Scaling-Up Techniques for the Nanofabrication of Cell Culture Substrates via Two-Photo Polymerization for Industrial-Scale Expansion of Stem Cells

Stem-cell-based therapies require a high number (106–109) of cells, therefore in vitro expansion is needed because of the initially low amount of stem cells obtainable from human tissues. Standard protocols for stem cell expansion are currently based on chemically-defined culture media and animal-derived feeder-cell layers, which expose cells to additives and to xenogeneic compounds, resulting in potential issues when used in clinics. The two-photon laser polymerization technique enables three-dimensional micro-structures to be fabricated, which we named synthetic nichoids. Here we review our activity on the technological improvements in manufacturing biomimetic synthetic nichoids and, in particular on the optimization of the laser-material interaction to increase the patterned area and the percentage of cell culture surface covered by such synthetic nichoids, from a low initial value of 10% up to 88% with an optimized micromachining time. These results establish two-photon laser polymerization as a promising tool to fabricate substrates for stem cell expansion, without any chemical supplement and in feeder-free conditions for potential therapeutic uses

Therapeutic effect of neural progenitor cells expanded in the 3D nano-engineered Nichoid substrate in a Parkinson’s disease preclinical model

3D microscaffoldsare becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiologicalniche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able to proliferate. In this work,we investigated the proliferation and stemness properties of Er-NPCswhen grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease.3D microscaffolds are becoming more and more relevant in regenerative medicine, as they lead to the creation of a structure similar to a physiological niche. An example is the nano-engineered Nichoid, a 3D structure in which the cells are able to proliferate. In this work, we investigated the proliferation and stemness properties of Er-NPCs when grown inside the Nichoid, and their potential therapeutic application in the treatment of Parkinson\u2019s Disease

Two-photon polymerized "nichoid" substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions

BACKGROUND: The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages. METHODS: We developed an innovative three-dimensional culture substrate made of “nichoid” microstructures, nanoengineered via two-photon laser polymerization. The nichoids limit the dimension of the adhering embryoid bodies during expansion, by counteracting cell migration between adjacent units of the substrate by its microarchitecture. We expanded mouse embryonic stem cells on the nichoid for 2 weeks. We compared the expression of pluripotency and differentiation markers induced in cells with that induced by flat substrates and by a culture layer made of kidney-derived extracellular matrix. RESULTS: The nichoid was found to be the only substrate, among those tested, that maintained the expression of the OCT4 pluripotency marker switched on and, simultaneously, the expression of the differentiation markers GATA4 and α-SMA switched off. The nichoid promotes pluripotency maintenance of embryonic stem cells during expansion, in the absence of a feeder layer and exogenous conditioning factors, such as the leukocyte inhibitory factor. CONCLUSIONS: We hypothesized that the nichoid microstructures induce a genetic reprogramming of cells by controlling their cytoskeletal tension. Further studies are necessary to understand the exact mechanism by which the physical constraint provided by the nichoid architecture is responsible for cell reprogramming. The nichoid may help elucidate mechanisms of pluripotency maintenance, while potentially cutting the costs and risks of both feed-conditioning and exogenous conditioning for industrial-scale expansion of stem cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-016-0387-z) contains supplementary material, which is available to authorized users

Implantable medical device

An implantable medical device obtained by means of two- photon laser polymerisation of a resin to form a three-dimensional matrix, wherein: said three-dimensional matrix comprises a number of levels distributed in height; and said three-dimensional matrix comprises reference means designed to uniquely identify the height of each level from a pre-set reference, by means of a multiphoton fluorescence-excitation microscope; said implantable medical device being characterised in that said reference means comprise a solid having a cross section that varies with height