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Validation of a High Throughput Wound Healing Assay using 3D Cell Patterning and Automated, Kinetic ImagingСкачать
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May 06, 2016
Wound healing, both acute and chronic, involves a complex internal and external choreography of signaling, in addition to interactions with neighboring cells and the surrounding environment or external stimuli, and cell migration. This synergistic relationship can also vary depending on the wound type. Therefore, characterizing the mechanisms therein is of great interest for many applications, including wound dressing, burn and ulcer healing, scar elimination, anti-aging and aesthetic cosmetics and much more. Historically, most wound healing assays use a scratch technique, where a confluent two-dimensional (2D) cell layer is mechanically injured, and cell migration is measured. Major limitations of this method are the lack of biomimetic environment, in vivo-like architecture and multi-cellular network, and as scratching methods vary, results are difficult to replicate. Newer three-dimensional (3D) methods allow cells to self-aggregate in the absence of a solid substrate. Vital cell-cell and cellextracellular matrix (ECM) communication networks are able to be reestablished. In this way, both cellular morphology and behavior more closely mimic that found in the body.
Here, we demonstrate a novel 3D wound healing assay model that can overcome 2D assay limitations. The method incorporates magnetic levitation where cells are first incubated with a nontoxic, magnetic nanoparticle assembly which does not induce an inflammatory cytokine response. The cells are then placed into a microplate well and levitated by placing a magnet above the well. The cells aggregate and form ECM within a few hours, the magnet is removed, and pipetting is performed to break up the aggregate, once again creating a single cell suspension. Appropriate cell numbers are then transferred to a 384-well assay plate and a ring magnet is positioned below the plate, allowing the cells to be patterned into a new 3D ring shape. Wound healing rates are determined by monitoring ring closure. Two individual fibroblast cell models, HT-1080 fibrosarcoma cells and primary dermal fibroblasts, were tested to compare wound healing rates between cancer cell line and primary cell models. Co-cultures containing fibroblasts and keratinocytes were also examined to ascertain whether more in vivo-like cell models have an effect on wound healing rates. Automated kinetic imaging was performed using a novel cell imaging multi-mode reader to track ring closure at regular intervals during the incubation period. The combination provides an easy-to-use, robust method to generate accurate and repeatable results of the effect that new test molecules have on important wound healing applications.