Chung-Yao Yang

Using surfaces to modulate the morphology and structure of attached cells – a case of cancer cells on chitosan membranes

This paper describes the development of physically and/or chemically modified chitosan membranes to probe cellular behaviors and molecular-level structural responses of NIH-3T3 fibroblasts (normal cells) and Ha-ras-transformed cells (abnormal cells) adhered onto these modified membranes. To prepare chitosan membranes with nanometrically scaled physical features, we have demonstrated an inexpensive and easy-to-handle method that could be easily integrated with IC-based manufacturing processes with mass production potential. These physically or chemically modified chitosan membranes were examined via scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and water contact angle measurement, in order to gain a better understanding of chitosan membrane surface characteristics including surface morphology, stiffness, functional groups, and surface hydrophobicity/hydrophilicity. NIH-3T3 fibroblasts and their Ha-ras-transformed progeny were cultured on these modified chitosan membranes. After 12, 24 and 48 h of culture, these cells were investigated to decipher cellular behaviors. We found that NIH-3T3 fibroblasts and their Ha-ras-transformed progeny exhibited distinct structurally based responses attributable to chitosan membrane surface chemical or physical properties that we demonstrate as possibly applicable, for drug screening applications. Secondarily, but crucially to this study, we developed a chitosan-based micropatterning procedure that allowed us to re-arrange mammalian cells (i.e., HeLa cells in this study, for cancer drug screening) at the desired locations (with a single-cell array format). This procedure was based on cell affinity to different surface topographies of chitosan membranes that we prepared. This cell-based patterning approach has the potential for use in a wide range of applications including use as a promising platform for drug discovery, cytotoxicity studies, functional genomics, and investigations of cellular microenvironment. We believe that this study would provide further understanding of naturally derived biomaterials, lay the foundation for broadening the applications of chitosan, and facilitate the development of new biomedical devices (i.e., artificial stents, implantable artificial tissues, and sustainable implantable biosensors) with unique cell–material interface properties and characteristics, such as in vitro cell culture and diagnostic platforms.

Micropatterning of mammalian cells on inorganic-based nanosponges.

Developing artificial scaffolding structures in vitro in order to mimic physiological-relevant situations in vivo is critical in many biological and medical arenas including bone and cartilage generation, biomaterials, small-scale biomedical devices, tissue engineering, as well as the development of nanofabrication methods. We focus on using simple physical principles (photolithography) and chemical techniques (liquid vapor deposition) to build non-cytotoxic scaffolds with a nanometer resolution through using silicon substrates as the backbone. This method merges an optics-based approach with chemical restructuring to modify the surface properties of an IC-compatible material, switching from hydrophilicity to hydrophobicity. Through this nanofabrication-based approach that we developed, hydrophobic oxidized silicon nanosponges were obtained. We then probed cellular responses-examining cytoskeletal and morphological changes in living cells through a combination of fluorescence microscopy and scanning electron microscopy-via culturing Chinese hamster ovary cells, HIG-82 fibroblasts and Madin-Darby canine kidney cells on these silicon nanosponges. This study has demonstrated the potential applications of using these silicon-based nanopatterns such as influencing cellular behaviors at desired locations with a micro-/nanometer level.

Integrated Circuit-Based Biofabrication with Common Biomaterials for Probing Cellular Biomechanics

Recent advances in bioengineering have enabled the development of biomedical tools with modifiable surface features (small-scale architecture) to mimic extracellular matrices and aid in the development of well-controlled platforms that allow for the application of mechanical stimulation for studying cellular biomechanics. An overview of recent developments in common biomaterials that can be manufactured using integrated circuit-based biofabrication is presented. Integrated circuit-based biofabrication possesses advantages including mass and diverse production capacities for fabricating in vitro biomedical devices. This review highlights the use of common biomaterials that have been most frequently used to study cellular biomechanics. In addition, the influence of various small-scale characteristics on common biomaterial surfaces for a range of different cell types is discussed.

Probing cellular behaviors through nanopatterned chitosan membranes

Abstract This paper describes a high-throughput method for developing physically modified chitosan membranes to probe the cellular behavior of MDCK epithelial cells and HIG-82 fibroblasts adhered onto these modified membranes. To prepare chitosan membranes with micro/nanoscaled features, we have demonstrated an easy-to-handle, facile approach that could be easily integrated with IC-based manufacturing processes with mass production potential. These physically modified chitosan membranes were observed by scanning electron microscopy to gain a better understanding of chitosan membrane surface morphology. After MDCK cells and HIG-82 fibroblasts were cultured on these modified chitosan membranes for various culture durations (i.e. 1, 2, 4, 12 and 24 h), they were investigated to decipher cellular behavior. We found that both cells preferred to adhere onto a flat surface rather than on a nanopatterned surface. However, most (> 80%) of the MDCK cells showed rounded morphology and would suspend in the cultured medium instead of adhering onto the planar surface of negatively nanopatterned chitosan membranes. This means different cell types (e.g. fibroblasts versus epithelia) showed distinct capabilities/preferences of adherence for materials of varying surface roughness. We also showed that chitosan membranes could be re-used at least nine times without significant contamination and would provide us consistency for probing cell–material interactions by permitting reuse of the same substrate. We believe these results would provide us better insight into cellular behavior, specifically, microscopic properties and characteristics of cells grown under unique, nanopatterned cell-interface conditions.

Fabricating millimeter-scale polymeric structures for biomedical applications via a combination of UV-activated materials and daily-use tools

This paper describes an easy-to-handle approach to create three-dimensional millimeter-scale (or submillimeter-scale) polymeric structures on various substrates that have been used as molds in order to develop a polymeric-based manufacturing procedure for making in vitro diagnostic devices with mass production capacity and portability. These polymeric structures were made by using UV-activated materials, adhesive tapes as the mask, and a UV-LED flashlight as the portable light source. This straightforward approach can be easily performed and has great potential for use in resource-limited settings. The ability to conduct three common metabolic assays – for glucose, total cholesterol, and nitrite ions in this study – in both a buffer system and human serum (analytical validation, US FDA regulations) with clinically relevant sensitivity has been demonstrated using these polymeric-based in vitro diagnostic devices. This study, we believe, would provide for a wide range of potential applications such as the development of in vitro diagnostic devices, and a large-scale, low-cost, and easy-to-handle fabrication procedure for either developing regions or resource-limited settings, and, ultimately, for the development of “zero-cost” diagnostic devices for global public health.

High-throughput physically based approach for mammalian cell encapsulation

Herein, we wish to tear down the traditional boundaries between physics and life sciences by demonstrating a physically based, flow-focusing method to encapsulate mammalian cells into alginate-based microspheres in a very short period of time. We paid particular attention to the physical properties of the alginate solution as it was critical to create a physiologically relevant environment within the alginate microspheres. The cells we cultured when re-culturing them on Petri dishes could still be maintained for at least 4 days after microsphere encapsulation. We believe that this study would provide interesting insight in biophysics, polymer physics, and applied physics.

Probing the dynamic responses of individual actin filaments under fluidic mechanical stimulation via microfluidics

Herein, we demonstrate an easy-to-handle approach that employs a combination of microcurvilinear flow and fluorescence microscopy for probing the dynamic responses of individual synthesized actin filaments. We observed morphological changes of single actin filaments with different spatiotemporal responses when they were elongated with rotation or underwent significant bending during fluidic shear stress, and found that they may initially increase their curvature but then start releasing the external force immediately thereafter. Our approach allowed us to visibly examine the dynamic responses of individual actin filaments under simultaneous forces of rotation and elongation, as well as bending resulting from fluidic shear stress.

Cellular behaviors on chemically/physically modified SiO 2 surfaces

This paper describe an easy-to-handle approach to probe cellular behaviors via using silicon dioxide nanotextures with various functional groups. The silicon dioxide nanotextures were performed through using metal assisted chemical etching and wet oxidation. The pitch of nanotextures can be adjusted by controlling etching durations. The results showed that cells preferred to spread out on nanotextures with longer pitch rather than on nanotextures with shorter pitch. In addition, cells also preferred to adhere on planar surface rather than on nanotextured surface. We believe, this study can help us to get more insights of cell biology and biomedical-relevant researches.

Influences of locally mechanical stimulation on cellular behaviors via microfabrication approach

This paper describe an easy-to-handle approach to probe cellular behaviors via applying a force on microstructures (mechanical stimulation) with mammalian cells. The external mechanical stimulation on cells was performed through applying various forces on the polydimethylsiloxane (PDMS) microstructures after cells adhered onto it. After mechanical stimulation on PDMS microstructures for 2 hours using 100 grams (0.98 N), the cells were washed with PBS and then stained with Annexin V and PI. The results show NIH-3T3 fibroblasts adhered on the top layer, incline and bottom layer of trapeziums. However, the results show that the cell apoptosis mostly took place at top layer and incline of trapeziums, which is different with the results of simulation. We believe, this study can help us to get more insights of cell apoptosis and biomedical-relevant researches.

Silicon substrate strength enhancement depending on nanostructure morphology

Silicon nanostructures are extensively being researched for many different applications for industries. Here we present two different types of nanostructures, silicon nanoplates and nanoholes fabricated by electroless metal assisted wet etching for enhancing the bending strength by ~3.7 fold and ~6 fold respectively as compared to polished silicon samples which emphasize the dependence of bending strength on nanostructure morphologies. Roughness at the nanostructure bottom cause stress concentration to increase which degrades the bending strength. Moreover, this technology can open a pathway of flexible silicon substrates for flexible and bendable electronics.