96 Well High-Throughput Wound Assay for Cell Migration

Objective: Smoking is a risk factor for many oral diseases. We have demonstrated that nicotine decreases human gingival fibroblast cell migration. Our aim was to develop a high-throughput cell culture assay that could screen pharmacological compounds for effects on wound closure in a reliable and efficient manner.

Methods: Primary human fibroblasts from healthy gingival tissue were seeded into 30mm dishes and 96 well plates at 10,000 cells per well and grown to confluence. Cells were treated with nicotine (1mM) or TGFB1 (1ng/ml) 2 hours before wounding and compared to untreated controls. A 96 well plate pin array was used to uniformly wound each well simultaneously. Plates fixed and stained at 0, 4, 7, 14, and 24 hours after wounding. Plates imaged with a light scanner. A Metamorph system was used to measure area of each wound. Areas at 4, 7, 14, and 24 hours compared to area at 0 hours to give percent closure which was used to evaluate effects of nicotine and TGFB1.

Results: The pin array wounded cells in a uniform and predictable manner (0 time point). After 4 hours, all wounds closed 50-56%. Controls and TGFB1 treated cells were 43% of the original wound, but nicotine treated were 55% of the original wound at 7 hours. At 14 hours, control, TGFB1 treated, and nicotine treated were 2.52%, 1.88%, and 3.41% respectively of the original wound area. The wounds closed in all treatments at 24 hours.

Conclusion: A pin array for wounding was a reliable and efficient manner for a high throughput cell culture wound assay. A light scanner was adequate for imaging the plates. Nicotine slowed wound closure at 7 and 14 hours compared to controls and TGFB1.

Funding acknowledgements: Baylor Oral Health Foundation and the Texas A&M Health Science Center Vice President for Research Development Grant