Fabrication of a Cell-adhesive Microwell Array for 3-dimensional in vitro Cell Model

다국어 초록 (Multilingual Abstract)

Purpose Microarrays of single living cells facilitate elucidation of the biological effects of a drug on a single cell providing large amounts of information regarding cellular chemistry at the molecular level in quantitative manner. Compared to 2D cultures, 3D in vitro cell models provide a more realistic cellular environment and permit the reproduction of in vivo cellular phenotypes.
Methods We fabricated a microwell array for single cell analysis that could preserve the native morphology and functionality of HeLa cells by creating microwells with a cell-adhesive inner surface. Dopaminergic inorganic-organic hybrid resin (HR4-DOPA) was used for cell-adhesive layer and fabricated by spin-coating and micro contact printing methods. The physical dimensions of the microwells were designed to achieve high single-cell occupancy.
Results Microwells was 25 × 25 μm2 in size from the diameter of a HeLa cell. An optimal depth of microwells and cell concentration for HeLa single-cell occupancy was investigated; 25-μm-deep microwells at 1.0 × 106 cells/ml demonstrated the highest single-cell occupancy of 67.5% while providing a sufficient cell-adhesive surface area for long-term cell culture. We obtained singly occupied microwells with HeLa cells in 1072 microwells from 8 microwell arrays in the 2 × 2mm2 area and with only 5.9% array-to-array variation.
Conclusion The number of singly occupied microwells and array-to-array variation provides adequate throughput for accurate quantification in advanced single-cell analysis.

참고문헌 (Reference)

• 1 Wang Y, "Trapping cells on a stretchable microwell array for single-cell analysis" 402 (402): 1065-1072, 2012
• 2 Mueller-Klieser W, "Three-dimensional cell cultures: from molecular mechanisms to clinical applications" 273 (273): C1109-C1123, 1997
• 3 Dai J, "The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells" 110 (110): 1-10, 1997
• 4 Kim G, "The biocompatability of mesoporous inorganic-organic hybrid resin films with ionic and hydrophilic characteristics" 31 (31): 2517-2525, 2010
• 5 Kim BY, "Solventresistant pdms microfluidic devices with hybrid inorganic/organic polymer coatings" 19 (19): 3796-3803, 2009
• 6 Leong K, "Single-cell patterning and adhesion on chemically engineered poly (dimethylsiloxane)surface" 25 (25): 4615-4620, 2009
• 7 Jen CP, "Single-cell chemical lysis on microfluidic chips with arrays of microwells" 12 (12): 347-358, 2012
• 8 Park JY, "Single cell trapping in larger microwells capable of supporting cell spreading and proliferation" 8 (8): 263-268, 2009
• 9 Huang WH, "Recent advances in singlecell analysis using capillary electrophoresis and microfluidic devices" 866 (866): 104-122, 2008
• 10 Jing G, "Precise cell patterning using cytophobic self-assembled monolayer deposited on top of semitransparent gold" 12 (12): 935-948, 2010
• 11 Smitskamp-Wilms E, "Postconfluent multilayered cell line cultures for selective screening of gemcitabine" 34 (34): 921-926, 1998
• 12 Lindstrom S, "Overview of single-cell analyses:microdevices and applications" 10 (10): 3363-3372, 2010
• 13 Spudich JL, "Non-genetic individuality: chance in the single cell" 262 (262): 467-471, 1976
• 14 Lindström S, "Miniaturization of biological assays—Overview on microwell devices for single-cell analyses" 1810 (1810): 308-316, 2011
• 15 Wlodkowic D, "Microfluidic single-cell array cytometry for the analysis of tumor apoptosis" 81 (81): 5517-5523, 2009
• 16 Yang ST, "Microbioreactors for highthroughput cytotoxicity assays" 11 (11): 111-127, 2008
• 17 Ochsner M, "Micro-well arrays for 3D shape control and high resolution analysis of single cells" 7 (7): 1074-1077, 2007
• 18 Lidstrom ME, "Life-on-a-chip" 1 (1): 158-164, 2003
• 19 Rettig JR, "Large-scale single-cell trapping and imaging using microwell arrays" 77 (77): 5628-5634, 2005
• 20 Zhao L, "Intracellular water-specific MR of microbead-adherent cells: the HeLa cell intracellular water exchange lifetime" 21 (21): 159-164, 2008
• 21 Chen CS, "Geometric control of cell life and death" 276 (276): 1425-1428, 1997
• 22 Givan AL, "Flow cytometry: first principles" Wiley 41-, 2001
• 23 Liu C, "Fabrication of microwell arrays based on two-dimensional ordered polystyrene microspheres for high-throughput single-cell analysis" 82 (82): 9418-9424, 2010
• 24 Broderick AH, "Fabrication and selective functionalization of amine-reactive polymer multilayers on topographically patterned microwell cell culture arrays" 12 (12): 1998-2007, 2011
• 25 Di Carlo D, "Dynamic single cell culture array" 6 (6): 1445-1449, 2006
• 26 Bacakova L, "Cell adhesion on artificial materials for tissue engineering" 53 (53): S35-S45, 2004
• 27 Kobayashi H, "Acquired multicellular-mediated resistance to alkylating agents in cancer" 90 (90): 3294-3298, 1993
• 28 Deutsch M, "A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells" 6 (6): 995-1000, 2006