Controlled particle production by membrane emulsification for mammalian cell culture and release

Existing commercially available microcarriers are very efficient at encouraging cell attachment and proliferation. However, recovery of the cells is problematic as it requires the use of proteolytic enzymes which are damaging to critical cell adhesion proteins. From this perspective, temperature responsive polymers appear to be a valid option. The current innovative study is to produce and engineer microcarriers in terms of particle size, surface coating and properties, as well as thermo-responsiveness for cell release. All these benefits are based on particle production by membrane emulsification to provide a highly controlled particle size. The polymer of choice is poly N-isopropylacrylamide (pNIPAM) because of the sharpness of its phase transition, biocompatibility and transition temperature close to the physiological value. These characteristics make pNIPAM a very attractive material for Tissue Engineering applications. Cells are cultured on the hydrophobic surface at 37°C and can be readily detached without using proteolytic enzymes from the surface by lowering the temperature to room temperature. The Dispersion Cell (MicroPore Technologies Ltd, UK) was successfully employed for the production of W/O emulsions. The generated monomer droplets were additionally solidified by applying a free radical polymerisation to manufacture solid pNIPAM microspheres. Additionally, calcium alginate particles were also generated and further functionalised with amine terminated pNIPAM to form temperature responsive core-shell particles by simply taking advantage of the electrostatic interactions between the carboxyl groups of the alginate and amino groups of the modified pNIPAM. Controlled particle production was achieved by varying process parameters and changing the recipe formulation (e.g. monomer concentration, surfactant concentration, pore size and inter-pore spacing, injection rate, shear stress applied at the membrane s surface). The manufactured particles were then analysed in terms of particle size and size distribution, chemical composition, surface analysis, shrinkage ratio and thermo-responsiveness and further sterilised and used for cell culture and release experiments. Swiss Albino 3T3 fibroblastic cells (ATCC, USA) were utilised to show proof-of-concept for this technology. Cell attachment and proliferation were assessed and successfully demonstrated qualitatively and quantitatively. pNIPAM solid particles, uncoated and with different protein coatings were shown to allow a limited degree of cell attachment and proliferation compared to a commercially available microcarrier. On a different approach, uncoated core-shell structures demonstrated improved capabilities for cell attachment and proliferation, similar to commercially available microcarriers. Having in mind the potential of temperature responsive polymers and the aim of this innovative study, cell detachment from the generated microcarriers was evaluated and compared to a commercially available temperature responsive surface. Necessary time for detachment was recorded and detached cells were recovered and reseeded onto tissue culture plastic surfaces in order to evaluate the replating and reattachment capabilities of the recovered cells. Successful cell detachment was achieved when using the core-shell structures as cell microcarriers, but the necessary time of detachment was of an order higher than that for the commercial temperature responsive surface.