Replicating the In Vivo Environment: Organotypic and Submerged Three-Dimensional Culture Methods

Abstract

In the embryo, soluble molecules are expressed in gradients to regulate tissue morphogenesis and direct cell lineage. Yet most tissue culture methods rely on modified two-dimensional (2D) substrates. When a cell is attached to a flat surface, the cellular structure is limited by adhesion to the substrate, and the cell is unable to acquire the in vivo morphology and possibly function. In most tissue culture applications, extracellular matrix (ECM) molecules are presented on the basal planar 2D substrate. In addition, cell-secreted ECM deposits on the 2D substrate or is released into the liquid media. Establishing concentration gradients of soluble factors, such as growth factors, within the liquid media is extremely difficult. Gradients generated by the cells are readily disrupted during routine movement for microscopic observation and media exchange procedures. A better way to study the role of soluble factors and ECM in vitro is to recapitulate the in vivo environment with three-dimensional (3D) tissue culture constructs. The encapsulated cells or the organotypic tissue explants maintain much of the native extracellular environmental with the ease of performing manipulations in vitro, i.e., transfections of plasmids for gain or loss of function studies. In this chapter, we will outline two methods for establishing explant culture of rodent embryonic brain regions to study neurite outgrowth and cell migration. The first method is similar to tissue organotypic culture in which the cells in the 3D construct are placed on a membrane that can suspended or floated on a medium source to generate gradients, whereas the second method submerges the 3D construct to have uniform medium exposure.