Perfusion culture systems to engineer 3D biomimetic bone marrow tissues

Haematopoiesis is the process of blood production in the bone marrow (BM) tissue within the bones and is essential for every human. Most research in the haematopoietic field has been done with murine models, due to limited accessibility and ethical concerns related to the use of human material. However murine models can only give limited insight into the human haematopoiesis due to major differences between the species. Most current in vitro systems to model human haematopoiesis fail to recapitulate the cellular diversity of native BM microenvironments or niches. The vascular component is a key part of native bone marrow that has been traditionally ignored when designing in vitro BM biomimetic niches. Moreover, those systems including vasculature often rely on the use of human umbilical vein endothelial cells and require an additional cell source to generate perivascular cells.
In this thesis a novel fully humanized in vitro model with endothelial, perivascular and osteoblastic cells was engineered in a perfusion bioreactor system using human adipose tissue-derived stromal vascular (SVF) cells to generate the vascular component. The model is based on a previously published osteoblastic niche that is created by seeding human BM mesenchymal stromal cells in hydroxyapatite scaffolds and inducing their osteogenic differentiation. The SVF cells were used as a single and easily-accessible cell source to successfully form endothelial-perivascular structures reassembling arteriolar vessels. This vascularized bone marrow niche was able to significantly improve the preservation of undifferentiated cord blood haematopoietic stem and progenitor cells under physiological-like conditions. Furthermore, the vasculature contributed to maintaining the niche osteogenic features.
Nevertheless, the macro-scale perfusion bioreactor system used to generate this vascularized osteoblastic tissue requires large amounts of cells/medium and applies a suboptimal flow to the tissue. For this reason, new flow-optimized macro- and mini-scale perfusion bioreactors were engineered in the context of this thesis. Computational fluid dynamic modeling confirmed the improved flow parameters in the new systems. Finally, we validated the functionality of these new bioreactors by engineering angiogenic niches that were characterized by flow cytometry and gene expression analyses.
In conclusion, the work presented in this thesis contributes to improving in vitro systems to model human hematopoiesis at two different levels. At biological level, full vascular structures composed by endothelial and perivascular cells were added to engineered osteoblastic niches using adipose-derived cells. At engineering level, the bioreactors used to generate these niches were miniaturized and flow-optimized.