The main goal of my work was to investigate different multicellular spheroids generated with different three-dimensional in vitro cell culture techniques in order to demonstrate their advantages over the two-dimensional culture method widely used today. In the current work, the results of the experiments carried out on the established spheroids are presented, which led to four main findings:

i. The use of RAFT TM provided excellent results in preserving islet spheroid viability, structure integrity and insulin, glucagon production for at least 18 days ex vivo.
Based on our data, RAFT can be a promising tool both in research and therapy as well, as there is a great demand in the clinic for therapies where diabetic patients can replace their insulin supply with insulinproducing xenograft islets implanted in their bodies. To the best of our knowledge, our group’s findings was first to report the successful maintenance of pancreatic islets using RAFT TM technique in vitro.

ii. Gene expression analysis by nanocapillary qRT-PCR (624 genes) and 1536 well high-throughput qRT-PCR (62 genes) resulted in the selection of lung cancer markers associated with higher (TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24) or lower (EGFR) expression in vivo or in 3D in vitro models compared to 2D monolayer cultures.

iii. The implemented multidimensional single cell proteomic profiling revealed that 3D (Cytodex3 and Nutrisphere) cultures represented a transition from 2D to in vivo situation by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66,GLUT1, CD274. In 3D systems CA9, CD24, EGFR showed higher expression than in vivo.

iv. Using human MCF-7 and mouse 4T1 mammary tumor cells, we
demonstrated the different chemoresistance of 2D and 3D cell cultures. The performed screening investigated that 6 out of the 16 active small imidazo[1,2-b]pyrazole-7-carboxamides molecules had different antitumor activity with minimum two and a half fold difference in potency using 2D and 3D in vitro cultures of same cell lines. In these cases, 3D cultures were less sensitive to the agent of interest, including the widely used and well-known doxorubicin. Our findings showed the reduced chemosensitivity of multicellular spheroids.