Periodic Reporting for period 2 - MicroNICHE (Microfluidic-assisted fabrication of artifical microniches for bone marrow stem cells)
Periodo di rendicontazione: 2018-07-01 al 2019-06-30
The main objective of the project is the microfluidic reconstruction of an artificial SCN. For this approach, a droplet-based microfluidic technique is proposed in which a bone marrow stem cell (BMSC) microniche with tunable size, material and topography will be developed. Two physical requisites would be necessary to reconstruct normal HSC function in a artificial stem cell niche, the control of the niche geometry, and the functional reconstruction of the artificial SCN in a mechanically adapted support. These requisites are considered in both presented models comprising various layers of biocompatible hydrogels containing MSCs and HSCs.
This approach for the artificial reconstruction of these niche components represents an efficient method to simulate the in vivo situation of HSCs with the goal to study stem cell behavior in vitro under controlled conditions. This tailored coating of an inner hydrogel core extendable to a third coating layer represents a promising technology to reconstruct more complex cell arrangements and mimic better the complexity of the stem cell niche.Therefore the presented device is a valuable and unique tool towards gaining insights in understanding the regulation factors influencing the stemness maintenance of HScs and more generally, in understanding stem cell behavior in 3-dimensional environments.
A precise characterization of the empty hydrogel structures volume and shape using confocal laser scanning microscopy (CLSM) was also performed. Both empty and cell laden structures were characterized using a Confocal Microscope by testing of different fluorescent beads/markers on alginate to be visible under the requested set-up. Cell encapsulation viability assays were also performed in order to asses the good direction of the proposed methodology. Several cell concentrations, hydrogel scaffolds and cell types ( human Mesenchymal stem cells and human Hemaopoietic stem cells) were used.Life dead assays were performed on the extracted beads. Results of this initial research were promising as human Mesenchymal stem cells were maintained and cultured for three days with high viability, and human Hematopoietic stem cells were maintained after encapsulation, suggesting that the platform can provide a novel and useful tool for the further studies to be carried out on the third year.
The work package 2 pursues the delivery of a functional high-throughput microfluidic platform to construct multi-layered microstructures by droplet technologies based in WP1. This work was done from 01/08/2017 to date.
In this task, a new microfluidic design and operational method has been developed, upgrading the previously manufactured two layered device to an additional third layer.As a result of this research, two different microfluidic geometries have been designed, manufactured, tested and characterized, and three layered beads in alginate-alginate-Puramatrix hydrogel have been successfully generated. Viability of this structure for stem cell encapsulation was performed successfully enclosing human Mesenchymal stem cells.
The expected results until the end of the project include the transfer to Europe the MicroNICHE technology developed in USA. The platform will be assessed for different cell types and concentrations. First, a HSC-niche proof-of-concept will be assessed using endothelial cells, covered by mesenchymal cells, which in turn will be covered by human hematopoietic precursor cell lines. A second proof-of-concept with primary HSCs will be also attempted.It is expected that HSCs stemless will be maintained successfully for several days on the proposed structures.
The possibility to deliver a MicroNICHE platform for improved culture of Bone Marrow Stem Cell (BMSC) products will mean an important step forward for the interest of the health of the European population. The completion and succeed of MicroNICHE as a model of stem cell niche will represent a paradigm shift in cell Technology and Regenerative Medicine, opening new ways to understanding important physiological processes. Ex vivo expansion of human HSCs in the proposed MicroNiche structure could also represent a new perspective in HPT. The developed and versatile technology which has been designed towards the recapitulation of the hematopietic stem cell niche, could also represent a novel tool to recreate other biological entities such as organs or tumours, opening insights in understanding in general 3D stem cell behavior.