| Popis: |
Background and objectives of the study: This master’s thesis consists of three parts involving induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs). iPSC-HLCs provide an alternative cell model for primary human hepatocytes (PHHs) and hepatoma cell lines. They have an unlimited cell source, while simultaneously having similar characteristics to PHHs, which make them excellent candidates for hepatic in vitro research. Multiple iPSC-HLC differentiation protocols, many of which involve three separate stages, have been published so far. The first part of this thesis investigates whether this continuous hepatic differentiation could be divided into two sections separated by a cryopreservation period for more time-efficient studies in the future. In order to better mimic the in vivo physiological environment, three-dimensional (3D) cell cultures have been developed alongside conventional two-dimensional (2D) cell cultures. Different 3D cultures have shown their potential with hepatic cells as well, as they seem to improve the liver-specific characteristics of the cells. Thus, the second and third part of the thesis concentrate on 3D culturing of iPSC-HLCs. To investigate whether 3D culture conditions support the state of hepatic maturity better than corresponding 2D conditions, mature iPSC-HLCs were cultured in a novel hydrogel consisting of gelatin and gellan gum (GG). In the third part, microfluidic chips were tested together with Geltrex to investigate the effect of gravitational flow and perfusion for the maturation of HLCs while simultaneously testing the suitability of the hydrogel for 3D cultures. Materials and methods: In the first part, three different methods (M1-M3) were used to differentiate definitive endoderm (DE) cells from iPSCs. Their differentiation efficiency was confirmed by flow cytometry. After cryopreservation and thawing, further hepatic differentiation was carried out with one of the methods (M1), which was consequently used in the remainder of the experiments. In the second part, a combination of gelatin and GG was used as a hydrogel to compare 3D culturing of mature iPSC-HLCs to two 2D culture conditions: 2D gelatin-GG and 2D Geltrex. Lastly, in the third part, custom-made chips received from Pasi Kallio group and commercial chips from AIM Biotech were used together with Geltrex for culturing maturing iPSC-HLCs. Perfusion of medium was also added to some of these microfluidic devices. Live/dead assays were used for evaluating cell viability during the experiments. Finally, both RT-qPCR and immunofluorescence were used to determine the stage of hepatic differentiation and cell maturation. Results: Based on immunostaining results and cell confluencies, the first studied DE differentiation method (M1) proved to be the most successful in terms of cell viability and differentiation efficacy after the cryopreservation period. Even though all the three methods successfully generated differentiated cells, the cell amount and morphology were the most similar to that of normal hepatocytes in M1. Cells that were cultured on top of gelatin-GG (2D) had the highest expression of liver-specific genes while simultaneously having evident albumin staining, indicating that these cells had kept their hepatic phenotype during the culture period better than cells cultured in the other two conditions. 3D conditions seemed to have lower liver-specific gene expression compared to the 2D culture conditions. However, as most of the 3D hydrogel was washed away during the immunostaining process, no definite conclusions could be drawn for the suitability of gelatin-GG for 3D cultures. Commercial chips proved to be more suitable for 3D culturing of maturing iPSC-HLCs compared to custom-made chips. The hydrogel used in the experiments, Geltrex, did not seem to hinder the maturation of the cells but at the same time, was not stiff enough to support 3D cell cultures for extended periods. Conclusions: M1 seems to be the most suitable method for producing DE cells that can be cryopreserved and later used for further hepatic differentiation and future studies. Additionally, gelatin-GG should be investigated more to verify its suitability for 3D cultures, as it proved to be rather suitable for 2D cell culturing. Moreover, the chip experiments suggest that especially the commercial chips are suitable for hepatic differentiation provided that the hydrogel in which the cells are embedded is stiff enough to support the 3D structure. Taken together, these findings give a platform for future studies involving 3D culturing of iPSC-HLCs. |
