Extracellular Matrix Production by Mesenchymal Stromal Cells in Hydrogels Facilitates Cell Spreading and Is Inhibited by FGF-2

In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell-derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell-derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material-presented cell adhesion motifs, cell-derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF-2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell-derived ECM is a guiding cue in 3D hydrogels and that FGF-2 is a potentially important ECM regulator within bioengineered cell and tissue systems.

2D Stem Cell Culture on Microfluidic Generated Hydrogel Beads

Tanja Cloé Hausherr

Stem cells capacity to self-renew and differentiate into specialized cell types, endow them with great promises in regenerative medicine. It is believed that the stem cells microenvironment plays a significant role in regulating their fate. However, with exception to embryonic stem cells, there is currently no tractable methods to control stem cells expansion in vitro. Microcarriers were shown to yield enhanced cell expansion compared to conventional cell culture. Nevertheless, the materials used for microcarriers fabrication do not offer the possibility to tailor their physicochemical properties to cell-specific requirements. To fill this technological gap, we propose to develop versatile hydrogel microcarriers with tunable mechanical properties and tailored surface modification. The combination of microfluidic droplet generation and poly(ethylene glycol)-based hydrogels allow the generation of monodisperse hydrogel microcarriers. Chemical moieties on the microcarriers surface enable versatile plug-n-play surface modifications. Microcarriers functionalized with thiolated adhesion ligand were successfully used for cell culture. Furthermore, magnetic particle incorporation within hydrogel microcarriers facilitated their handling during cell culture. To validate our hydrogel microcarriers, we characterized the culture of fibroblasts and HEK cells over 5 days. An increased number of cells were recovered with the hydrogel microcarriers compared to conventional culture. Finally, we were able to culture human mesenchymal stem cells on our hydrogel microcarriers. Altogether, we demonstrated the proof-of-principle experiments for the generation of customized hydrogel microcarriers for stem cell expansion in vitro. The presented technology will offer great promises in the future.

2011

Recapitulating macro-scale tissue self-organization through organoid bioprinting

Jonathan Brassard, Moritz Hofer, Matthias Lütolf, Mikhail Nikolaev

A 3D bioprinting approach has been developed to facilitate tissue morphogenesis by directly depositing organoid-forming stem cells in an extracellular matrix, with the ability to generate intestinal epithelia and branched vascular tissue constructs. Bioprinting promises enormous control over the spatial deposition of cells in three dimensions(1-7), but current approaches have had limited success at reproducing the intricate micro-architecture, cell-type diversity and function of native tissues formed through cellular self-organization. We introduce a three-dimensional bioprinting concept that uses organoid-forming stem cells as building blocks that can be deposited directly into extracellular matrices conducive to spontaneous self-organization. By controlling the geometry and cellular density, we generated centimetre-scale tissues that comprise self-organized features such as lumens, branched vasculature and tubular intestinal epithelia with in vivo-like crypts and villus domains. Supporting cells were deposited to modulate morphogenesis in space and time, and different epithelial cells were printed sequentially to mimic the organ boundaries present in the gastrointestinal tract. We thus show how biofabrication and organoid technology can be merged to control tissue self-organization from millimetre to centimetre scales, opening new avenues for drug discovery, diagnostics and regenerative medicine.

NATURE RESEARCH2020

Defined matrices for epithelial organoid culture and disease modelling

Saba Rezakhani

Advances in our understanding of the biology of stem cells and their niches have provided the necessary foundation to create in vitro complex self-organizing three-dimensional (3D) structures called organoids. While organoids represent a highly promising new class of biological model systems, the translational potential of these systems is currently limited by their dependence on animal-derived matrices, particularly Matrigel, an extracellular matrix (ECM) derived from mouse tumors. Apart from their immunogenicity and batch-to-batch variability, these matrices do not allow modulation of ECM composition and, accordingly, systematic adaptation of properties to specific cells and tissues. To address this issue, new biomaterial-based approaches have focused on the development of synthetic alternatives to Matrigel. This work focuses on the development of chemically defined matrices to elucidate the specific effects of ECM, with the goal of further increasing the translational relevance of epithelial organoids for regenerative medicine and disease modeling applications. Given the critical functionality of the intestine and the liver, and emergence of debilitating diseases due to damage to these organs, I have mainly focused on intestinal and liver organoids. First, I developed a new family of synthetic poly(ethylene glycol) (PEG)-based hydrogels, so-called low-defect thiol-Michael addition (LDTM) hydrogels, by modifying the conventional crosslinking strategy of existing PEG-co-peptide hydrogels formed by Michael addition of bis-cysteine peptides and vinyl sulfone-terminated PEG macromers. These mechanically tunable hydrogels are chemically defined and can be prepared with minimal batch-to-batch variability. They also allow the derivation of mouse and human intestinal and liver organoids, the latter without any animal components. Furthermore, I leveraged the modularity of synthetic matrices and derivation of liver ductal organoids in these matrices, to model some aspects of liver fibrosis, a global health problem affecting millions of people, and for which organ transplantation is the only medical solution. I demonstrated the role of tissue stiffness, a feature usually neglected in fibrosis models, on a regenerative mechanism associated with liver fibrosis called ductular reaction (DR). Human ductal liver organoids showed reduced proliferative capacity in LDTM gels with stiffness values similar to those of fibrotic liver tissue. In addition, transcriptomic analysis revealed impaired stemness potential, as well as concomitant induction of inflammatory responses. Overall, the newly developed hydrogels provide a reproducible, chemically defined environment that can be readily prepared with minimal batch-to-batch variability for multiple stem cell types and for organoid culture. Successful derivation of human intestinal and liver organoids in these matrices may open exciting prospects for clinical applications of organoids. In addition, the modularity of these matrices to mimic the ECM in pathological tissues, particularly the aberrant mechanical properties of fibrotic tissues, will enable the development of powerful disease models and predictive drug screening platforms and promote the development of new therapies.

EPFL2021

Defined matrices for epithelial organoid culture and disease modelling

Saba Rezakhani

EPFL2021