Engineered three-dimensional microenvironments as functional in vitro models of stromal tissues

Currently, the majority of current cultures is still carried out with long-established techniques like the exploitation of 2D supports, the use of tissue-derived immortalized cell lines, and the administration of un-physiological doses of soluble factors to induce a biological response. However, the lack of structural and physical cues often leads to biological artifacts, from the total loss of cellular function to the lack of correlation between the predicted and actual results when the experimental model shifts from in vitro to in vivo.
Hence, in this work I test the hypothesis that recapitulating in vitro crucial chemo-physical components of the native cell environment can uniquely maintain the original function and the phenotype of cultured cells. Therefore, the critical aspects are (i) the choice of a suitable source of cells, and (ii) the engineering of the culture conditions. In first instance, it is proposed that freshly isolated adult cells, as opposed to cell lines, are needed to mimic physiological and pathological processes occurring in animal tissues and organs. Secondly, in vitro culture conditions need to be adapted to support cell viability, function, and growth. In particular, the proposed approach relies on the combination of the cells with a suitable biomaterial able to provide a 3D environment for cell adhesion and suitable to allow complex spatial interactions with neighboring cells. The concept of the third dimension as a critical parameter able to influence cell physiology is challenged in different contexts. The complexity of the proposed culture systems, due to the high number of variables among 2D and 3D experimental groups, is such that the precise dissection of the single contributions is not obvious. However, we propose that the combination of a physiological 3D architecture with a suitable biomaterial provide technological and biological advantages able to trigger further investigations.
Notably, the material itself can be chosen so to mimic the native organ, e.g. the mineralized matrix of bone substituted in vitro by a ceramic material. Additionally, we suggest that the use of bioreactors as supportive technologies can exploit the full potential of 3D cell cultures.
Despite implying an increase in the complexity of the procedures required to execute experiments based on 3D cell cultures, it is proposed that the relevance of the results surpasses the efforts required to implement new culture models.
In the first chapter of my thesis, I focused on the validation of a platform for the expansion of bone-marrow derived stromal cells (MSC). As a result, the bioreactor-based platform was validated not only as a streamlined approach to expand MSC that maintain at a higher extent progenitor features, but also as a valuable tool to recreate in vitro an engineered stromal niche.
In the second chapter of the thesis the focus was moved to exploit the unique features of 3D cultures on the recapitulation of the thymic stroma in vitro. This chapter describes the evolution of a culture system able to manufacture in vitro a thymic organoid constituted by TEC that can suits as a model to investigate thymus physiology.
Finally, in the third chapter of the thesis, the concept of 3D stromal tissue engineering is applied to the hematopoietic niche, a specialized microenvironment devoted to regulate hematopoietic stem cells (HSC) quiescence and activity through a wide array of chemo-physical cues. Starting from previous reports in which freshly harvested bone marrow- or adipose tissue-derived cells can be cultured within porous scaffolds, allowing the formation of an organized 3D stromal tissue, we propose that cellularized constructs can be cultured in perfusion bioreactors to reconstruct the HSC niche through the controlled modulation of several parameters.
Taken together, these results highlight that an increase in the complexity of the traditional culture systems is crucial to better recapitulate the functional microenvironment of stromal and stroma-dependent cells or stem cells. Growing and handling cells in a 3D structure combined with a compliant biomaterial and bioengineering tools can dramatically increase the relevance of scientific data, enable unpredecented modalities to control the artificial microenvironment, and decrease the need of costly, time consuming, and ethically debated in vivo experiments.