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

Summary

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.

Abstract

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.

Protocol

1. Preparation of Functionalized Slides

1. 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.

NOTE: Do not use Kim-Wipes to wipe the slides as it would generate fine paper dust.

2. Immerse the slide rack containing the slides in a staining jar filled with concentrated sulphuric acid and incubate at room temperature overnight.

NOT....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-aminopropyltriethoxysilane (APTES)	Sigma-Aldrich	440140
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
Amphotericin B	Sigma	A2411
RPMI-1640	Mediatech, Inc.	50-020-PC
Ceramic Tip 190 μm orifice	Digilab	60020441-00
GraphPad Prism Software	GraphPad Software, Inc.
Genepix Pro V4.1	Molecular Devices