Abstract
Abstract 1331
Human mesenchymal stromal cells (MSC) maintain “stemness” of human hematopoietic stem cells (HSC) by cell-cell contact when used as feeder-layer. We previously demonstrated the presence of specific cadherin-catenin-based junctions between HSC and MSC in a two-dimensional coculture-setting. To develop a more physiological in vitro model for the hematopoietic stem cell niche, we have established a novel 3D-coculture-system based on the so-called 3D-KITChip capable of accommodating up to 1×107 MSC on an area of ∼2cm2. For a precise reproduction of the microenvironment of the niche, the 3D-KITChip is inserted in a microfluidic bioreactor, allowing active nutrient and gas supply.
MSC were derived from bone marrow aspirates of healthy voluntary donors and grown to confluence following standard protocols. MSC were then trypsinized and reseeded (3×105 cells) on a unique microchip with defined microwell cavities, developed by the Karlsruhe Institute of Technology (“3D-KITChip”). After 48–72h, 2×105 HSC isolated from umbilical cord blood were added and the chip was inserted in a microfluidic bioreactor. The closed circulation system comprising the bioreactor, a cassette pump, and a medium reservoir was run for up to 7 days. In this closed loop setup, oxygen concentration, medium composition and medium flow could be controlled precisely. After 1–7d of coculture, the bioreactor was re-opened and the two cell populations were analyzed by immunostaining, RT2-PCR, genomics and functional assays such as colony forming assays (CFA).
The MSC form a complex 3D mesh in the microcavities of the 3D-KITChip which is very stable for the time of the experiments and could be cultured in this way for up to 6 weeks. It could be demonstrated that HSC are distributed three dimensionally inside this MSC mesh. HSC could be kept alive in this environment for at least 7 days and a defined proportion of CD34+-HSC adhered to the MSC in the microcavities and built up direct cellular connections to the surrounding MSC. A direct comparison of the adhering vs. the non-adhering cell-fraction is currently underway. By means of RT2-PCR, we could demonstrate that the proportion of CD34+/nestin+/p21+/CXCR4+ cells could be maintained throughout the whole culture period of 7 days. Moreover, no CD38 or CD44-expression could be detected, whereas c-kit-expression continuously decreased during the culture period.
This novel setup allows the precise adjustment of the key elements in the hematopoietic stem cell niche that govern stem cell homing, engraftment and mobilization. Further experiments aim to elucidate the role of the 3D-environment, oxygen tension, sheer stress, composition of the medium and perfusion conditions on the above mentioned stem cell behavior. The impact of junctional complexes comprising alpha-/beta-catenin, p120 and N-cadherin in this context will be determined.
Disclosures:
Ho: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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