Impact of long term propagation in 3D culture on the phenotype of existing human cell lines

Tissue-specific architecture, mechanical and biochemical cues and cell-cell interactions are altered or lost under the simplified conditions of two-dimensional (2D) cell culture. Three-dimensional (3D) cell cultures attempt to mimic the in vivo situation by preserving the 3D integrity of individual cells and enabling cells to create their own niche in conjunction with the extracellular matrix. There is good evidence to show that 3D models can be used to study physiology where tissue-like function can be maintained. It is common practice however to expand populations of cells in 2D culture initially (particularly cell lines) prior to seeding such cells into 3D culture models. In such circumstances, cells are simultaneously: a) adapting to their new surroundings (2D to 3D growth transition); and b) being challenged and/or examined as part of the 3D cell culture assay (for example, a drug cytotoxicity test). This makes it difficult to decipher the precise outcome of the assay given that cells are concurrently exposed to two such major variables.

We hypothesise that cell populations initially propagated and expanded within a 3D environment are more likely to adapt to their surroundings when seeded into a 3D culture model or transplanted in vivo (i.e. without the need for 2D to 3D transition). In this project we will collaborate with ECACC Culture Collections, an organization that supplies cell lines to researchers. The propagation of popular cell lines that are grown routinely as adherent 2D monolayers (e.g. A549 lung epithelium; MCF7 breast tumour; both established and widely understood), will be maintained in a novel system that enables continual cell passaging in 3D to convert their phenotype to a more native 3D state. Specifically, the student will: 1) (0-18 mths) optimize methods to continually passage A549 and MCF7 human cell lines in 3D culture; 2) (6-24 mths) Characterize the cell phenotype by detailed examination of cell morphology, cytoskeletal structure, expression of integrin/caherins and focal adhesion proteins, expression of markers associated with 3D phenotype known to be lost in 2D culture; 3) (18-30 mths) conduct a 3-way comparison of 2D vs 3D vs in vivo (e.g. MCF7 tumour tissue) examining key structural cell proteins known to be differentially regulated in 2D and 3D culture; 4) (24-36mths) test the ability of the 2D vs 3D propagated cells to produce 3D structures when grown in alternative technologies designed to support 3D cell growth in vitro, e.g. scaffolds, and spheroid cultures; 5) (30-36 mths) Assess the ability of 2D & 3D propagated cells to form xenograft tumours when transplanted into immune deficient mice. The growth rate, size and tissue composition will be compared. The project will provide new information of how the growth environment plays an important role in the basic biology of cultured cells. The student will gain knowledge of cell science and training in cellular and molecular techniques with academic and industrial partners. It is anticipated that these new approaches for maintaining cells will be immediately beneficial to biologists looking to improve their in vitro model.