Self-assembled cell aggregates for tissue engineering

Tissue engineering aims to replace damaged or lost tissues or organs with functional constructs that have been developed in the laboratory. Cells, ideally from the patient themselves, are isolated and grown in culture flasks before being transferred to a three-dimensional polymer scaffold. Here, the continued growth and development of the cells into a functional tissue replacement is supported until the construct is ready for implantation into the patient. This, of course, is an idealized scenario and there are still many technical hurdles that must be overcome if routine fabrication of tissue replacements is to become a reality. One major problem encountered within this area is the nature of 3D scaffolds that cells are grown on. While they can provide a suitable environment for cells by promoting their adhesion and releasing factors that help their growth, their internal scale and structure means that cells behave as if grown on a flat surface. In natural tissues, cells receive signals in three dimensions, not only from their neighbours, but from the protein matrix that surrounds them. These signals are vital for the maintenance of correct tissue function and cell survival, so there is a need in tissue engineering and, more broadly, in biological research as a whole, to develop novel methods to grow cell structures with a true 3D organization. This research proposes a novel method of 3D cell culture where cells and cell-sized polymer microparticles are rapidly cross-linked together in suspension to effectively self-assemble a polymer scaffold around the cells. Not only will this provide the cells with a 3D signalling environment, but it also benefits from the ability to release growth factors from the particles to assist cell and tissue development. In this system, the particles are biodegradable and disappear over time to leave a natural tissue. The research programme will initially optimize methods to cross-link the cells with polymer microparticles and proceed to determine the best conditions for the 3D formation of aggregate structures. To demonstrate its potential benefits, adult stem cells from bone marrow will be isolated and aggregated with polymer microparticles. These cells have the potential to develop into a number of different tissues depending on the culture conditions used. For this work, the cells will be aggregated with microparticles that release a growth factor that promotes bone formation. The success of this approach will be determined by examining bone-specific markers within the aggregates in comparison to aggregates that contained no growth factor. It is apparent that this technique has huge potential for the generation of replacement tissue for medical applications. Not only is it useful for bone engineering, but can also be applied to any cell or tissue type and relevant growth factors. Therefore, this could find widespread use in helping to overcome the huge socio-economic cost caused by injury and disease. This technology could also be used to develop more realistic 3D tissue models for fundamental biological research. For example, the ability to construct realistic tumour models in the laboratory offers the prospect of a greater understanding of cancer and the development of more effective treatments. In addition, this research could be applied to construct tissues for drug testing applications, decreasing the requirement to test pharmaceutical products on animals.

This project aims to form functional tissue structures in 3D culture by cross-linking surface-modified cells and polymer microparticles. The protein avidin has four high-affinity binding sites for the biomolecule biotin. Hence, by incubating biotinylated cells and microparticles, addition of avidin will cause their cross-linking into large aggregates. Initial work will involve preparation of porous PLGA microparticles of a similar size to suspended cells by spray drying or solvent evaporation. Chemical modification will then be performed by aminolysis or hydrolysis to introduce groups for the covalent coupling of biotin. Biotinylation will be assessed by flow cytometry after staining with fluorescent avidin. Cells will be biotinylated by oxidation of surface sialic acids and ligation of biotin hydrazide to the resulting aldehydes. Parameters for aggregation of the two biotinylated species will be optimized in stirred and rotary culture with aggregate size measured using light microscopy. Using optimized conditions, aggregates will be isolated at different times and embedded in paraffin or resin. Sections will be obtained with a microtome and analyzed for cell proliferation, microparticle degradation and the effect of particle porosity on cell viability. This will be achieved by microscopic analysis of histological stains, prior incubation with LIVE/DEAD stain and immunofluorescent detection of matrix proteins. This technique will then be applied to the osteogenic differentiation of rat mesenchymal stem cells (MSCs) in 3D using microparticles loaded with bFGF, an osteogenic growth factor. Initially bFGF will be encapsulated into microparticles and its release measured by a protein assay. Activity of released bFGF will be assessed by a specific bioassay. Following aggregation, osteogenic MSC differentiation will be analyzed by characterizing specific bone markers in aggregate sections or dissociated cells using light and fluorescence microscopy and flow cytometry.