Browsing by Subject "suspensio"

Polyakryyliamidille, maailman käytetyimmälle maan stabilointiaineelle, on vuosien ajan pyritty löytämään korvaava biopolymeerivalmiste. Tässä tutkimuksessa tutkittiin UPM:n tuottamien anionisen, kationisen sekä kemiallisesti muokkaamattoman koivuperäisen mikrofibrilloidun selluloosan (MFS) stabilointi- ja flokkulointiominaisuuksia. Tuloksia verrattiin Kemiran tuottamien anionisen tai kationisen polyakryyliamidin (PAM) stabilointi- ja flokkulointikykyyn. Kokeet aloitettiin esikokeilla, joilla hiottiin varsinaisten stabilointi- ja flokkulointikokeiden menetelmiä. Esikokeiden perusteella varsinaisten stabilointikokeiden murukooksi valittiin 1-2 mm murut. Stabilointikokeita (45 tai 90 kg/ha) tehtiin myös hienojakoisille 0,06-0,2 mm ja 0,2-1 mm seulotuille kahdelle hiesusavimaalle. Esikokeet hienojakoisella maalla kertoivat, että MFS-valmisteet toimivat PAM:ia heikommin testatuissa olosuhteissa. Suspensiokokeiden esikokeissa testattiin ulkopuolisen kalsiumlähteen vaikutusta polymeerivalmisteiden flokkauskykyyn. Kalsiumin voimakkaan flokkaavan vaikutuksen vuoksi tämä jätettiin pois varsinaisista kokeista. Myös anioniset polymeerivalmisteet jätettiin pois varsinaisista suspensiokokeista heikon tehon vuoksi. Varsinaisissa aggregaattikokeissa ilmakuivia 1-2 mm:n kokoluokkaan seulottuja hiesusavi- ja hiesumuruja käsiteltiin esikokeissa käytetyillä MFS-valmisteilla sekä anionisella PAM:lla (45 tai 90 kg/ha), minkä jälkeen muruja kuivatettiin 3 vuorokauden ajan +21 °C tasalämpöhuoneessa. Kuivatuksen jälkeen maamurujen kestävyys äkillistä kostumista vastaan testattiin muruanalysaattorilla. Maanäytteet ajettiin laitteella kahdesti siten, että murut ehtivät kuivua kahden vuorokauden ajan huoneenlämmössä. Maanäytteet testattiin myös mekaanisen rasituksen kestävyyden suhteen 10 minuutin kuivaseulonnalla. Kationisoitu ja kemiallisesti muokkaamaton MFS soveltuivat biopolymeereistä parhaiten murujen mekaanisen rasituksen kestävöittäjiksi. Kationisoitu ja anionisoitu MFS suojasivat muruja parhaiten veden dispergoivalta vaikutukselta. Suspensiokokeissa 0,1 g ilmakuivaa <0,06 mm:n ja 0,06-0,2 mm:n kokoluokkiin seulottuja kahta hiesusavi- ja yhtä hiesumaata suspendoitiin 30 ml:aan deionisoitua vettä. Suspensioon lisättiin MFSvalmisteita sekä kationista PAM:ia siten, että suspension polymeerikonsentraatioksi saatiin 2 tai 6 mg/l. Suspension sameutta seurattiin 10 minuutin ajan ja viimeisen kerran vuorokauden jälkeen turbidimetrin avulla. MFS-valmisteista parhaiten toimi flokkulanttina kationinen valmiste, joka kirkasti suspensiota lähes yhtä tehokkaasti kuin PAM.

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.

The physical stability of plant-based beverages is often weak and is used to be improved by food additives. β-glucan and dextran are hydrocolloids that have been found to increase fluid viscosity and therefore may have the potential to replace E-coded hydrocolloids. The aim of this master's thesis was to investigate the effects of oat β-glucan and microbiologically produced dextran on the physical stability of a drinkable oat snack. In addition, the aim was to determine the importance of the molecular size and concentration of β-glucan for its stabilizing ability. The oat base without any added hydrocolloids served as a control. β-glucan and dextran were added to the oat base as extracts so that they replaced a certain volume of oat base water. To determine the concentration effect of β-glucan, an oat beverage containing a high molecular weight β-glucan extract extracted from oat bran was prepared in three different extract concentrations. To determine the effect of the molecular weight, an oat beverage containing an enzymatically hydrolyzed low molecular weight β-glucan extract was prepared. The effects of dextran were studied by preparing oat beverages containing exopolysaccharide extract (EPS) (dextran positive and negative sample). Samples were stored in a refrigerator for 14 days, during which time their stability was characterized by observing phase separation and measuring viscosity, particle size and turbidity. The stability of the beverage samples decreased because of sedimentation, leading to the visual phase separation at the top of the samples. The high molecular weight β-glucan extracted from the oat bran reduced sedimentation and phase separation, and stability and viscosity increased as the extract concentration increased from 5% to 7.5%, with almost invisible phase separation in the latter. Thereafter, increasing the extract concentration to 10% no longer reduced the phase separation, but resulted in a decrease in viscosity during storage. Low molecular weight β-glucan (extract content 10%) did not increase the stability of the oat base, and the viscosity of the sample decreased by about 60% during storage. The decrease in viscosity in these samples may be due to the aggregation of proteins and / or β-glucan due to the higher β-glucan content. The effects of dextran on the stability of the oat beverage remained unclear, as the dextran-positive and negative samples did not differ significantly in viscosity or phase separation, so further studies are needed. Based on this thesis, high molecular weight β-glucan appeared to be a potential substitute for food additives in oat-based beverage applications.