UNIVERSITÀ DEGLI STUDI DI MILANO-BICOCCA
DOTTORATO DI RICERCA IN TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi) – XXXIII CICLO
Project Supervisor: Prof. Francesco Nicotra
Project Title: Glycan Functionalized Synthetic Etracellular Matrices (Glyco-ECMs)
Possible support for Phd students w/o a University fellowship: Not at the moment
Introduction
Glycans are ubiquitous in all living cells and organisms, where they serve essential functions ranging from acting as structural components to regulate physiological and pathological processes [[i]]. Recent studies have challenged the classical view of protein glycosylation as an intracellular event by demonstrating that glycans may experience further structural remodelling by extracellular enzymes [[ii]]. This makes the glycome a highly dynamic molecular entity that mirrors a particular biological milieu and confer to cell microenvironment an important regulatory role. Cell niche microenvironment in fact is rich in glycosylated extracellular matrix (ECM) proteins that provide biochemical signals interpreted by cell surface receptors, such as integrins[[iii],[iv]] or galectins [[v]]. This initiate signalling cascades controlling cell survival, cell proliferation, differentiation and stem cell state [[vi]]. Hence, glycan appear as invaluable biomolecular cues for the functionalization of 3D ECM mimetics in order to modulate cell-ECM and cell–cell interactions and to guide cell behaviour.
Reproducing in vitro the same environmental situation presents in vivo, allows the cells to develop morphology and physiology like that occurring in vivo. This mimicry can be performed culturing cells as 3D aggregates, or as suspensions inside 3D hydrogels composed of extracellular matrix (ECM) proteins[[vii]]. Conventional methods to produce 3D system for cell cultures allow you to obtain 3D hydrogels for cell encapsulation tailoring the stiffness [[viii],[ix]]. Today advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to developin vitromodels with complexity approaching that of thein vivocell microenvironment.
Objective of the project
The project aims to develop a new in vitro platform of tailor made new 3D in vitro model systems (ECM and glyco-ECM mimics) mimicking cell microenvironment, as easy-to-use tools for personalized medicine. Synthetic ECM mimics platform will be designed to cover the gap between in vitro and in vivo biological studies. The obtained glyco-ECM mimics will be able to recapitulate the natural microenvironment to characterize cell-cell and cell ECM interaction and to test drugs kinetic or nanodrugs delivery and activity.
Methodologies
· ECM and 3D Glyco-ECM mimetics production will be first optimized without cells, and then performed directly with cells. The 3D tools will be produced using conventional procedure with commercial PEG-star-linkers and 3D bioprinting procedure. Different stiffness and morphology will be selected mimicking the native cell microenvironment in pathological and physiological processes.
· The produced 3D ECM and Glyco-ECM tools will be evaluated to characterize the contribute of selected glycans in cell differentiation. Biological evaluation will be conducted in term of cell response (cell viability, differentiation, quiescence) and biomolecular interaction considering both glycan identities, morphology and stiffness of microenvironment. The obtained 3D mimetics will be also useful in the assessment of in vitro models for personalized medicine applications (drug/nanoparticles screening and in vitro tumor models)
[i][]Nairn AV et al. Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis. J Biol Chem. 2012; 287: 37835–56. doi: 10.1074/jbc.M112.405233
[ii][]Wang YC et al. Protein post-translational modifications and regulation of pluripotency in human stem cells. Cell Res. 2014; 24(2):143-60. doi: 10.1038/cr.2013.151.
[iii][] Campbell ID et al. Integrin structure, activation, and interactions Cold Spring Harb. Perspect. Biol. 2011; 3:a004994. DOI: 10.1101/cshperspect.a004994
[iv][] Streuli CH. Integrins as architects of cell behavior. Mol Biol Cell. 2016; 27(19): 2885–2888.
doi: 10.1091/mbc.E15-06-0369
[v][] Ochieng J et al. Extracellular functions of galectin-3. Glycoconj J 2002; 19:527-535.doi:10.1023/B:GLYC.0000014082.99675.2f
[vi][] Naba A et al. The extracellular matrix: Tools and insights for the "omics" era. Matrix Biol. 2016; 49:10-24. doi: 10.1016/j.matbio.2015.06.003. Epub 2015 Jul 8.
[vii][] Cukierman et al. Taking cell-matrix adhesions to the third dimension. Science. 2001; 294(5547):1708-12.
[viii][] Russo et al, Gelatin hydrogels via thiol-ene chemistry Monatshefte für Chemie - Chemical Monthly,2016, 147:587–92. DOI: 10.1007/s00706-015-1614-5
[ix][] Occhetta P, Russo et al. MVA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding. J Biomed Mater Res A. 2015, 103:2109-17. doi: 10.1002/jbm.a.35346