title: Microscale 3D collagen cell culture assays in conventional flat-bottom 384-well plates.
abstract: Three-dimensional (3D) culture systems such as cell-laden hydrogels are superior to standard two-dimensional (2D) monolayer cultures for many drug-screening applications. However, their adoption into high-throughput screening (HTS) has been lagging, in part because of the difficulty of incorporating these culture formats into existing robotic liquid handling and imaging infrastructures. Dispensing cell-laden prepolymer solutions into 2D well plates is a potential solution but typically requires large volumes of reagents to avoid evaporation during polymerization, which (1) increases costs, (2) makes drug penetration variable and (3) complicates imaging. Here we describe a technique to efficiently produce 3D microgels using automated liquid-handling systems and standard, nonpatterned, flat-bottomed, 384-well plates. Sub-millimeter-diameter, cell-laden collagen gels are deposited on the bottom of a ~2.5 mm diameter microwell with no concerns about evaporation or meniscus effects at the edges of wells, using aqueous two-phase system patterning. The microscale cell-laden collagen-gel constructs are readily imaged and readily penetrated by drugs. The cytotoxicity of chemotherapeutics was monitored by bioluminescence and demonstrated that 3D cultures confer chemoresistance as compared with similar 2D cultures. Hence, these data demonstrate the importance of culturing cells in 3D to obtain realistic cellular responses. Overall, this system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure.
authors.affiliation: Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Brendan M
authors.initials: BM
authors.lastname: Leung
authors.affiliation: Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA Department of Chemical Engineering, McGill University, Montreal, Canada.
authors.forename: Christopher
authors.initials: C
authors.lastname: Moraes
authors.affiliation: Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Cavnar
authors.affiliation: Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Luker
authors.affiliation: Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA Center for Molecular Imaging, Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Luker
authors.affiliation: Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA Department of Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, USA Division of Nano-Bio and Chemical Engineering WCU Project, UNIST, Republic of Korea takayama@umich.edu.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Takayama
mesh_terms: D000970:Antineoplastic Agents / Q000633:toxicity; D057205:Automation, Laboratory / Q000295:instrumentation / Q000379:methods; D045744:Cell Line, Tumor; D003094:Collagen / Q000378:metabolism*; D005782:Gels / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements; D046509:Tissue Culture Techniques / Q000295:instrumentation / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D005782:Gels; D003094:Collagen
keywords: 3D culture; aqueous two-phase system; high-throughput screening