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The cornea is a transparent tissue protecting the most anterior part of the eye and if it is damaged, it may result in a partial or complete loss of vision. Millions of patients are in need of donor cornea, but there is a substantial shortage of good-quality corneas and there is always a risk of tissue rejection when allogeneic tissues are transplanted. Tissue engineering offers a way to develop an artificial cornea using biomaterials together with the patients’ own cells to provide an alternative for conventional tissue transplantation. The objective of this thesis was to study and comprehend the current state of corneal tissue engineering research and investigate which biomaterials could be suitable for the purpose of creating a biomaterial-based corneal substitute. In this thesis, the experimental part concentrated on studying hydrogels with corneal cells. In the experimental part, the behaviour of immortalised human corneal epithelial cells (HCE) and stromal keratocytes (HCK) in 2D and 3D cultures were studied. The aim of the 2D cell culture study was to test how these two cell lines behave in chosen different culture medium options, three commercially available media (CnT-20, CnT-30 and CnT-Prime-CC) and HCE and HCK maintenance media. The best suited media were then used in 3D cell culture with hydrogels. There were hydrogels A, B, C and D and one commercial hydrogel. These were tested in 3D cell culture where keratocytes were mixed with the hydrogel and epithelial cells were seeded on top. The hydrogel-cell samples were cultured in CnT media for 7 to 14 days and air-lifted for 7 days for epithelial cell stratification. Cell proliferation in 2D culture was evaluated with PrestoBlue™ assay and cell behaviour and expression of relevant corneal proteins were analysed with immunofluorescence staining (IF). These results indicated CnT-20 to be the best option for both cell lines, but all three CnT media were tested also in 3D cell culture with hydrogels. The hydrogel experiments were also evaluated with IF staining. Interestingly, the most suitable medium for 3D cell culture was CnT-Prime-CC. The best suited hydrogel option in this study was hydrogel A and resulting cell-hydrogel samples were thin and soft but had clear, transparent and even structure.

Retinal diseases, especially age-related macular degeneration (AMD), are becoming a large issue as life expectancy increases. Blood-retinal barrier (BRB) is located adjacent to the retina and has many important functions in normal vision and it is the main site associated with AMD pathogenesis. BRB consists of retinal pigment epithelium (RPE), Bruch's membrane (BrM) and choriocapillaris endothelium. Mathematical modeling provides a tool to study the properties of the BRB and thus new treatment options for AMD. In experimental research, the BRB barrier function is usually measured with a so-called diffusion chamber. In this thesis, two models are constructed: 1) a passive diffusion model across the BRB based on the physicochemical properties of the BRB (BRB model) and 2) a model of a miniaturized half-perfusion chamber concept to study the effects of different parameters to the functionality of the system (chamber model). The predictive BRB model was constructed by combining physical theories from the literature to describe the hindered diffusion within a certain type of geometry. The main objectives were to construct the model, to study the behavior of the permeability of the whole BRB and its parts as a function of different molecular properties as well as to validate the model by using experimentally measured permeabilities. The chamber model was done by using finite element method (FEM) and by utilizing Comsol Multiphysics FEM modeling software. The main interest was on the effects of certain system dimensions and other chamber parameters to the error caused by the measurement system. The results of the BRB model indicate that it predicts the correct magnitudes of the BRB permeabilities, but fails to predict the more accurate behavior based on the molecular properties of the diffusing molecule, such as molecular size and lipophilicity. As assumed, the model confirms that RPE forms the most significant barrier in the BRB, as the BrM has affects only the very lipophilic molecules. With the chamber model, the results show that the functionality of this kind of half-perfusion chamber is limited by the free diffusion rate. The chamber dimensions and other parameters are less significant, as they mainly just change the diffusion distances. The proposed BRB model is the first model of this kind for the BRB. It can be utilized to study the barrier properties in normal function and in disease, especially with little refinements. There are several directions towards which the model can be taken in the future, such as dynamic regulation of the permeability. The chamber model clearly suggests that the free diffusion rate is the main issue with this kind of half-perfusion chamber. The ability of the diffusion chambers to measure the actual absolute values of permeability, and to provide reliable results, should be viewed with caution.

The number of people affected by degenerative diseases of the retina increases notably due to the longer life expectancy. The weakening of the sight concerns especially older people, who suffer from degeneration of the retina and its retinal pigment epithelium (RPE) monolayer. Currently, these degenerative diseases of the retina are incurable. Cell transplantation of healthy RPE cells is considered to be a potential treatment for these diseases. Due to the unlimited self-renewal capacity and pluripotency human pluripotent stem cells (hPSCs) are considered as possible cell sources for transplantation purposes. However, the current differentiation protocols for RPE cells from hPSCs are inefficient and result in low yields. The aim of this thesis was to study whether biodegradable poly(L-lactide) (PLLA) microparticles and commercial biostable cellulose anion exchange diethylaminoethyl (DE) 53 microparticles could be exploited in increasing the efficacy of differentiation of hPSCs into RPE cells. In addition, the potential of the microparticle culture system for stem cell maintenance in feeder-free conditions was evaluated. The attachment and proliferation of hPSCs on microparticles were analyzed by immunofluorescence analysis and microscopy. The success of differentiation of RPE from stem cells in microparticle cultures was evaluated by inspecting the appearance of pigmentation in cultures as well as by investigating the expression of RPE maturation markers on gene expression levels by RT-PCR. The PLLA microparticles did not support the attachment and proliferation of hPSCs in feeder-free conditions. The poor performance of PLLA particles in cell culture studies might be due to the hydrophobic nature of the particle surfaces. In contrast, hPSCs attached, proliferated and maintained their pluripotency on DE 53 particles. Furthermore, the hPSCs were successfully differentiated into RPE cells on these DE 53 particles. However, the differentiation efficacy did not outperform the conventional differentiation protocol. This was the first study in which the hPSCs were successfully differentiated into RPE cells on microparticles. Microparticles are far from being routinely used in hPSC maintenance and differentiation to mature cell types. More long-term and large-scale studies need to be conducted in the future. Nevertheless, at the moment they seem appealing and promising with a possibility to grow stem cells in large-scale and cost effectively. The microparticle culture systems set challenges for the cell analysis due to their 3-dimensional structure and autofluorescence property. Therefore, novel methods for characterization, analysis and imaging of microparticle culture systems should be developed. /Kir12

Age-related macular degeneration (AMD) and some other retinal diseases are associated with degeneration of retinal pigment epithelium (RPE) cells. A potential cure could involve cell transplantation therapy using RPE cells differentiated from pluripotent human embryonic stem cells (hESCs). A big challenge is to produce a homogeneous population of hESC-derived RPE (hESC-RPE) cells, and coating materials are one potential alternative to promote the differentiation. The aim of this study was to evaluate the cell attachment and differentiation of hESC-RPE cells on different coating materials and culture conditions. hESCs were derived and maintained without serum and animal feeder cells. This study consisted of three parts. In the first part, hESCs were cultured on different xeno-free coating materials and in a culture medium that stimulated the differentiation towards RPE cells. CELLstart™, collagen IV, and VitroCol™ were the best coating materials to support the cell attachment. The differentiation of the hESCs towards RPE cells was analyzed by visual pigment observation, immunocytochemistry, and quantitative real time PCR. There were no significant differences in the differentiation of the cells cultured on CELLstart™, collagen IV, or VitroCol™. In the second part, confocal microscopy wells were tested. hESCs differentiating to RPE cells and enzymatically degraded hESC-RPE cells attached and grew best in collagen IV coated Ibidi wells. In the third part, hESC-RPE cells were cultured in BD BioCoat™ collagen IV cell culture inserts in culture mediums containing basic fibroblast growth factor (bFGF) or not. RPE cells attached and grew well in the inserts with or without bFGF. The good attachment of hESCs differentiating towards RPE cells to CELLstart™, collagen IV, and VitroCol™ coatings was a remarkable result, since to my knowledge no publication of hESC differentiation towards RPE cells with a xeno-free coating material and a serum-free culture medium in adherent cell culture exists. Collagen IV can be recommended as a coating material for hESCs differentiating towards RPE cells, since it is cheaper than CELLstart™ and VitroCol™ and easy to use. In the future, the effects of growth factors and other supplements on RPE differentiation could be studied. Collagen IV can also be recommended for Ibidi confocal microscopy wells for hESC-RPE cells, at least for short-term experiments. Longer-term effects should be further studied. BD BioCoat™ collagen IV cell culture inserts can be utilized in research purposes, but they are not optimal since they contain xeno-products. In the future, the ability of bFGF to promote RPE cell cluster distribution and to transdifferentiate RPE cells could be further studied. /Kir10