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Moving Upwards: A Simple and Flexible <em>In Vitro</em> Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

University of Minnesota - Twin Cities

Methods to generate murine cardiomyocytes from pluripotent stem cells (PSCs) in vitro are resource and time intensive. All current protocols require exogenously provided soluble factors and almost all utilize embryoid body formation to modulate pathways associated with mesoderm specification and cardiomyocyte differentiation. Here, we developed a simple protocol without EBs and without exogenous soluble factors that enabled cardiomyocyte differentiation of a murine induced PSC line based on controlled nutrient deprivation in 2D monolayer cultures. We showed that this protocol reproducibly imposed metabolic stress and consequently modulated active β-catenin levels to yield functional cardiomyocytes. The yield of cardiomyocytes and calcium handling kinetics were comparable to existing approaches. However, this approach did not produce consistent results between murine PSC lines suggesting signaling pathways linking nutrient deprivation to β-catenin activation are not universally conserved and may be a remnant of the parent population from which the induced PSCs were derived.

Simple Monolayer Differentiation of Murine Cardiomyocytes via Nutrient Deprivation-Mediated Activation of β-Catenin.

The ability to quantify cell migration and invasion is critical in the study of cancer metastasis. Current invasion assays, such as the Boyden Chamber, present difficulties in the measurement of the invasion of cells that are few in number and are intrinsically tied to the cell microenvironment. There exists a need for a three-dimensional invasion assay that is easily reproduced, accessible for most laboratories, and requires no displacement of cells from their original microenvironment. Here we present a simple design for an inverted vertical invasion assay able to assess the invasion capabilities of cells in a three dimensional, extracellular matrix-based environment without displacement from the original culture location. We used the assay to determine the migratory capacity of hybrids between mesenchymal/multipotent stem/stroma cells (MSCs) and breast cancer cells MCF7. These hybrids are formed reliably but rarely (1 in 1,000 cells) and for this reason require an invasion assay that does not involve extensive cell manipulation. Using this assay, we found that MSCs, breast cancer cells, and corresponding fusion products are able to migrate and invade through the extracellular matrix and that hybrids invade in a manner more similar to stromal cells than cancer cells. Thus, this assay can aid the study of the invasive capacity of both cancerous cells and associated fusion hybrids and could augment testing of therapeutic strategies to inhibit metastatic spread.

Tanner J. McArdle

Department of Biomedical Engineering

University of Minnesota - Twin Cities

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Tanner J. McArdle

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University of Minnesota - Twin Cities

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Department of Biomedical Engineering