Towards the achievement of NEVADA’s goals, we presented several designs of 3D microscaffold arrays, fabricated by 2-PP photolithography, to mimic tissue-specific architecture, enabling cell-to-matrix interaction and cell-to-cell communication in vitro. To prove that cell culture in 3D microscaffold arrays, better mimics the in vivo states and thus maintain a more homogenous cell population, we used a model of pre-implantation embryos, in which pluripotent mESC populations were maintained under controlled culture conditions. We performed several experiments to demonstrate that 3D microscaffolds reinforce the pluripotency gene expression program. First, we used light microscopy to assess the morphology of the mESC colonies growing on 2D and 3D scaffolds. Second, we used alkaline phosphatase (AP) staining to prove that cell colonies are expressing stem cell marker. Finally, to support our preliminary observations, we performed quantitative immunofluorescence analysis of three key pluripotency markers - NANOG, OCT4 and ESRRB. Our data demonstrated that in comparison to 2D surface, mESCs in 3D microscaffold arrays exhibit enhanced expression levels of these three pluripotency markers and a homogenous expression of two pluripotency markers - NANOG and ESRRB. We thus hypothesized that mESCs in 3D microscaffold arrays might have a stronger self-renewal ability resembling more closely the pre-implantation embryos.
NEVADA project has achieved some of its objectives and milestones. It has faced disruptions and delays because of COVID containment measures. Due to the coronavirus epidemic, unprecedented actions have been undertaken against COVID-19 at ETH Zurich. This has greatly impact experimental lab-based research activity and have brought a massive disruption to our project.
