Investigating Bioprintable Hydrogels, Glycosylation Modulation, and Synthetic Tethering Strategies for Heterotypic Spheroid Models

This work was conducted within subproject B05 of the Collaborative Research Centre (CRC) 225 “From the Foundations of Biofabrication to Functional Tissue Models” and carried out in collaboration between the Department of Musculoskeletal Tissue Regeneration (Prof. Dr. Regina Ebert), the Institute of Organic Chemistry (Prof. Dr. Jürgen Seibel), and the Department of Macromolecular Chemistry (Prof. Dr. Lutz Nuhn) at the Julius-Maximilians-Universität Würzburg. The overarching aim of this thesis was to investigate complementary strategies for controlling the cellular microenvironment, focusing on (i) thermoresponsive hydrogels for bioprinting, (ii) metabolic modulation of glycosylation, (iii) preliminary polymer-based surface anchoring, and (iv) engineered cell-cell tethering for heterotypic spheroid formation with multiple myeloma (MM) cells. First, POx/POzi hydrogel was evaluated as an alternative to Pluronic F127. Both supported the short-term viability of human mesenchymal stromal cells with telomerase reverse transcriptase overexpression (hMSC-TERT) after bioprinting. Gene expression analysis indicated that transient shear stress during extrusion was less influential than the post-printing microenvironment, highlighting that future optimization should prioritize post-printing stabilization and controlled cell-matrix interactions rather than minimizing shear alone. Second, peracetylated MG glucosamine analogues were tested for their ability to reduce tissue-nonspecific alkaline phosphatase (ALP) activity, a marker enzyme of osteogenic cells, by interfering with β-1,4-glycosylation. However, no significant reduction in ALP activity was observed. The most plausible explanation is inadequate metabolic incorporation and subsequent quality-control removal in the endoplasmic reticulum (ER) (EDEM/ERAD). While ineffective in hMSC-TERT, such analogues may hold relevance in cancer models, where glycosylation regulation is frequently altered. Third, membrane-anchoring polymers were examined to establish optimal staining concentrations, cytocompatibility conditions, and their degree of membrane association. These results define practical working parameters and provide a foundation for future functionalization, in which additional reactive groups could be incorporated to enable controlled cell-cell or cell-matrix interactions. The final section focused on engineering artificial cell-cell tethering to enable stable heterotypic MSC-MM spheroid formation. hMSC-TERT formed partially mixed spheroids with INA6, whereas U266 remained segregated, preventing mechanically stable spheroid formation. Biotin-streptavidin tethering was tested but did not achieve robust attachment, likely due to aIII stoichiometric mismatch at the cellular surface. Therefore, a metabolic glycoengineering strategy using newly synthesized mannosamine analogues and bifunctional linkers designed for orthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC) and inverse electron-demand Diels-Alder (IEDDA) click reactions was pursued. While Ac4ManNCyoc successfully integrated into the glycocalyx and reacted with tetrazine-bearing fluorophores, all linker variants (2, 5, and 7 nm) exhibited insufficient aqueous solubility and failed to mediate selective staining or cell-cell tethering. In conclusion, this work establishes essential methodological groundwork for the development of engineered MSC-MM coculture and spheroid models. Future progress depends primarily on the design of hydrophilic, bifunctional linkers with defined spacing. Once such a tether is established, the resulting spheroids will provide a platform to study microenvironmental interactions, disease progression, and therapeutic response in multiple myeloma.