December 1, 2014 (Vol. 34, No. 21)

Leyna Zhao, Ph.D. global marketing manager ACEA Biosciences

New Approach for Automated Continuous Real-Time Quantitative In Vitro Analysis

Cell migration and invasion are fundamental components of embryogenesis, vasculogenesis, immune responses, and pathophysiological events such as cancer cell metastasis. Cell migration and invasion involve morphological changes resulting from actin cytoskeleton rearrangement and the emergence of protrusive membrane structures followed by contraction of the cell body, uropod detachment, and secretion of matrix-degrading enzymes. These processes are influenced by extracellular and intracellular factors and signaling events via specialized membrane receptors.

The xCELLigence® Real-Time Cell Analysis (RTCA) DP Instrument in combination with CIM-Plate® 16 devices (ACEA Biosciences) allows label-free, automated quantification of cell migration and invasion in real time under physiological conditions (Figure 1A). Each well in the CIM-Plate 16 is a modified Boyden chamber. Figure 1B demonstrates how the impedance microelectric sensor on the porous membrane detects cells as they migrate through the porous membrane and attach to the impedance microelectrode in the lower chamber.

Below are summaries of how this method has been applied to study inflammation, wound healing, and cancer metastasis.


Figure 1. The xCELLigence RTCA DP system and the CIM-Plate 16 devices. The RTCA DP system can hold up to three CIM-Plate 16 devices, resulting in a 48-well throughput.

Detecting Macrophage Chemotaxis

Macrophage recruitment, retention, and activation are critical factors in the promotion of plaque progression, which can lead to myocardial infarction or stroke. Understanding this process is an active area of research. Chemotaxis assays are invaluable for studying a range of mediators, such as chemokines, implicated in inflammatory pathologies. However, conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of data generated.

Iqbal et al. (PLoS ONE. 2013 Mar; 8(3): e58744) optimized and validated the impedance-based xCELLigence RTCA technology to measure the migration and adhesion of murine macrophages in response to CC chemokines and other chemoattractant signaling molecules.

Figure 2A is a cell migration profile of mouse macrophages migrating from the top to the bottom chamber following murine CCL5 (red) gradient. Negative control with no chemokine (green) in the bottom chamber, or with no macrophages and no chemokine (blue), was also shown. Figures 2B–F are representative images of macrophages adhered to the underside of porous membrane in response to murine CCL5 (B and C), no chemokine (D and E), or control where no cells or chemokine were added (F). The number of cells adhered to the underside of the membrane (seen as light grey regions in the scanning electron microscopy images) was quantitated (Figure 2G). The results agree with those observed with the RTCA technology.


Figure 2. Real-time chemotaxis of Bio-Gel elicited macrophages to murine CCL5. (A) Representative trace from the RTCA software of 4 × 105 Bio-Gel elicited macrophages from C57BL/6J in the top chamber with murine CCL5 (5 nM) (red) or no chemokine (green) in the bottom chamber. A control of no macrophages and no chemokine was also run (blue). Scanning electron microscopy (SEM) images are shown of macrophages adhered to the underside of filters in response to 5 nM murine CCL5 (B, C); no chemokine (D, E); or control where no cells or chemokine were added (F). The number of cells adhered to the underside of the membrane (seen as light grey regions in the SEM images) was quantitated (n = 1 experiment with five to six technical replicates of each condition analyzed) (G). Figures modified from PLoS One. 2013 Mar; 8(3): e58744.

Analyzing Keratinocyte Migration

CDCP1 is a transmembrane glycoprotein expressed by keratinocytes in native human skin and in primary cultures. McGovern et al. (Br J Dermatol. 2013 Mar; 168(3): 496–503) used xCELLigence real-time cell migration assays and an in vitro human skin reconstruct to investigate CDCP1 expression during epidermogenesis and its role in keratinocyte migration.

The peak rate of cell migration of primary keratinocytes from different patients varies from 12 to 22 hours. As a result, the use of single time-point transwell assays is unreliable, costly, and time-consuming. The xCELLigence system offers real-time, continuous monitoring of cell migration rates. This removed the variability from experiments and provided fast, high-quality results with reduced consumable costs.

Keratinocytes from three individuals were seeded in serum-free medium alone or serum-free medium supplemented with anti-CDCP1 function-blocking antibody. The rate of cell migration was easily quantified by measuring the slope of each cell migration curve during the linear phase using the RTCA system software. Anti-CDCP1 was shown to reduce keratinocyte migration by 50%, 64%, and 68% of untreated primary keratinocytes.


Monitoring Ovarian Cancer Spheroid Transmigration

Ovarian cancers metastasize by shedding cells into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment. Cancer cells inherently aggregate into multicellular structures, which contribute to the metastatic process by invading the peritoneal lining to form secondary tumors.

To mimic the peritoneal microenvironment encountered by tumor cells, a spheroid-mesothelial co-culture model was established. Bilandzic and Stenvers (J Vis Exp. 2014 May; 20(87): e51655) developed a method using the xCELLigence RTCA DP system to conduct quantitative real-time measurements of the invasive capacity of ovarian cancer cell lines grown as spheroids. This approach allowed for the continuous measurement of invasion over time, offering advantages over traditional endpoint assays and real-time microscopy image analyses.

Figure 3A shows the CIM-Plate well arrangement. Preformed human ovarian cancer spheroids (KGN) were plated on top of a monolayer of an LP9 mesothelial layer/matrix barrier in the upper chamber; media with or without fetal bovine serum was added to the lower chamber. Electrodes underneath the two chambers measure increasing electrical impedance as more cells enter the lower chamber. Results from an invasion assay conducted with and without FBS in the bottom chamber of a CIM-Plate well are shown; ovarian cancer spheroids (KGN) cell invasion is compared to that of LP9 mesothelial cells (Figures 3B and 3C). This method enabled a rapid determination of factors that regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time. 


Figure 3. Real-time ovarian cancer spheroid transmigration. (A) Schematic showing the CIM-Plate well. Representative results from an RTCA invasion assay conducted with and without FBS in the bottom chamber of a CIM-Plate well. KGN cell invasion is compared to LP9 mesothelial cells. Results are shown as mean ± SD cell index from triplicate wells at 24 hours (B) and over an entire two-day assay period (C). Figures modified from J Vis Exp. 2014 May; 20(87): e51655.

Conclusion

Cell migration techniques utilizing standard and transwell Boyden chambers are labor intensive, producing results that can be difficult to reproduce. The CIM-Plate 16 combines the benefits of continuous, label-free, impedance-based technology with the classic Boyden chamber. This approach enables automated, real-time, and quantitative in vitro analysis of cell migration and invasion. These studies demonstrate use of the xCELLigence RTCA DP system with CIM-Plate 16 devices in assessing epidermogenesis, immune responses, and cancer cell metastasis. The continuous real-time data also identifies optimal time points for performing parallel imaging studies and other functional analyses of cell migration/invasion.



























Leyna Zhao, Ph.D. (lzhao@aceabio.com), is global marketing manager at ACEA Biosciences.

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