Investigation of 3D Bioprinted Cocultures as Liver Tissue Models
Typ
Examensarbete för masterexamen
Program
Biotechnology (MPBIO), MSc
Publicerad
2019
Författare
Mohammed, Maram
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Liver fibrosis is a strong risk factor for liver cancer and has become a major concern worldwide. All chronic liver diseases (CLD) can lead to liver fibrosis. Nonalcoholic fatty liver disease (NAFLD) and its more severe form nonalcoholic steatohepatitis (NASH) are of particular interest to pharmaceutical companies. One obstacle in the development of efficient therapies is the lack of robust, representative, and biologically relevant in vitro models of human liver fibrosis to aid the development phase of pharmaceuticals. This is due to the fact that most in vitro models used today are 2D mono layer cultures of stellate cells. These models ignore the importance of the interplay between hepatocytes and stellate cells in liver fibrosis as well as the role played by spatial organization of the different cells.
Bioprinting allows the production of tissue-like structures that mimic the 3D architecture and complexity of native tissue. In this project two different hepatocyte to stellate cell ratios (4:1 and 2:1) in addition to a hepatocyte monoculture were 3D bioprinted to produce liver tissue models. Fibrosis was then induced by addition of the growth factor TGF-β resulting in a fibrotic liver tissue model. Auxiliary proteins supporting liver specific cells were incorporated in the bioink used. The produced fibrotic liver tissue model’s viability and functionality were assessed using different assays. Namely, Live/Dead analysis, Celltiter-Blue viability assay, Lactate dehydrogenase (LDH) cytotoxicity assay, Bicinchoninic acid (BCA) protein assay and Actin/NucBlue staining. These assays were originally designed to be used on cells in 2D and not on cells imbedded in a bioink/hydrogel. These assays’ applicability for use on cells bioprinted in 3D was therefore evaluated before they were used to analyze the produced bioprinted fibrotic liver tissue models.
It was possible to adapt the mentioned assays to be used with 3D bioprinted constructs. Viability, cytotoxicity and protein deposition results indicated that the 2:1 coculture ratio of HepG2 (Hepatocytes) to LX-2 cells (Hepatic stellate cells) showed greater potential in simulating liver fibrosis compared to the 4:1 ratio of HepG2 to LX-2 cells. There did not seem to be a significant difference between the group in which fibrosis was induced by TGF- β compared to the control group in any of the conditions investigated. This indicates that fibrosis may have in fact not been induced despite the addition of TGF- β as both groups were behaving in a similar manner. Further investigation is needed in this regard.
Beskrivning
Ämne/nyckelord
Liver fibrosis , Cocultures , Bioprinting , HepG2 , LX-2 , 3D model