Enrico Domenico Lemma

Studying Cell Mechanobiology in 3D: The Two-Photon Lithography Approach

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates.

Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential

Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.

Nanofabricated 3D cage-like structures for cancer cell discrimination

Some cellular mechanisms such as migration and invasion, are determined and regulated by cell mechanical properties, especially during embryonic development or tumorigenesis. In these cases, cells attitude to undergo deformation and to displace their whole body is a crucial point for survival and metastasis formation. Extracellular environment three-dimensionality is a key feature influencing cell mechanics, and micro- and nano-fabricated structures represent a promising tool to study cell behavior. Here we engineer 3D- cage-like microstructures realized by two-photon polymerization to analyze cell migration through pores significantly smaller than the cell nucleus. We found that cells spontaneously interact with the cages by applying force at the cell-cage contact points while rearranging their cytoskeleton. The ability to traverse pores of different area depends on the invasiveness of the cell lines, allowing to discriminate between cells with different invasive potential.