Glycoengineering as a tool to control the behavior of bone marrow-derived mesenchymal stromal cells in biofabrication processes

Introduction:

3D Bioprinting is a promising and innovative technique in the field of tissue engineering allowing the generation of highly precise constructs for different purposes. During that process, cells are facing different challenges to survive in the 3D hydrogel. Our project investigates shear stress impact and adherence behavior of human mesenchymal stromal cells (hMSC) by applying metabolic glycoengineering to alter the cell glycocalyx and the bioinks.

Materials and methods:

For metabolic glycoengineering, hMSC were first incubated with different azido sugars for 48h followed by a click-reaction. Cells were either incubated with DBCO-Cy3 as click molecule for 1h or in presence of Cu ions with alkyne-Cy3 for 5 min. For galectin-1 studies, cells were incubated with an artificial galectin-1 ligand or seeded onto a ligand coated glass slide.

Results:

The azido sugar expression in the glycocalyx could be microscopically detected up to 48h identifiying the mannosamine variant as superior regarding cell viability and incorporation efficiency. The glycochip assay resulted in the appearence of non-adherent cell spheroids, but revealed no cell adhesion toward the galectin-1 ligand coated regions. Analysis of galectin-1 gene expression showed no elevated mRNA level after incubation with the ligand.

Discussion:

Since the metabolic glycoengineering is working, suitable molecules can now be identified to be introduced into the glycocalyx and evaluated for cell rigidity-increasing effects before and after 3D bioprinting. The adherence assay needs to be redesigned since cell adhesion was expected and the glycochip functionalization might impede surface-cell interaction. The galectin-1 ligand impact on cell functions will be further characterized.

Key words:

Bioprinting, glycoengineering, galectin-1, shear stress