Periodic Reporting for period 1 - ELECTRO NEEDLE (In situ stem cell monitoring system based on conductive nanoneedle devices for tracking cell fates in invasive manner)
Reporting period: 2018-10-01 to 2020-09-30
In recent years there has been an explosion of interest in cell/tissue research, given its promising medical applications in cell-based tissue regeneration, drug testing, and basic research. Nerve tissue repair is an exciting and high impact area of research to pursue as it directly impacts on the quality of human life, because the adult central nerve system cannot be regenerated on its own after trauma or disease, such as Alzheimer's disease, and spinal cord injuries.
Guiding nerve tissue regeneration is a timely, exciting, and high impact area of research as it directly impacts on the quality of human life. To handle these concerns, reliable control of stem cell differentiation into nervous tissues is being important. There have been many creative approaches to properly guide the stem cells to differentiate into neural cells, but one of the most promising ones is nanoneedle (nN) arrays. These arrays are known as guiding cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, and further prompt the cells on arrays to cause morphological changes and reduce migration rates. This directly affects stem cells into a neuronal lineage with varying lengths.
In this fellowship, we have aimed to present a new microfabrication approach in which a combination of reactive ion etching protocols. With this new fabrication technique, we could produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm.
Guiding nerve tissue regeneration is a timely, exciting, and high impact area of research as it directly impacts on the quality of human life. To handle these concerns, reliable control of stem cell differentiation into nervous tissues is being important. There have been many creative approaches to properly guide the stem cells to differentiate into neural cells, but one of the most promising ones is nanoneedle (nN) arrays. These arrays are known as guiding cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, and further prompt the cells on arrays to cause morphological changes and reduce migration rates. This directly affects stem cells into a neuronal lineage with varying lengths.
In this fellowship, we have aimed to present a new microfabrication approach in which a combination of reactive ion etching protocols. With this new fabrication technique, we could produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm.
To investigate the changes in cell morphology that occurred from the new nanoneedle arrays, we cultured human mesenchymal stem cells (hMSCs) on flat silicon (as a control), nanopillars, two different blunt nanoneedles, and one set of sharp nanoneedles. We fixed the cells for immunostaining at four time-points (6, 12, 24, and 72 h after seeding). We then used image-based cell profiling to analyze 5,372 immunofluorescent microscopy images and extract single-cell morphological and protein localization features for over 100,000 cells. From this high-content image analysis, we were able to quantify pronounced, systematic morphological changes as a function of nanoneedle sharpness. In particular, decreasing the tip diameter reduced the spread area of both cells and nuclei, promoted cell body elongation, and decreased the protrusion ratio, which is the area of cell protrusions divided by the total cell area. In addition, nuclear solidity (a measure of nuclear perimeter tortuosity) visualized as slight scalloping in the in-plane nuclear membrane around the nanoneedles, decreased with increasing nanoneedle sharpness. Background-corrected and batch-normalized intensities of cytoskeletal proteins (F-actin, α-tubulin) were also influenced by changing tip diameter. Local cell density, determined by Voronoï tessellation, was also greater on nanoneedles than flat surfaces or nanopillars.
We next looked at the impact of nanoneedle tip diameter on gene expression. hMSCs were cultured for 6 and 24 h on flat silicon substrates or nanoneedles with varying tip diameters, before measuring the expression of a wide portfolio of genes, including focal adhesions proteins (PXN), nuclear lamins (LMNA, LMNB). Interestingly, LMNA expression was significantly influenced by both the presence of a nanostructured substrate and as a function of increasing nanoneedle tip diameter. LMNA codes for lamin A, a major structural component of the nuclear lamina, and our observation is consistent with previous studies showing a strong correlation between nuclear deformation and lamin expression. This finding was consistent with the reported increase in LMNA expression in cells on porous nanoneedles. We did not observe any significant changes in LMNB, the gene encoding lamin B.
Moreover, we were also able to significantly reduce the gene expression of PXN after 6 h of culture on the sharp nanoneedles, compared to the flat substrates (Figure 2c). This result was consistent with previous studies performed on porous nanoneedles, however, our non-degradable arrays enabled us to investigate gene expression beyond a 6 h time point. This analysis revealed a return to baseline expression levels after 24 h, which was expected given that PXN codes for the paxillin, a protein that is expressed at focal adhesions of during cell attachment. Indeed, immunostaining for paxillin showed a reduced intensity and reduced focal adhesion points for the hMSCs cultured on sharp nanoneedles at 24 h.
Finally, we used focused-ion-beam scanning electron microscopy (FIB-SEM) to image cross-sections through the cell-nanoneedle interface in order to visualize how biological membranes were perturbed by the nanotopography. For both the sharp nanoneedles and nanopillars, the plasma cell membrane was strongly perturbed by the vertical arrays, wrapping conformably around the silicon nanostructures after just 6 h. However, the depth of impingement of the plasma membrane was far greater on the sharp needles, moreover, the nuclear deformation was highly dependent upon tip diameter, with only the sharp nanoneedles able to perturb the nuclear membrane. The degree of cellular and nuclear membrane deformation increased with culture time, an important insight for the design of non-degradable nanoneedle arrays for long-term culture, intracellular delivery and sensing. We further investigated these structural changes for the 12 h timepoint by reconstructing consecutive FIB-SEM slices into a volumetric map, which allowed us to fully visualize the cell-nanoneedle interface in 3D. This reconstruction analysis showed that the impingement behavior was consistent across the entire cell and nuclear area.
We next looked at the impact of nanoneedle tip diameter on gene expression. hMSCs were cultured for 6 and 24 h on flat silicon substrates or nanoneedles with varying tip diameters, before measuring the expression of a wide portfolio of genes, including focal adhesions proteins (PXN), nuclear lamins (LMNA, LMNB). Interestingly, LMNA expression was significantly influenced by both the presence of a nanostructured substrate and as a function of increasing nanoneedle tip diameter. LMNA codes for lamin A, a major structural component of the nuclear lamina, and our observation is consistent with previous studies showing a strong correlation between nuclear deformation and lamin expression. This finding was consistent with the reported increase in LMNA expression in cells on porous nanoneedles. We did not observe any significant changes in LMNB, the gene encoding lamin B.
Moreover, we were also able to significantly reduce the gene expression of PXN after 6 h of culture on the sharp nanoneedles, compared to the flat substrates (Figure 2c). This result was consistent with previous studies performed on porous nanoneedles, however, our non-degradable arrays enabled us to investigate gene expression beyond a 6 h time point. This analysis revealed a return to baseline expression levels after 24 h, which was expected given that PXN codes for the paxillin, a protein that is expressed at focal adhesions of during cell attachment. Indeed, immunostaining for paxillin showed a reduced intensity and reduced focal adhesion points for the hMSCs cultured on sharp nanoneedles at 24 h.
Finally, we used focused-ion-beam scanning electron microscopy (FIB-SEM) to image cross-sections through the cell-nanoneedle interface in order to visualize how biological membranes were perturbed by the nanotopography. For both the sharp nanoneedles and nanopillars, the plasma cell membrane was strongly perturbed by the vertical arrays, wrapping conformably around the silicon nanostructures after just 6 h. However, the depth of impingement of the plasma membrane was far greater on the sharp needles, moreover, the nuclear deformation was highly dependent upon tip diameter, with only the sharp nanoneedles able to perturb the nuclear membrane. The degree of cellular and nuclear membrane deformation increased with culture time, an important insight for the design of non-degradable nanoneedle arrays for long-term culture, intracellular delivery and sensing. We further investigated these structural changes for the 12 h timepoint by reconstructing consecutive FIB-SEM slices into a volumetric map, which allowed us to fully visualize the cell-nanoneedle interface in 3D. This reconstruction analysis showed that the impingement behavior was consistent across the entire cell and nuclear area.
We reported the design and application of cytocompatible, solid silicon nanoneedle arrays with precisely tunable tip diameters (ACS Nano 2020, 14 (5), 5371-5381). By varying the nanoneedle tip diameter, we were able to directly influence cell morphology, gene expression, and differentiation capacity in primary human mesenchymal stem cells (hMSCs). While previous studies with high-aspect-ratio structures have shown that cell protrusions can be controlled using nanostructures with different densities (Adv. Mater. 32 (9), 1903862), our study demonstrates that nanoneedle tip diameter can also be used to regulate stem cell morphological heterogeneity. We hope our solid silicon nanoneedle arrays will be applicable to control other types of stem cell fates, as our study proved that how nanoneedle sharpness can be used to control the mechanical microenvironment around the hMSCs. Further investigation on stem cell differentiation, especially focused on human induced pluripotent stem cell-derived neural stem cells (iPSC-NSCs) and mouse embryonic stem cells (mESC) is on-going.
Also, our nanoneedle system can be utilized to efficiently deliver small molecules and gene delivery, by impinging plasma and nuclear membrane. To achieve this, we are going to combine surface engineering technique based on layer-by-layer (LbL), to functionalize the nanoneedle surface and load the molecules/genes to be delivered inside the cells. We expect the combination of surface chemistry and nanoneedle geometry will enhance the efficiency of cell growth, gene transfection to the cell/tissue, and so on.
Also, our nanoneedle system can be utilized to efficiently deliver small molecules and gene delivery, by impinging plasma and nuclear membrane. To achieve this, we are going to combine surface engineering technique based on layer-by-layer (LbL), to functionalize the nanoneedle surface and load the molecules/genes to be delivered inside the cells. We expect the combination of surface chemistry and nanoneedle geometry will enhance the efficiency of cell growth, gene transfection to the cell/tissue, and so on.