Automated high-throughput wound healing assay

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Study of the migration of multi-cellular sheets is significantly important in tissue development and repair after injury (DiPietro and Burns 2003). The in vitro wound healing process involves a complex and orderly sequence of events involving cell migration and proliferation. Currently, it is difficult to use the classic scratch-wound assay for large-scale applications, such as screening libraries of small molecules or siRNAs. In this type of assay, a confluent monolayer is “injured” by forcibly removing a strip of cells, and the remaining monolayer “heals” through some combination of cell migration, spreading, and proliferation. The wound is typically created by scraping with a needle, pipette tip, or razor blade. This crude process is limiting in a number of ways, including being very time consuming for a large number of samples, difficulty in delivering reproducible wounds over a large number of samples, potentially damaging the plate surface, and limited implementation in formats smaller than 96-well plates.

The Laser-Enabled Analysis and Processing (LEAP™) system has been developed to address current limitations in live cell analysis and manipulation. LEAP has previously been used for high-throughput laser-mediated cell elimination for general cell purification (Koller et al. 2004). The LEAP Wound Healing Application was designed for laser-mediated in situ elimination of a region of a cell monolayer. LEAP overcomes limitations in conventional wound creation by providing reproducible wounds of any size and shape in various multi-well plate formats, including 384-well, in a fully automated fashion. The application monitors closure of the wound area over time. LEAP adds speed, reproducibility, versatility, flexibility, and easy translation to higher density plate formats, all without damaging the plate (Fig.1).

The results shown here establish that the LEAP Wound Healing Application generates consistent and reproducible wounds in cell monolayers in a high-throughput format, demonstrating the capability of LEAP to address key needs in high-throughput discovery biology and screening.

Validation Approach

HEK293, HUVEC, and A431 cells were used as model systems for cre¬ating wounds in cell monolayers and monitoring the healing process in response to specific peptides. The LEAP Wound Healing Application was used to automatically create rectangular wounds (Fig. 1) throughout a plate. Cells were seeded at 2000-5000 cells per well in 384-well C-lect™ plates. Cells were allowed to attach and grow to near confluence and placed in 0.2% serum for 18 hours. Cells were stained with 2.5 M Cell¬Tracker™ Orange and, just before processing, Cyntellect’s Phototherm™ sensitizer was added to each well. The LEAP Wound Healing Applica¬tion reproducibly created wounds across a plate. Cells were washed and placed into low serum-containing media. Wound closure was monitored at multiple time points in different concentrations of serum or peptide factors. The area of wound at each time point relative to the first time point was assessed.

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  Fig. 1. Wound created in a 96-well plate of HEK293 cells (A) and wounds of different shapes created in a 384-well plate of HUVEC (B) and A431 cells (C-F). All cells are stained with CellTracker™ Orange.

Results

The LEAP Wound Healing Application can create wounds of arbitrary sizes, shapes, and positions within a well (Fig. 1). This capability enables adaptation of the assay to differing rates of closure, including the use of multiple sizes of wounds within the same well to ensure a wide range of compound potencies can be accommodated within a screen.

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  Fig. 2. Wounds were created in monolayers of HEK293 cells grown in a 384-well plate. Statistical significance between points at 1% and either 5% or 20% serum varied from Z’ = 0.5 and Z’ = 0.9, respectively; n=3.

Wounds generated by the laser are highly consistent and reproducible. The average width of rectangular wounds in wells of a 384-well plate varied less than 7% well to well, while average area varied less than 9%..

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  Fig. 3. Wounds were created in monolayers of HUVEC, A431, and HEK 293 cells seeded in 384-well plates and placed in 0.2% serum. Wound healing was monitored in the presence of 50 ng/ml VEGF for the HUVEC, 50 ng/ml EGF for the A431, and 250 pM Peptide X in screening medium for the HEK293. The 24-hour time point is shown. Control is medium without peptide added (n = 3).

HUVEC, A431, and HEK cells were used to test the capability of the LEAP Wound Healing Application to quantify the rate of healing in response to serum (Fig. 2) and specific peptides known to affect migration and propagation of specific cell lines (Fig. 3). Wound closure rate over a 72-hour period showed a marked difference among medium containing 0.1%, 5%, or 20% serum (Fig. 2). VEGF, a known inducer for HUVEC (Hughes et al. 2005), was observed to increase wound closure rate in HUVEC by 2.6-fold. EGF, a known inducer for A431 cells (Diaz Miqueli et al. 2007), was observed to increase wound closure rate in A431 by 2.3-fold. An experimental molecule (Peptide X) was tested on a HEK293 cell line used for small molecule screening. Peptide X increased the wound closure rate in HEK293 cells by 1.6-fold.

Conclusion

The LEAP Wound Healing Application brings consistency, high-throughput, and automation to the classic scratch-wound assay, enabling the high-throughput generation and monitoring of wounds in cell monolayers for screening of small molecule libraries and peptides. Using a laser to produce wounds in cell monolayers provides distinct advantages in addition to high-throughput capacity. These other advantages include the generation of highly consistent wounds from well-to-well, and also control over the shape, size, and position of the wound within a sample well.

References

1. Koller et al, Cytometry, Part A 61A:153–161, 2004
2. DiPietro, LA, and AL Burns, Wound Healing, Methods and Protocols, Human Press, 2003
3. Hughes et al, Ann Biomed Eng., 33(8):1003-1014, 2005
4. Diaz Miqueli et al, Hybridoma, 26(6):423-431, 2007