Research

The microenvironment of cells, which determines their biological behavior, such as the packing and shedding of EVs, is the result of a multitude of physical, chemical and biological parameters. Thus, the biological functionalities of MSC-EVs are affected by the culture conditions of the parental cells. Especially, physiologic oxygen concentrations and physiologic cell-cell or cell-substrate interactions using 3D cell culture approaches have been shown to influence the number of secreted EVs, their surface marker signature, their cargo, and ultimately their biological function. Although it is clear that oxygen concentration and 3D cell culture during extracellular vesicles (EV) generation affect the secretion and cargo of MSC-EVs, it is not well understood if and how these parameters affect the immunomodulatory properties of MSC-EVs. Based on available scientific data and our preliminary results our central hypotheses are: 1) Major characteristics, such as number, size distribution, surface markers, and intracellular and membrane-bound proteins of MSC-EVs are affected by the culture conditions (2D vs. 3D) of MSCs. 2) Therefore, different culture conditions of MSCs affect the biological functions of MSC-derived EVs, in particular their procoagulant potential (exposure of TF) and their immunomodulatory properties (influence on monocyte subset distribution). To test these hypotheses, we will generate MSC-EV preparations from hydrogel, spheroid and flat surface culture from physioxic conditions and characterize these preparations towards their immunomodulatory properties and procoagulant potential. Here, we focus first on characterization of the EVs and EV cargo to understand how the abovementioned parameters affect formation and loading of MSC-EVs. Second, we will investigate the interplay between MSC-EVs from different culture conditions and cells of the adaptive and innate immune system. This will result in a detailed understanding of MSC-EV formation and loading and ultimately enable to manufacture MSC-EVs with defined immunomodulatory properties for the treatment of inflammatory conditions.

HUMAN PLACENTA Collagen-I from THT Biomaterials GmbH is a novel biomaterial that due to his human source alleviates the downstream limitations associated with the use of animal-derived materials in research. Although the intrinsic fibrillogenesis capacity of THT HUMAN PLACENTA Collagen-I has shown to be sufficient for 2D coating applications, his polymerization ability is limited for the formation of stable 3D hydrogel structures that are indispensable for physiologically relevant cell culture strategies. In this regard, Prof. Cornelia Kasper´s research lab from the Universität für Bodenkultur Wien BOKU has the necessary expertise to support THT in adjusting the mechanical properties of HUMAN PLACENTA Collagen-I to obtain stable and functional hydrogels. Prof. Cornelia Kasper´s research lab suggests to functionalize the HUMAN PLACENTA Collagen-I with methacrylate groups, a common strategy used to modify of different proteins or sugar-based biopolymers. The presence of methacrylate-groups will enable the introduction of covalent bonds upon exposure to UV in the presence of photoinitiators, thus forming hydrogels that can be used subsequent used for different 3D applications. The newly functionalized product (HUMAN PLACENTA Collagen-I methacrylate) will expand THT portfolio allowing his straightforward the use for customers working in different 3D biological applications such as 3D cell culture (e.g. organoids culture), lab-on-a-chip, bioprinting and thus broaden the current applicability of HUMAN PLACENTA Collagen-I. Significantly, the envisioned biomaterial can also be used as ready-to-use bioink for trendy technologies such as light-based 3D bioprinting. Apart from possible publications and co-authorships abstracts, THT & Universität für Bodenkultur Wien BOKU can potentially obtained IP on HUMAN PLACENTA Collagen-I methacrylate generating value for both project partners. If for any reason the innovation check should be cancelled, BOKU reserves the right to charge for services provided in the meantime.

Osteoarthritis (OA) represents a considerable societal and economic burden in today’s society. Despite the tremendous developments in the field of articular cartilage tissue engineering (AC TE) in the recent decade, none of the TE-based approaches has been able to regenerate the cartilage to levels of native tissue. The established paradigm of AC TE involves employment of undifferentiated MSCs in combination with 3D scaffolds/hydrogels and appropriate growth factors to induce chondrogenic differentiation of cells and deposition of ECM components like collagen and glycosaminoglycans. Once successful tissue has been formed in vitro, engineered cartilage grafts can be studied in vivo in large animal models to assess safety and efficacy of such grafts. Unfortunately, a considerable amount of grafts fails in vivo, which indicates the overall unsuitability and immaturity of the engineered tissues to function in the mechanically demanding environment of the joint in vivo. More importantly, there is no incentive to publish or submit for publication unsuccessful studies, which indicates that the number of failed studies employing large animal models could be considerably higher. Therefore, there is a need for novel strategies to screen and identify in vitro engineered cartilage grafts that have higher chances of success in vivo. In addition to increasing the success rate of such studies, this approach would have a great potential to reduce the number of animals utilized in such studies. In this context, there is evidence suggesting that chondrogenically differentiating MSCs respond anabolically to mechanical stress at later stages of differentiation by producing ECM components like glycosaminoglycans. Interestingly, the differentiation of MSCs is also associated with metabolic changes, where glycolysis is reduced and oxidative phosphorylation is enhanced as maturation progresses. The goal of this project is to develop a platform that could be used to assess such metabolic changes by sampling metabolites in- and outside the developing cartilage grafts to make statements concerning the maturity. By establishing such platform an additional readout would be available, in addition to commonly used biochemical and histological techniques within AC TE, that would facilitate a more informed decision making prior to an in vivo transition of a potential cartilage graft.