New hydrogel based 3D cell micro systems: optimization of valuable tools in tissue repair and cancer

In drug development, cancer research, as much as in stem cell and tissue engineering field, 3D cell cultures are widely used in vitro systems, providing more valuable and advanced information compared to traditional 2D cultures, and are importantly expected to bridge the gap between over-simplified bidimensional cultures and in vivo models. In fact, 3D cell culture systems can offer physiologically relevant insights, representing more accurately the in vivo extracellular matrix (ECM) and the microenvironment where cells reside in tissues. In the context of stem cell and regenerative medicine field, biodegradable and biocompatible scaffolds play a key role, providing a temporal and spatial environment for stem cells proliferation, tissue in-growth and differentiation. Firstly, in this thesis work, the photo-polymerizable semi-synthetic protein hydrogel scaffolds PEG-fibrinogen (PFHy) and PEG-silk fibroin (PSFHy) were produced and used to design microspheres embedding human cardiac progenitor stem cells (Sca1+ LincMSC) as 3D micro-systems able to allow proper stem cells growth and that could find application as potential cell delivery carriers for tissue repair. These 3D systems were produced and optimized both in terms of the biomaterial mechano-physical properties (stiffness) and biochemically, performing a treatment and/or a preconditioning of the embedded cells using hydrogen sulfide (H2S) slow-releasing donors. H2S is an endogenous gasotransmitter whose production in mammalian cells occurs through tightly regulated pathways and plays a key role in the central nervous, respiratory, and cardiovascular systems. Multiple cellular targets sulfhydrylated by H2S, such as Cytochrome C oxidase, Nrf2/ARE-regulated genes, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, are involved in the anti-hypoxic, antioxidant, and anti-apoptotic cellular response. Given the importance of H2S physiological role, many studies in recent years have been aimed at investigating the possibility of using exogenous H2S donors for the treatment of cardiovascular and neurodegenerative diseases related to both the reduction of endogenous H2S levels and the activation of oxidative and pro-inflammatory processes. Although some preliminary studies show that the cell pretreatment with slow-release H2S donors improves the survival of mesenchymal stem cells after implantation at the injury site, the effects of exogenous H2S on stem cell growth, proliferation, migration, and differentiation are still not understood. Especially, the use of H2S-releasing compounds for the optimization of stem cell growth and delivery systems is an almost totally unexplored field. In view of these considerations, in our 3D stem cell microspheres, the effects of GSGa, a glutathionylated garlic extract produced in our laboratory and able to slowly release hydrogen sulfide, were assessed. Interestingly, in our previous works, the treatment with GSGa was found to increase cMSC viability, proliferation and resistance to the oxidative stress. Herein, we found that H2S treatment exerts protective effects on cMSC from the photo-polymerization damage, that occurs when cells are encapsulated in photopolymerizable hydrogels which, although are ideally suited for stem-cell based tissue regeneration and 3D bioprinting, require the use of potentially toxic photoinitiators, exposure to UV radiation, formation of free radicals that trigger the cross-linking reaction, and other events whose effects on cells are not yet fully understood. These 3D PEG-protein hydrogel microspheres represent a versatile and wideranging tool, since, in the context of cancer research, 3D cell cultures in the form of spheroids/tumoroids are widely used, because: first, the spherical organization is very similar to what is observed in vivo in avascular tumor masses; next, the sphere is able to recapitulate different gradients of oxygen, nutrients and drugs present in a real tumor mass. These considerations prompted us to produce tumoroids microspheres using a triple-negative MDA-MB-231 breast cancer cell line with the aim of evaluating whether our PFHy and PSFHy 3D micro-systems could be good artificial tumoroids useful as in vitro models to investigate the physical and biochemical pathways involved in the disease and as potential screening platformsfor more reliable in vitro testing of anticancer drugs and compounds. Our experiments showed that, when incorporated into the 3D systems, having PFHy lower stiffness and PSFHy higher stiffness, the gene expression of breast cancer cells changed in stiffness-dependent manner. Furthermore, in PSFHy-based tumoroids with high stiffness, a decreased multi-drug resistance was found, along with an increased expression of osteocalcin protein, indicating the breast cancer cells transdifferentiation toward osteoblast-like cells induced by the physical stimulus of the hydrogel stiffness increase (PiT Physically induced trans-differentiation). Moreover, the effects of the H2S donor GSGa were investigated in the tumoroids systems on cancer cell viability, proliferation and invasion. In general, the anticancer activity of H2S slow-release donors is well documented in 2D cell culture systems and herein we sought to expand upon this knowledge using 3D culture experiments. H2S donors exert antitumor activity by acting on the EGFR/ ERK/ MMP-2, PTEN/ Akt, PI3K/Akt/mTOR pathways and by inhibiting NF-kB. Our experiments notably showed that the concentration of H2S able to cause MDA cell death in 2D cultures, in the 3D systems, on the contrary, exerted pro-proliferative and promigratory effects. These findings indicate that the use of optimized 3D cell culture systems which, compared to 2D cultures, more closely mimic the in vivo conditions, is of crucial importance in the cancer research field and in the evaluation and development of new potential antitumor therapies. In our investigation, particular attention was focused on 3D cell migration using the “gel-in-gel” assay based on the production of a cell hydrogel microsphere (inner gel) and of an outer gel in which cells can migrate. To improve the feasibility of this assay, during this thesis work, we have designed and optimized a “lab on a chip” named “3D Cell Migration chip” (3D-CMchip) which offers many advantages compared to the traditional method and characterized by a removable superhydrophobic nanostructured surface to optimize the cellular microsphere in situ production. 3D-CMchip provides a platform to analyse the effects of chemico-physical stimuli on migration/invasion of different cell types (mesenchymal stem cells, fibroblasts and tumor cells). In particular, PFHy microspheres with MDA-MB-231 breast cancer cells were used for the analysis of H2S donors effects on cancer cells migration in the 3D-CMchip. Furthermore, since the 3D-CMchip allows the easy development of more complex co-culture systems, herein, MDA breast cancer cells were co-cultured with fibroblasts to create even more predictive tumoroids models by considering the network of interactions between cancer cells and cell components of the tumor microenvironment (TME), which is an indispensable element for a deep and complete understanding of the tumor disease.