 2.5D cell cultures are similarly grown on top of a thin layer of ECM, and diluted structural ECM proteins are mixed to the medium added above the cells and substrate.Although most of the seminal work in TFM was performed in 2D, or 2.5D, many cell types require the complex biophysical and biochemical cues from a 3D ECM to behave in a truly physiologically realistic manner within an "in vitro" environment.When the rotation or stretch of a sub volume is large, errors can be introduced into the calculation of cell surface tractions since most TFM techniques employ a computational framework based on linear elasticity.