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The aim of this work was to develop a 3D microenvironment for glioblastoma brain tumor engineering based on alginate hydrogels as a matrix for cell immobilization followed by cultivation in a biomimetic perfusion bioreactor. Alginate microfibers with immobilized cells were obtained by a simple extrusion technique. We have examined the influence of the needle diameter (22G - 28G), cell density in alginate solution (1 x 106 - 8 x 106 cells/ml) and different cancer cell lines (rat C6 and human U251 and U87) on cell immobilization efficiency and viability. The best alginate microfibers (500 µm in diameter) with all immobilized cells were obtained by applying a 25G needle with a minimal cell density of 4 x 106 cells/ml. The obtained microfibers with immobilized cells (C6 and U87) were cultivated in a perfusion bioreactor at the continuous medium flowrate in the range 0.05-0.30 ml/min over short- and long-term cultivation periods. The results have shown that the flowrate of 0.30 ml/min, corresponding to the superficial velocity of 100 µm/s, in combination with the C6 cell density of 8 x 106 cells/ml in short-term studies yielded higher cell viabilities and proliferation as compared to the control static culture. In addition, U87 cells immobilized in alginate microfibers at the density of 4 x 106 cells/ml after long-term cultivation at the medium flowrate of 0.05 ml/min (superficial velocity of 15 µm/s) stayed viable. The overall results have shown potentials of the applied approach for tumor engineering provided optimization of cultivation conditions for each cell type.

Glioblastoma is the most common and aggressive malignant brain tumor in adults. Existing treatment choices that include surgery, radiation and chemotherapy are not successful in long-term survival, while development of new anticancer drugs is being held back by the lack of adequate model systems for anticancer drug testing. Namely, in traditionally used two-dimensional (2D) monolayer cancer cell cultures the native cell morphology, polarity and interactions between both cells and cells and extracellular components are either changed or absent, while studies on animals often produce misleading results due to interspecies differences. Hence, there is a pressing need for new glioblastoma model systems that provide more in vivo-like environment for investigation and development of new anticancer drugs. The aim of this work was to develop a biomimetic 3D environment for cultivation of glioblastoma cells based on alginate microfibers as cell carriers and perfusion bioreactors. Previous studies have shown that static cultures of cervical cancer cells SiHa immobilized in alginate microfibers may be diffusion limited while perfusion, which enhanced mass transport, has induced negative effects on human embryonic teratocarcinoma cells NTERA-2 in superficial zones of alginate microbeads by hydrodynamic shear stresses. Thus, in the present study, the specific focus was on optimization of cell concentration within microfibers and regimes of cultivation to achieve beneficial effects of fluid flow in perfusion bioreactors. A series of experiments were conducted in which the concentration of rat glioma cells C6 was varied between 2 and 8 × 10 6 cell cm -3 at several flowrates and regimens of static and perfusion culture periods. Mixed results were obtained implying that efficient mass transport has a higher effect in microfiber cultures at lower cell concentrations (i.e. ~2 × 10 6 cell cm -3 ). In specific, medium flow at the superficial velocity of 100 µm s -1 induced considerable cell proliferation as compared to control static cultures, which maintained the initial cell numbers. Mathematical modelling indicated that the convective transport of substances with low diffusion coefficients (~10 -19 m 2 s -1 ) may have induced the observed positive effects. Still, exact relations of cultivation conditions and cell responses in terms of viability, proliferation and metabolic activity should be further investigated.

Bilayer scaffolds based on gellan gum (GG) and nanoparticulate bioactive-glass (BAG) were developed by an integrative approach based on engineering principles and characterization in biomimetic bioreactors. The osteo-inductive GG-BAG layer containing 2 % w/w GG and 2 % w/w BAG (composition: 70 n/n % SiO2, 30 n/n % CaO) was produced by gelation followed by freeze-drying to obtain open porosity in axial and radial directions. The chondral layer was obtained by dispensing a warm 2 % w/w GG solution at 60˚C over the frozen macroporous GG-BAG layer at -25˚C. The temperatures were optimized by applying a one-dimensional unsteady-state heat transfer model so to obtain a thin integration zone, 0.5 – 1 mm thick. The scaffolds were evaluated regarding bioactivity in a biomimetic bioreactor with specially designed chambers to provide supply of two media relevant for chondral and bone tissues. In the present experiment, simulated body fluid (SBF) was supplied countercurrently continuously during 14 days of the experiment (1.1 ml min-1 flowrate), while dynamic compression (5 % deformation, 0.68 Hz frequency, 337.5 µm s-1 loading rate, 1 h / day) was applied on the chondral layer, from day 7 to day 14. SEM analyses have confirmed the retained integrity of the scaffolds, as well as formation of hydroxyapatite (HAp) uniformly throughout the osteo-layer of the scaffolds.Significantly higher bioactivity under biomimetic conditions compared to static controls resulted in slightly but significantly increased compression modulus. These results indicated a high potential of the applied integrative strategy for the development of biomimetic bilayer scaffolds.

Cancer cell immobilization in polymer hydrogels serving as extracellular matrices and cultivation in perfusion bioreactors that provide appropriate chemical signals, efficient mass transfer and hydrodynamic shear stresses is a promising strategy for development of physiologically relevant tumor models. In this work, perfusion cultures of 2 cancer cell types (C6 rat glioma and embryonal carcinoma NT2/D1 cells) immobilized in alginate microgels were established, while static cultures served as controls. Continuous perfusion had different effects on the cultured cells inducing enhanced proliferation of the glioma cells immobilized in microfibers (8x10^6 cell/ml), while reducing the viability of the NT2/D1 cells immobilized in microbeads (1x10^6 cell/ml). In order to elucidate the observed effects, chemical engineering principles were applied to assess mass transfer and hydrodynamic conditions. The second Fick’s law was solved analytically while the diffusionadvection-reaction equation was solved numerically to model mass transport in the static and bioreactor cultures, respectively. Moreover, Reynolds numbers, pressure drops and shear stresses in bioreactor cultures were calculated for assessment of flow regime and hydrodynamic conditions. The modeling results have indicated that oxygen transport is diffusion-controlled through the alginate hydrogel, while medium perfusion improves mass transfer of larger compounds having smaller diffusion coefficients (∼10^(-13) m^2/s), which possibly stimulated glioma cell proliferation. On the other hand, the obtained shear stress (~50 mPa) in the perfused packed bed of microbeads was above physiological levels, which provided the explanation of the poor NT2/D1 cell survival. This study stresses the importance of multidisciplinary approach in addressing such multifactorial diseases as cancer.

Scaffolds used for osteochondral tissue engineering should comprise two distinct regions: a bottom region with characteristics corresponding to bone tissue, such as a porous structure with mineral components (predominantly hydroxyapatite), and a top region with characteristics of articular cartilage, which is gelatinous with high water content. In this work, we have investigated possibilities to formulate and optimize a procedure for obtaining such biphasic scaffolds based on gellan gum (GG). A porous base layer of the scaffold was obtained by lyophilization of the 2 % GG hydrogel with dispersed bioactive glass nanoparticles, as hydroxyapatite precursors. Next, different procedures were investigated to produce the upper GG hydrogel such as partial immersion of the porous layer in the GG solution and pouring the GG solution over the porous layer at different moisture conditions and temperatures. A simple mathematical model was derived and subsequently experimentally validated to find optimal temperatures of the porous layer, GG solution and the surrounding environment to provide adequate gelation rate to form the GG hydrogel on top of the porous layer with a thin interfacial zone.