A culture substrate made of synthetic structures called “nichoids”, able to maintain the function of stem and progenitor cells during expansion, was designed and patented at Politecnico di Milano with the collaboration of CNR and Fondazione Cariplo.
This innovative culture substrate mimics the geometric constraints perceived by stem cells in their native microenvironment, the so-called “niche”, thus allowing the maintenance of cell stemness during expansion without a feeder layer. The nichoid substrate is fabricated by two-photon laser polymerization of a photo-sensitive resin, with a procedure which permits to extend the niche-patterned culture substrate up to an area compatible with large-scale biological research and with the clinical application of the cells expanded on the substrate as a therapeutic product. The nichoid substrate can be incorporated into a culture system that can be industrialized and commercialized, such as a mono- or multi-well plate, or a culture flask.
The nichoid substrate has been extensively validated with human bone marrow-derived mesenchymal stem cells and with mouse-derived embryonic stem cells.
The advantages of the nichoid substrate, over traditional flat substrates, after prolonged cell expansion in the total absence of a cell-feeder layer and of soluble factors (including the “leukemia inhibitory factor” or LIF) are:
- cell density increases by 70%;
- cell expression of stemness markers is maintained comparable to non-expanded control cells;
- thanks to the feeder-free and xeno-free culture, the cells cultured in the nichoid substrate would be suitable for a clinical application in the context of a stem cell therapy.
Moreover, all the standard biological analytical techniques (including trypsinization, staining, immunofluorescence, optical microscopy, histology, histochemistry, etc.) can be applied on cells expanded in the nichoid substrate because this substrate is fully three-dimensional for the cells, but it is only 30 microns thick, thus optically accessible.