepare a working solution in RPMI- 1640 medium. Typical maximum concentrations of the working solution are 1,024 &mu;g/mL for fluconazole and 16 &mu;g/mL for amphotericin B5. Other concentrations may be used for different agents.</li>
 <li>Prepare eight different concentrations of the compound by diluting the stock solution in two-fold dilutions, spanning a range with the estimated IC50 of the compound in the middle.</li>
 <li>After 24 hr of biofilm growth, use the microarrayer to print 50 nL of eight different concentrations of the drugs, along with positive (no drug i.e. cells in only media) and negative (treated with sodium hypochlorite for 20 min) controls, in at least six replicates on top of the biofilms.</li>
</ol>
NOTE: Maintain a relative humidity of 100% to prevent the drying of the spots while adding the drugs.
<ol start="4">
 <li>Soon after adding the drugs, incubate the chip with the drugs in a humidified chamber at 37 &deg;C for 24 hr.</li>
 <li>Wash the drugs off by dunking the CaBChip 3-5 times for 2 min every time in PBS and stain by incubating the CaBChip with 0.5 &mu;M FUN1 at 37 &deg;C for 30 min9.</li>
 <li>Wash the stain off by dunking the CaBChip 3-5 times in PBS for 1-3 min every dunk, and dry the CaBChip using a nitrogen stream. Read fluorescence intensity using a microarray scanner (GenePix 4100A, Axon Instruments, CA) at a wavelength of 532 nm with a PMT gain of 380.</li>
 <li>Set the fluorescence intensity of the positive control (no drug) and dead (bleach-treated) biofilm spots at 100% and 0%, respectively.</li>
 <li>Determine the percentage inhibition by the reduction in the fluorescence intensity (F) relative to the average of the control spots using the following equation</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/3845/3845eq2.jpg" /> 
where F, Fmax and Fo are raw fluorescence intensities of drug-treated, no-drug control and bleach-treated spots, respectively. The scanner settings were adjusted to obtain Fmax and Fo of 30000 and 4000 RFU, respectively.
<ol start="9">
 <li>Calculate the 50% inhibitory concentration or IC50 by fitting the variable slope Hill equation using GraphPad Prism Software (La Jolla, CA).</li>
</ol>
5. Representative Results
A representative CaBChip, consisting of a 48&times;16 array of nano-biofilms of C. albicans, is shown in Figure 2. The bright field microscopy shows an overall architectural feature of the nano-biofilm. The scanning electron microscopic images of the biofilm show that the fungal hyphae are embedded within the matrix of collagen fibers, which are approximately 2 &mu;m and 100 nm in diameter, respectively. The FUN1-stained microarray scanner images show the yeast and hyphal forms, which are characteristic of fungal biofilms. The 2D- and 3D-confocal fluorescence images of FUN1-stained biofilms can be seen to have spatial heterogeneity, with regions of metabolically active cells interspersed within the extracellular matrix (composed of collagen as the encapsulating material and most likely also of exopolymeric material produced by biofilm cells), which is not stained by the metabolic dye. The thickness of the biofilm was estimated to be approximately 50 &mu;m. The CaBChip was used to estimate the antifungal susceptibility of two drugs, fluconazole and amphotericin B, and the results are shown in Figure 3. Consistent with published reports on industry standard 96-well plate assays, the biofilms are resistant against fluconazole10 and the calculated IC50 for amphotericin B is 0.3 &mu;g/mL11.
<img alt="Figure 1" src="/files/ftp_upload/3845/3845fig1.jpg" /> 
Figure 1. Flow chart for the fabrication of CaBChip.
<img alt="Figure 2" src="/files/ftp_upload/3845/3845fig2.jpg" /> 
Figure 2. A picture of the high-thoughput CaBChip, printed using a robotic microarrayer and containing 768 spots on PS-MA-coated slides. Each hemispherical spot is 50 nL in volume, approximately 700 &mu;m in diameter, and with a 1.2 mm separation between spots. Also shown (clockwise) are light microscopy, microarray scanner, 2D- and 3D-fluorescence microscopy, and scanning electron microscopy images of the individual biofilms on the CaBChip. The SEM figure at high magnification of 25,000x shows a hyphal filament of width 2 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3845/3845fig3.jpg" /> 
Figure 3. Results of antifungal susceptibility testing and determination of IC50 values for (A) fluconazole and (B) amphotericin B using CaBChip. The fluorescence microscope images of amphotericin B-treated biofilms are shown in (C). The results are mean &plusmn; standard error mean for two separate chips containing 10 replicates for each condition.

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <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+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

Jose L. Lopez-Ribot and Anand K. Ramasubramanian own equity in MicrobeHTS Technologies, Inc., which is developing antifungal agents. MicrobeHTS Technologies, Inc. provided no financial support for these studies.

We have developed a cell-based high-density microarray, CaBChip, consisting of nanoliter volumes of Candida albicans biofilms. The microarray was printed on modified glass substrates, which allowed for robust attachment of collagen gel spots while providing hydrophobicity necessary for a non-spreading, hemispherical 3D gel. A single CaBChip can replace approximately eight 96-well plates, and several chips can be printed and processed at the same time. The chip utilizes nano-scale cultures...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>3-aminopropyltriethoxysilane (APTES)</td>
 <td>Sigma-Aldrich</td>
 <td>440140 </td>
 </tr>
 <tr>
 <td>Polystyrene-Co-Maleic Anhydride (PS-MA) </td>
 <td>Sigma-Aldrich </td>
 <td>426946</td>
 </tr>
 <tr>
 <td>Glass microscopy slides </td>
 <td>Fisher Scientific </td>
 <td>12-549-3</td>
 </tr>
 <tr>
 <td>Rat Tail collagen type I</td>
 <td>BD Biosciences </td>
 <td>354236</td>
 </tr>
 <tr>
 <td>Robotic Microarrayer</td>
 <td>Omnigrid Micro </td>
 <td>MICROSYS4000/4100A</td>
 </tr>
 <tr>
 <td>Microarray Scanner </td>
 <td>Genepix Personal 4100A </td>
 <td>GENEPIX4100A</td>
 </tr>
 <tr>
 <td>Hybridization Cassette</td>
 <td>ArrayIt Corporation </td>
 <td>AHCXD</td>
 </tr>
 <tr>
 <td>FUN1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo&#x200B;-1,3-thiazol-2-yl)-methylidene)-1-phenyl&#x200B;quinoliniumiodide] </td>
 <td>Invitrogen Corp. </td>
 <td>F-7030</td>
 </tr>
 <tr>
 <td>Fluconazole </td>
 <td>Sicor Pharmaceuticals, Inc. </td>
 <td>J02AC01 </td>
 </tr>
 <tr>
 <td>Amphotericin B</td>
 <td>Sigma</td>
 <td>A2411 </td>
 </tr>
 <tr>
 <td>RPMI-1640</td>
 <td>Mediatech, Inc.</td>
 <td>50-020-PC</td>
 </tr>
 <tr>
 <td>Ceramic Tip 190 &mu;m orifice</td>
 <td>Digilab</td>
 <td>60020441-00</td>
 </tr>
 <tr>
 <td>GraphPad Prism Software </td>
 <td>GraphPad Software, Inc. </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Genepix Pro V4.1</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

candida albicans biofilm chip (cabchip) for high-throughput antifungal drug screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Watch this Scientific Journal Video about Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening at JoVE.com

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

candida-albicans-biofilm-chip-cabchip-for-high-throughput-antifungal

Research

JoVE Journal

Bioengineering

13.6K Aufrufe.  University of Texas at San Antonio. Wir haben eine hohe Dichte Microarray-Plattform bestehend aus 3D-Nano-Biofilme von entwickelten C albicans Genannt Ca BChip. Die Anfälligkeit Profil von Medikamenten auf ein getestet Ca BChip ist vergleichbar mit dem konventionellen 96-Well-Platte-Modell, was darauf hindeutet, dass der Pilz-Chip ist ideal für echtes High-Throughput-Screening von Antimykotika geeignet.

Serie: Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

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

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

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

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

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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.

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

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...