Development of Advanced Technology to Enhance the Growth and Maintenance of Liver Cells and Tissue In Vitro

Abstract

In vitro cell culture models are widely used for much of the research carried out into human diseases and treatments as well as more basic biological research. Due to animal testing becoming increasingly undesirable, the access to accurate in vitro models is of utmost importance. For many
applications the models used lack physiological relevance, often consisting merely of a single cell line grown in a plastic dish. Techniques such as three-dimensional cell culture have started to bridge the gap between the in vitro and in vivo cellular microenvironment. To take this a step further, perfusion bioreactors can be used to provide dynamic fluid flow to cultures and mimic the vasculature in vivo. These systems commonly suffer from drawbacks such as high cost and complexity as well as large size.

In this project a novel perfusion bioreactor system was developed for use with three-dimensional cell culture models. The aim was to create a system which could provide the same levels of physiological complexity as other systems currently in use whilst reducing cost and complexity and therefore maximising accessibility. To this end, the liver was chosen as a model organ for testing the efficacy of this system due to its high levels of vascularisation suggesting it would benefit greatly from the addition of medium perfusion, as well as the large amount of literature based around perfusion systems for in vitro liver models. The system designed for this purpose utilises magnetic stirrer-based perfusion to keep the apparatus simple whilst allowing ease of use and high levels of scalability. An Alvetex® insert is held within the system, providing a complex three-dimensional microenvironment to support the cells and further enhance their structure and function. Extensive optimisation and characterisation of this system was performed to ensure a detailed understanding of the conditions experienced by cultured cells and tissues.

It was found that the novel bioreactor system could greatly increase the expression of key hepatic genes in the HepG2 cell line whilst improving the localisation of many important structural and function proteins. Further use of this system demonstrated the efficacy of this model for modelling drug toxicity. The Alvetex® support for the cells also allows for co-culture of multiple cell types in different organisations. The use of such a co-culture setup was demonstrated with the HepG2 models by successfully incorporating fibroblasts and endothelial cells.

In a different model, utilising precision-cut liver slices, the system demonstrated potential for maintaining the viability of the liver slices over prolonged periods over up to 2 weeks. Inclusion of an oxygenation system to function alongside the bioreactor has the potential to further promote the viability of precision-cut liver slices, however more research would be required to accurately measure any improvements due to this system. Such a model, if successful, could be of significant research value for the study of chronic diseases such as liver fibrosis.

The results found herein utilising the novel perfusion bioreactor show that complex tissue equivalents can be created using simple, low cost and user-friendly apparatus. Whilst the system was successful for the culture of liver models, the level of control over the properties of the system also gives it potential for use with other organ models and therefore could become a useful platform for a wide range of in vitro and ex vivo research in the future.