Biomaterial arrays with defined adhesion ligand densities and matrix stiffness identify distinct phenotypes for tumorigenic and nontumorigenic human mesenchymal cell types.

Here, we aimed to investigate migration of a model tumor cell line (HT-1080 fibrosarcoma cells, HT-1080s) using synthetic biomaterials to systematically vary peptide ligand density and substrate stiffness. A range of substrate elastic moduli were investigated by using poly(ethylene glycol) (PEG) hydrogel arrays (0.34 - 17 kPa) and self-assembled monolayer (SAM) arrays (~0.1-1 GPa), while cell adhesion was tuned by varying the presentation of Arg-Gly-Asp (RGD)-containing peptides. HT-1080 motility was insensitive to cell adhesion ligand density on RGD-SAMs, as they migrated with similar speed and directionality for a wide range of RGD densities (0.2-5% mol fraction RGD). Similarly, HT-1080 migration speed was weakly dependent on adhesion on 0.34 kPa PEG surfaces. On 13 kPa surfaces, a sharp initial increase in cell speed was observed at low RGD concentration, with no further changes observed as RGD concentration was increased further. An increase in cell speed ~ two-fold for the 13 kPa relative to the 0.34 kPa PEG surface suggested an important role for substrate stiffness in mediating motility, which was confirmed for HT-1080s migrating on variable modulus PEG hydrogels with constant RGD concentration. Notably, despite ~ two-fold changes in cell speed over a wide range of moduli, HT-1080s adopted rounded morphologies on all surfaces investigated, which contrasted with well spread primary human mesenchymal stem cells (hMSCs). Taken together, our results demonstrate that HT-1080s are morphologically distinct from primary mesenchymal cells (hMSCs) and migrate with minimal dependence on cell adhesion for surfaces within a wide range of moduli, whereas motility is strongly influenced by matrix mechanical properties.

A quantitative comparison of human HT-1080 fibrosarcoma cells and primary human dermal fibroblasts identifies a 3D migration mechanism with properties unique to the transformed phenotype.

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced ß1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with ß1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

A chemically-defined screening platform reveals behavioral similarities between primary human mesenchymal stem cells and endothelial cells.

Chemically defined substrates, which rigorously control protein-surface and cell-surface interactions, can be used to probe the effects of specific biomolecules on cell behavior. Here we combined a chemically-defined, array-based format with automated, time-lapse microscopy to efficiently screen cell-substrate interactions. Self-assembled monolayers (SAMs) of alkanethiolates bearing oligo(ethylene glycol) units and reactive terminal groups were used to present cell adhesion peptides while minimizing non-specific protein interactions. Specifically, we describe rapid fabrication of arrays of 1 mm spots, which present varied densities of the integrin-binding ligand Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). Results indicate that cell attachment, cell spreading, and proliferation exhibit strong dependencies on GRGDSP density for both human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs). Furthermore, relative spreading and proliferation over a broad range of GRGDSP densities were similar for both primary cell types, and detailed comparison between cell behaviors identified a 1 : 1 correlation between spreading and proliferation for both HUVECs and hMSCs. Finally, time-lapse microscopy of SAM arrays revealed distinct adhesion-dependent migratory behaviors for HUVECs and hMSCs. These results demonstrate the benefits of using an array-based screening platform for investigating cell function. While the proof-of-concept focuses on simple cellular properties, the quantitative similarities observed for hMSCs and HUVECs provides a direct example of how phenomena that would not easily be predicted can be shown to correlate between different cell types.

Patterned self-assembled monolayers: efficient, chemically defined tools for cell biology.

Self-assembled monolayers (SAMs) of alkanethiolates on gold can be used to carefully probe immobilized biomolecule interactions with cell-surface receptors. However, due to a lack of experimental throughput associated with labor-intensive production, specialized fabrication apparatus, and other practical challenges, alkanethiolate SAMs have not had widespread use by biological researchers. In this Minireview, we investigate a range of techniques that could enhance the throughput of SAM-based approaches by patterning substrates with arrays of different conditions. Here we highlight microfluidic, photochemical, localized removal, and backfilling techniques to locally pattern SAM substrates with biomolecules and also describe how these approaches have been applied in SAM-based screening systems. Furthermore we provide perspectives on several crucial barriers that need to be overcome to enable widespread use of SAM chemistry in biological applications.

Differential effects of a soluble or immobilized VEGFR-binding peptide.

Regulating endothelial cell behavior is a key step in understanding and controlling neovascularization for both pro-angiogenic and anti-angiogenic therapeutic strategies. Here, we characterized the effects of a covalently immobilized peptide mimic of vascular endothelial growth factor, herein referred to as VEGF receptor-binding peptide (VR-BP), on human umbilical vein endothelial cell (HUVEC) behavior. Self-assembled monolayer arrays presenting varied densities of covalently immobilized VR-BP and varied densities of the fibronectin-derived cell adhesion peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) were used to probe for changes in HUVEC attachment, proliferation and tubulogenesis. In a soluble form, VR-BP exhibited pro-angiogenic effects in agreement with previous studies, indicated by increases in HUVEC proliferation. However, when presented to cells in an insoluble context, covalently immobilized VR-BP inhibited several pro-angiogenic HUVEC behaviors, including attachment and proliferation, and also inhibited HUVEC response to soluble recombinant VEGF protein. Furthermore, substrates with covalently immobilized VR-BP also modulated HUVEC tubulogenesis when a matrigel overlay assay was used to provide cells with a pseudo-three dimensional environment. Taken together, these results demonstrate that the context in which ligands are presented to cell surface receptors strongly influences their effects, and that the same ligand can be an agonist or an antagonist depending on the manner of presentation to the cell.

Combinatorial screening of chemically defined human mesenchymal stem cell culture substrates.

Self-assembled monolayers (SAMs) of alkanethiolates on gold are chemically defined substrates that can be used to evaluate the effects of an immobilized biomolecule. However, the types of biomolecules that can influence stem cell behavior are numerous and inter-related, and efficient experimental formats are a critical need. Here we employed a SAM array technology to investigate the effects of multiple, distinct peptides and peptide combinations on human mesenchymal stem cell (hMSC) behavior. Specifically, we characterized the conjugation of peptide mixtures to SAM arrays and then investigated the combined effects of a bone morphogenic protein receptor-binding peptide (BR-BP), a heparin proteoglycan-binding peptide (HPG-BP), and varied densities of the integrin-binding ligand Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) on hMSC surface coverage and alkaline phosphatase activity. Results indicate that an amine reactive fluorescent probe can be used to characterize peptide composition after immobilization in SAM array spots. Furthermore, hMSC response to BR-BP and HPG-BP is dependent on GRGDSP density and at day 7, hMSC alkaline phosphatase expression is highly dependent on GRGDSP density. Taken together, we demonstrate how a SAM array approach can be used to probe the combinatorial effects of multiple peptides and motivate further investigations into potential synergies between cell adhesion and other bioactive peptides.

Surfaces that sequester serum-borne heparin amplify growth factor activity.

Surfaces presenting a heparin-binding peptide can non-covalently sequester heparin from culture supplements, such as fetal bovine serum. In turn, sequestered, serum-borne heparin can non-covalently localize growth factors at the cell-material interface, resulting in amplified growth factor bioactivity.

Harnessing endogenous growth factor activity modulates stem cell behavior.

The influence of specific serum-borne biomolecules (e.g. heparin) on growth factor-dependent cell behavior is often difficult to elucidate in traditional cell culture due to the random, non-specific nature of biomolecule adsorption from serum. We hypothesized that chemically well-defined cell culture substrates could be used to study the influence of sequestered heparin on human mesenchymal stem cell (hMSC) behavior. Specifically, we used bio-inert self-assembled monolayers (SAMs) chemically modified with a bioinspired heparin-binding peptide (termed "HEPpep") and an integrin-binding peptide (RGDSP) as stem cell culture substrates. Our results demonstrate that purified heparin binds to HEPpep SAMs in a dose-dependent manner, and serum-borne heparin binds specifically and in a dose-dependent manner to HEPpep SAMs. These heparin-sequestering SAMs enhance hMSC proliferation by amplifying endogenous fibroblast growth factor (FGF) signaling, and enhance hMSC osteogenic differentiation by amplifying endogenous bone morphogenetic protein (BMP) signaling. The effects of heparin-sequestering are similar to the effects of supraphysiologic concentrations of recombinant FGF-2. hMSC phenotype is maintained over multiple population doublings on heparin-sequestering substrates in growth medium, while hMSC osteogenic differentiation is enhanced in a bone morphogenetic protein-dependent manner on the same substrates during culture in osteogenic induction medium. Together, these observations demonstrate that the influence of the substrate on stem cell phenotype is sensitive to the culture medium formulation. Our results also demonstrate that enhanced hMSC proliferation can be spatially localized by patterning the location of HEPpep on the substrate. Importantly, the use of chemically well-defined SAMs in this study eliminated the confounding factor of random, non-specific biomolecule adsorption, and identified serum-borne heparin as a key mediator of hMSC response to endogenous growth factors.

Patterning discrete stem cell culture environments via localized self-assembled monolayer replacement.

Self-assembled monolayers (SAMs) of alkanethiolates on gold have become an important tool for probing cell-material interactions. Emerging studies in stem cell biology are particularly reliant on well-defined model substrates, and rapid, highly controllable fabrication methods may be necessary for characterizing the wide array of stem cell-material interactions. Therefore, this study describes a rapid method for creating SAM cell culture substrates with multiple discrete regions of controlled peptide identity and density. The approach uses a NaBH(4) solution to selectively remove regions of bioinert, hydroxyl-terminated oligo(ethylene glycol) alkanethiolate SAM and then locally replace them with mixed SAMs of hydroxyl- and carboxylic acid-terminated oligo(ethylene glycol) alkanethiolates. The cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) was then covalently linked to carboxylic acid-terminated mixed SAM regions to create cell adhesive environments within a bioinert background. SAM preparation and peptide immobilization were characterized using polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS), as well as assays to monitor conjugation of a fluorescently labeled peptide. This "localized SAM replacement" method was achieved using an array of microchannels, which facilitated rapid and simple processing. Results indicate that immobilized RGDSP promoted spatially localized attachment of human mesenchymal stem cells (hMSCs) within specified regions, while maintaining a stable, bioinert background in serum-containing cell culture conditions for up to 14 days. Cell attachment to patterned regions presenting a range of cell adhesion peptide densities demonstrated that peptide identity and density strongly influence hMSC spreading and focal adhesion density. These substrates contain discrete, well-defined microenvironments for stem cell culture, which could ultimately enable high-throughput screening for the effects of immobilized signals on stem cell phenotype.

Biological implications of polydimethylsiloxane-based microfluidic cell culture.

Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.