Biomimetic 3D scaffolds for the cultivation and differentiation of stem cells.

Background: Efficient and accurate differentiation of adult and embryonic stem cells remains an overriding research priority. Many stem cell differentiation protocols are limited by the non-physiological two dimensional nature of traditional cell culture methodologies, resulting in inefficient differentiation and the production of mixed populations of cells. The development of matrices and scaffolds allowing three dimensional biomimetic cellular interactions will promote enhanced stem cell growth and differentiation. Preliminary Work: In our preliminary studies in collaboration with Porvair, we have developed a novel three dimensional polymer scaffold derived from hydrophilic PEG disks. As our primary research interest is in the development of new sources of insulin-producing cells, our initial work centred on the growth and differentiation of pancreatic ductal cells, the adult stem cells of the pancreas. Our preliminary work established that PEG-based scaffolds could be modified and adapted to optimise the growth of specific cell types (e.g. fibroblasts showed competent growth on the unmodified surface of the disks, whereas adult stem cells had more complex optimal growth requirements). Disks were initially coated with bovine serum albumin, gelatine, or FBS (foetal bovine serum), each of which substantially improved the growth and differentiation of adult stem cells compared with uncoated disks. Our work with pancreatic ductal cells, mouse embryonic stem cells, liver cells and fibroblasts indicates that modification of the 3D scaffolds is a simple and efficient way to tailor the disks to the growth of specific cell types. Aims: In the differentiation of embryonic stem cells to a desired adult phenotype, many protocols initially favour the generation of three dimensional Embryoid Bodies (EBs) before addition of the specific differentiation stimuli. In parallel with our initial studies on the Porvair scaffolds, we have utilised a novel microgravity cell culture system to generate greatly enhanced EBs, with superior deposition of extracellular matrix, improved cell-cell communication and higher differentiation frequencies when utilising established differentiation protocols. In the present proposal we aim to utilise these optimised EBs in combination with our optimised and tailored three dimensional Porvair scaffold to develop a novel and enhanced biomimetic stem cell differentiation environment. We believe that these modifiable polymer scaffolds represent an exciting new platform technology on which to build bioresponsive materials to improve and refine broad-scale stem cell differentiation protocols. Methodologies: Our collaborators at Porvair are able to produce scaffold disks in a broad range of formats depending on the desired application. At present our preliminary work has been performed on 5mm x 10 mm circular disks. We plan to test a new 96-well format for the disks in the early stages of the proposed project, since this would allow differentiation protocols utilising minimal volumes of media and supplements (cutting the current high costs associated with stem cell differentiation work). Additional manufacturing templates for the disks include 96-well plates with flow-through capacity, allowing the collection of secreted proteins from differentiated cell populations, and allowing transient media supplementation for defined time periods. Our preliminary work has established that cells grown on these disks are amenable to H&E staining, cryosectioning for SEM and TEM analysis, fluorescence immunohistochemical staining for specific stem cell and ECM markers, and that cells can be retrieved from the scaffolds by simple trypsin digestion, allowing molecular analysis of the cell proteome and transcriptome. Modification of the disks at source to increase surface roughness (and surface area), and peptide functionalisation of the surface with defined differentiation peptides will be monitored by AFM.