Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips.

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 µm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 µm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Vero cells gain renal tubule markers in low-calcium and magnesium chemically defined media.

In this study, a chemically defined, animal component-free media was developed to promote Vero growth in suspension. Key media compounds were screened using Plackett-Burman styled experiments to create a media formulation to support suspension growth. Vero cells remained viable in suspension, but their growth rate was extremely low, conversely, other cell types such as CHO-K1, MDCK and HEK293T were able to grow in single cell suspension in the same media. To investigate the slow growth of Vero cells, RNA-seq analysis was conducted. Vero cells were cultured in three different conditions: adherently in serum-containing medium, adherently in in-house medium, and in suspension in low calcium and magnesium in-house medium. This study illustrates that adherent cells maintain similar gene expression, while the suspension phenotype tends to overexpress genes related to renal tubules.

Limitation in Controlling the Morphology of Mammalian Vero Cells Induced by Cell Division on Asymmetric Tungsten-Silicon Oxide Nanocomposite.

Engineered nanomaterials are often used in tissue engineering applications to influence and manipulate the behavior of cells. Recently, a number of tungsten-silicon oxide nanocomposite devices containing equal width (symmetric) tungsten and silicon oxide parallel line comb structures were developed and used by our group. The devices induced over 90% of seeded cells (Vero) to align within ±20° of the axes of 10 µm wide tungsten lines. Furthermore, a mathematical model was successfully developed to predict this alignment behavior and forecast the minimum width of isolated tungsten lines required to induce such behavior. However, the mechanism by which the widths of the symmetrical tungsten and silicon oxide lines induce the alignment behavior is still unknown. Furthermore, the model was never tested on more complex asymmetrical structures. Herewith, experiments were conducted with mammalian cells on complex asymmetrical structures with unequal tungsten and silicon oxide line widths. Results showed that the model could be extended to more complex pattern structures. In addition, cell morphology on the patterned structures reset during cell division because of mitotic rounding, which reduced the population of cells that elongated and aligned on the tungsten lines. Ultimately, we concluded that it was impossible to achieve a 100% alignment with cells having unsynchronized cell cycles because cell rounding during mitosis took precedence over cell alignment; in other words, internal chemical cues had a stronger role in cell morphology than external cues.

A flow cytometric granularity assay for the quantification of infectious virus.

A flow cytometry-based assay was developed to assess the infective titer of two recombinant viruses: a recombinant herpes simplex type 2 (rHSV-2) and a recombinant canary pox (rALVAC.gfp). This method uses granularity of infected Vero and QT-35 cells, respectively, and correlates this to the infectious titer of virus samples. The percent of the cell populations with a high level of granularity could accurately be correlated to viral titers obtained through a traditional plaque assay, with R2 values greater than 0.8 using a semi-logarithmic scale. This approach offers a rapid, high-throughput method for infectious virus titration with similar accuracy to a traditional plaque assay.

What's Happening on the Other Side? Revealing Nano-Meter Scale Features of Mammalian Cells on Engineered Textured Tantalum Surfaces.

Advanced engineered surfaces can be used to direct cell behavior. These behaviors are typically characterized using either optical, atomic force, confocal, or electron microscopy; however, most microscopic techniques are generally restricted to observing what's happening on the "top" side or even the interior of the cell. Our group has focused on engineered surfaces typically reserved for microelectronics as potential surfaces to control cell behavior. These devices allow the exploration of novel substrates including titanium, tungsten, and tantalum intermixed with silicon oxide. Furthermore, these devices allow the exploration of the intricate patterning of surface materials and surface geometries i.e., trenches. Here we present two important advancements in our research: (1) the ability to split a fixed cell through the nucleus using an inexpensive three-point bend micro-cleaving technique and image 3D nanometer scale cellular components using high-resolution scanning electron microscopy; and (2) the observation of nanometer projections from the underbelly of a cell as it sits on top of patterned trenches on our devices. This application of a 3-point cleaving technique to visualize the underbelly of the cell is allowing a new understanding of how cells descend into surface cavities and is providing a new insight on cell migration mechanisms.

Nanoscale-Textured Tantalum Surfaces for Mammalian Cell Alignment.

Tantalum is one of the most important biomaterials used for surgical implant devices. However, little knowledge exists about how nanoscale-textured tantalum surfaces affect cell morphology. Mammalian (Vero) cell morphology on tantalum-coated comb structures was studied using high-resolution scanning electron microscopy and fluorescence microscopy. These structures contained parallel lines and trenches with equal widths in the range of 0.18 to 100 µm. Results showed that as much as 77% of adherent cell nuclei oriented within 10° of the line axes when deposited on comb structures with widths smaller than 10 µm. However, less than 20% of cells exhibited the same alignment performance on blanket tantalum films or structures with line widths larger than 50 µm. Two types of line-width-dependent cell morphology were observed. When line widths were smaller than 0.5 µm, nanometer-scale pseudopodia bridged across trench gaps without contacting the bottom surfaces. In contrast, pseudopodia structures covered the entire trench sidewalls and the trench bottom surfaces of comb structures with line-widths larger than 0.5 µm. Furthermore, results showed that when a single cell simultaneously adhered to multiple surface structures, the portion of the cell contacting each surface reflected the type of morphology observed for cells individually contacting the surfaces.

Pattern-Dependent Mammalian Cell (Vero) Morphology on Tantalum/Silicon Oxide 3D Nanocomposites.

The primary goal of this work was to investigate the resulting morphology of a mammalian cell deposited on three-dimensional nanocomposites constructed of tantalum and silicon oxide. Vero cells were used as a model. The nanocomposite materials contained comb structures with equal-width trenches and lines. High-resolution scanning electron microscopy and fluorescence microscopy were used to image the alignment and elongation of cells. Cells were sensitive to the trench widths, and their observed behavior could be separated into three different regimes corresponding to different spreading mechanism. Cells on fine structures (trench widths of 0.21 to 0.5 µm) formed bridges across trench openings. On larger trenches (from 1 to 10 µm), cells formed a conformal layer matching the surface topographical features. When the trenches were larger than 10 µm, the majority of cells spread like those on blanket tantalum films; however, a significant proportion adhered to the trench sidewalls or bottom corner junctions. Pseudopodia extending from the bulk of the cell were readily observed in this work and a minimum effective diameter of ~50 nm was determined for stable adhesion to a tantalum surface. This sized structure is consistent with the ability of pseudopodia to accommodate ~4⁻6 integrin molecules.

Biocompatibility of hydrogel-based scaffolds for tissue engineering applications.

Recently, understanding of the extracellular matrix (ECM) has expanded rapidly due to the accessibility of cellular and molecular techniques and the growing potential and value for hydrogels in tissue engineering. The fabrication of hydrogel-based cellular scaffolds for the generation of bioengineered tissues has been based on knowledge of the composition and structure of ECM. Attempts at recreating ECM have used either naturally-derived ECM components or synthetic polymers with structural integrity derived from hydrogels. Due to their increasing use, their biocompatibility has been questioned since the use of these biomaterials needs to be effective and safe. It is not surprising then that the evaluation of biocompatibility of these types of biomaterials for regenerative and tissue engineering applications has been expanded from being primarily investigated in a laboratory setting to being applied in the multi-billion dollar medicinal industry. This review will aid in the improvement of design of non-invasive, smart hydrogels that can be utilized for tissue engineering and other biomedical applications. In this review, the biocompatibility of hydrogels and design criteria for fabricating effective scaffolds are examined. Examples of natural and synthetic hydrogels, their biocompatibility and use in tissue engineering are discussed. The merits and clinical complications of hydrogel scaffold use are also reviewed. The article concludes with a future outlook of the field of biocompatibility within the context of hydrogel-based scaffolds.