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SpeCCC Project - description and current current status

The aim of the SpeCCC project is to address the limitations in the current 3D cell culture systems that are preventing their widespread use. These limitations include: 1) Homogeneous scalable bioreactor systems have generally not been developed specifically for 3D microtissue cultures and the systems that are available (e.g. hollowfibre systems) are not ideal for 3D microtissue culture due to diffusion limitations; 2) Hydrogel carriers have been used for 3D microtissues in scalable bioreactors, but hydrogel carriers can have issues with inefficient diffusion of nutrients and waste metabolites, and lack of mechanical stability of the carrier; 3) Many of the liver cell lines currently used for 3D microtissues demonstrate poor retention of key liver specific functions. These limitations will be investigated in the SpeCCC project

The core objective of the SpeCCC project is to develop specialised functional liver cell carrier components for use in fluidised bed bioreactors, and 96 well plate formats. The other objectives are; 1) to develop a homogeneous fluidised bed bioreactor which has the potential to be scaled up for use in a bioartificial liver device (BAL) and scaled down for use in a multi microbioreactor system (for drug testing and); and 2) to develop a microwell plate toxicology/drug test kit based on the functional cell carrier component.

The cell carrier component is the central element of all these applications and therefore it is a prerequisite for their development. The two parts that make up the cell carrier component are 1) the true functional immortalised liver cell lines (human hepatocyte and human endothelial cells), and 2) the second generation porous silicone carrier, which has a defined and optimised geometry, diameter (i.e. defined and optimised diffusion distance), pore size, pore volume, surface (for cell attachment), specific gravity and mechanical stability.

Current status of projects:

Significant progress has been made towards the development of these cells lines in WP 2. Immortalised cell lines have been established from primary human hepatocytes and liver sinusoidal endothelial cells. Significant testing has been carried out on both cell types demonstrating that the novel liver sinusoidal endothelial cell lines display a primary-like endothelial cell phenotype. The results obtained for the novel hepatocyte cell lines indicate that these cell lines are in a premature state which can be further differentiated by modulating the culture conditions of these cells. Progress has also been made in WP 4 in developing a range of weighted and non-weighted carriers with different characteristics for testing in WP 5 and 6. Plasma treatment of the second generation carriers has been carried to make the surface of the silicone hydrophilic and thus more suitable for the cell growth.

Mathematical Modelling on mass-transfer effects has been carried out in work package 5 and 6. The assumptions on the cell numbers growing on the carriers were based on the results of the growth studies. This work is fully integrated with the carrier design and manufacturing work in WP 4.

Studies have been carried out on the growth of model hepatocytes (HEP G2) and immortalized hepatocytes (SpeCCC Hep) as well as endothelial (SpeCCC EC) cell lines (singly and in co culture) in the first generation carriers (Immobasil FS). For the second generation carriers, cell attachment, cell numbers and viability were followed until reaching the steady state. The immortalized cell lines provided by InSCREENeX were grown successfully in mono and co culture on improved carriers from WP 4. This will provide useful data for the experimental work on the second generation trial materials generated in Work Package 4. A 10 mL fluidized bed has been set-up and characterised with respect to fluidization of ImmobaSil FS and 2nd generation carriers. Different seeding protocols for the model cell line Hep G2 in the first generation silicone carriers (Immobasil HD) in a fluidised bed bioreactor were evaluated in Work Package 6. Further work is on-going to improve the design and performance of the fluidised bed bioreactor to achieve the SpeCCC target yields.