1Department of Biomedical Engineering, University of Texas at San Antonio, 2Department of Biology, University of Texas at San Antonio
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Srinivasan, A., Lopez-Ribot, J. L., Ramasubramanian, A. K. Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening. J. Vis. Exp. (65), e3845, doi:10.3791/3845 (2012).
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 °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.
1. Preparation of Functionalized Slides
NOTE: Do not use Kim-Wipes to wipe the slides as it would generate fine paper dust.
NOTE: To avoid contact with skin, use chemical-resistant gloves and safety goggles while working with concentrated acids and toxic and corrosive chemicals.
NOTE: Make the APTES solution in a plastic container since APTES deposits preferentially on the walls of glass container.
where h is thickness of film coating, e is rate of evaporation, η , C and ρ are viscosity, concentration and density of the coating solution, respectively, and ω is angular velocity.
NOTE: Toluene is harmful when inhaled for a long time and use of a fume hood is recommended.
2. Preparation of Yeast Inocula and Collagen Encapsulation
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.
3. Preparation of CaBChip
4. Susceptibility Testing of Preformed Biofilms in CaBChip Against Antifungal Agents
NOTE: Maintain a relative humidity of 100% to prevent the drying of the spots while adding the drugs.
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.
5. Representative Results
A representative CaBChip, consisting of a 48×16 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 μm 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 μm. 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 μg/mL11.
Figure 1. Flow chart for the fabrication of CaBChip.
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 μm 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 μm.
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 ± standard error mean for two separate chips containing 10 replicates for each condition.
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, allows for easy and fast handling, is amenable to automation, minimizes manual labor and costs and is fully compatible with standard microarray technology and equipment. Moreover, the technology is flexible and can be easily adapted to other microorganisms. Despite miniaturization, the nano-biofilms formed on CaBChip display properties that are characteristic of the C. albicans biofilm lifestyle, including 3D architecture and drug resistance. Thus, the CaBChip allows for true high-throughput screening, and has the potential for a paradigm shift in antimicrobial drug discovery.
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.
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 & Craniofacial Research (5R21DE017294).
|Polystyrene-Co-Maleic Anhydride (PS-MA)||Sigma-Aldrich||426946|
|Glass microscopy slides||Fisher Scientific||12-549-3|
|Rat Tail collagen type I||BD Biosciences||354236|
|Robotic Microarrayer||Omnigrid Micro||MICROSYS4000/4100A|
|Microarray Scanner||Genepix Personal 4100A||GENEPIX4100A|
|Hybridization Cassette||ArrayIt Corporation||AHCXD|
|FUN1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinoliniumiodide]||Invitrogen Corp.||F-7030|
|Fluconazole||Sicor Pharmaceuticals, Inc.||J02AC01|
|Ceramic Tip 190 μm orifice||Digilab||60020441-00|
|GraphPad Prism Software||GraphPad Software, Inc.|
|Genepix Pro V4.1||Molecular Devices|