Adaptation of Explanted Primary Cells to 2D and 3D Culture Environments

It is widely recognised that cells adapt to their environments through responding to local signals and physical cues. For example, cultured cells grown on conventional two dimensional (2D) polystyrene substrates adopt an unnatural geometry, remodel their cytoskeleton and change their growth, differentiation and functional characteristics, and become a poor proxy of their native counterparts. The vast majority of the surface area of individual cells grown in monolayer cultures is either exposed to the plastic substrate or incubating medium, with minimal opportunity for interaction with adjacent cells, which is in contrast to what occurs in a real tissue. These factors have a significant impact on cell performance and consequently influence the representation of the biological assay. It is recognised that many of the existing and popular cell lines used in research today have become far removed from their source of origin and are no longer truly representative as an effective model. Three-dimensional (3D) cell culture models have been shown to overcome many of these limitations and enable cells to grow and function in a more realistic manner. The biotechnology company, Reinnervate Limited (www.reinnervate.com), has developed Alvetex, a novel porous polystyrene scaffold that provides a 3D space in which cells can grow. The scaffold is engineered into a 200 micron thick membrane that is mounted within existing cell culture plates and dishes. Cells occupy the scaffold and form 3D structures in close union with adjacent cells and essentially produce a thin tissue layer in vitro. Alvetex is developed as a platform technology for widespread generic use and has been optimised through the co-development of devices such as well inserts to provide users with flexibility to design their own 3D culture systems. In this study, we propose to investigate and compare the growth, differentiation and function of primary cells when explanted and maintained onto polystyrene substrates in 2D (conventional plasticware) and 3D (Alvetex) formats. We will use explants of chick embryo as an established model to study tissue formation in different environments. The linked processes of chondrogenesis and osteogenesis have been demonstrated to occur from cranial neural crest explants under certain ex vivo conditions via the normal sequence of differentiation. For example, when explants of different facial processes are maintained ex vivo, chondrogenesis occurs with a morphology that correlates with the facial process from which they derive. Explants of embryonic limbs undergo similar spatial patterns of chondrogenesis. Our aim is to derive primary cultures from these sources in a 3D culture system employing Alvetex technology in comparison to explants on conventional plasticware. We seek to establish the differences between cell types maintained in primary cultures in 2D and 3D in systems where the same differentiation process occurs in different spatial patterns. These differences will highlight the factors limiting the potential of existing 2D culture techniques and indicate how the capabilities of cells in culture can be improved. To develop and test the culture system further the student will work on the following objectives: (1) Develop protocols for establishing primary cultures of chick embryo chondrocytes, limb bud mesenchyme and facial process mesenchyme in the novel polymer matrix; (2) By microarray analyse RNA from 2D and 3D cultures and markers that are characteristic of each culture type. (3) Compare the effects of maintaining established cell lines in 2D and 3D culture and the developmental potential of both equivalent cell types. The mouse embryonic cell line, ATDC5, is capable of cartilage formation and its capacity to differentiate will also be investigated in 2D and 3D culture subsequent to long term culture in these environments.