| Abstract [eng] |
The aim of this master thesis project was by using a human glioblastoma cell line to select optimal conditions for the cultivation of a 3D in vitro model of glioblastoma with a hypoxic gradient and to apply it for exosome-encapsulated anticancer medicine efficiency testing. To achieve this goal, the following objectives have been set: 1) to create in vitro 3D model of human glioblastoma and to evaluate the formation of a hypoxic gradient; 2) to isolate exosomes from human microglia and monocytes and identify them by size and specific markers; 3) to load temozolomide and doxorubicin hydrochloride into human microglial and monocyte exosomes and evaluate drug loading efficiency; 4) to determine the effect of temozolomide and doxorubicin hydrochloride loaded microglial and monocytic exosomes on the metabolic activity and migration of cells in the designed 3D glioblastoma in vitro model. Glioblastoma 3D model was created from HROG-36 immortalized cell line grown in a round-bottom well on a thin layer of agarose. The formed glioblastoma spheroids were encapsulated and cultured in the extracellular matrix hydrogel Geltrex. The formation of hypoxic regions in the glioblastoma 3D model was determined by confocal microscopy, by labelling thiol groups in hypoxic cells with pimonidazole hydrochloride and then staining with fluorescent RED 549 MAb1. Exosomes from HMC-3 microglial and U937 monocyte culture medium were isolated by polymer precipitation/centrifugation technique, evaluated for protein amount by spectrophotometric Bradford assay, characterized for particle size using the dynamic light scattering method, and for CD9 and CD81 markers by ELISA method. Temozolomide and doxorubicin were loaded into the exosomes by electroporation. The amount of the encapsulated medicines was assessed by high-performance liquid chromatography and fluorescence intensity (doxorubicin). The metabolic activity of glioblastoma cells was measured by fluorescent PrestoBlue assay. The migration of glioblastoma cells in the extracellular matrix was assessed by light microscopy, and the images were analysed by ImageJ software. Statistical data analysis was performed using SigmaPlot 13.0. software. Hydrogel incapsulated glioblastoma 3D model with hypoxic gradient was created using human HROG-36 cell line. The typical size of isolated monocyte exosomes was about 120 nm, and of microglial exosomes – about 90 and 140 nm; the exosomes from both cell types had CD9 and CD81. The loading efficiency of the drug into HMC-3 microglial exosomes was higher compared to U937 monocyte exosomes. Better loading efficiency was determined for temozolomide compared to doxorubicin hydrochloride. Exosomes from monocytes and microglia loaded with anticancer drugs significantly reduced metabolic activity and invasiveness of glioblastoma cells cultured in hydrogel-encapsulated spheroids. |
