Browsing by Subject "suspension"

Oat beta-glucan, β-glucan, is a soluble dietary fibre located in the endosperm and aleurone cell walls. It is a polysaccharide that has an ability to form viscous gel in aqueous solutions due to its high water binding capacity. The objective of this study was to determine the physical characteristics of commercial oat β-glucan with different purity in solution. Four commercial oat beta-glucan powders with varying β-glucan content were provided for this study. Solubility, water binding capacity, viscosity, suspension and emulsion stability, droplet particle size, particle distribution and zeta potential were determined from the samples. There were two phases in the experimentation part in which the first phase focused on suspensions and the second phase focused on emulsions. There were four different homogenising treatments used to solubilize the samples into water during the first phase of the experiment, which were stirring in room temperature, 80 ˚C, Ultra-Turrax and microfluidization. Megazyme exhibited highest solubility in water with a solubility rate of 100 % regardless of the treatment used. OatWell had the highest water binding capacity (11.7 g/g) after heat treatment. Heat treated OatWell had the highest viscosity (590 mPas). Megazyme emulsions had the highest emulsion stability (TSI ranging from 0.6 to 3.6) as it did not experience phase separation until fifth day of measurement. Megazyme emulsions also had the lowest particle sizes ranging from 0.4 μm to 1.5 μm. The results of this study highlight that the content of β-glucan has notable effect on its solubility and the homogenisation treatment used has an increasing effect on solubility and decreasing effect on water binding capacity and viscosity. Purity of the oat β-glucan also has an effect on emulsion stability as high purity oat β-glucan are able to stabilise emulsion system after homogenisation.

Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.