Controlled Self-assembly of Stem Cell Aggregates Instructs Pluripotency and Lineage Bias.

Stem cell-derived organoids and other 3D microtissues offer enormous potential as models for drug screening, disease modeling, and regenerative medicine. Formation of stem/progenitor cell aggregates is common in biomanufacturing processes and critical to many organoid approaches. However, reproducibility of current protocols is limited by reliance on poorly controlled processes (e.g., spontaneous aggregation). Little is known about the effects of aggregation parameters on cell behavior, which may have implications for the production of cell aggregates and organoids. Here we introduce a bioengineered platform of labile substrate arrays that enable simple, scalable generation of cell aggregates via a controllable 2D-to-3D "self-assembly". As a proof-of-concept, we show that labile substrates generate size- and shape-controlled embryoid bodies (EBs) and can be easily modified to control EB self-assembly kinetics. We show that aggregation method instructs EB lineage bias, with faster aggregation promoting pluripotency loss and ectoderm, and slower aggregation favoring mesoderm and endoderm. We also find that aggregation kinetics of EBs markedly influence EB structure, with slower kinetics resulting in increased EB porosity and growth factor signaling. Our findings suggest that controlling internal structure of cell aggregates by modifying aggregation kinetics is a potential strategy for improving 3D microtissue models for research and translational applications.

Surface functionalization and dynamics of polymeric cell culture substrates.

The promise of growing tissues to replace or improve the function of failing ones, a practice often referred to as regenerative medicine, has been driven in recent years by the development of stem cells and cell lines. Stem cells are typically cultured outside the body to increase cell number or differentiate the cells into mature cell types. In order to maximize the regenerative potential of these cells, there is a need to understand cell-material interactions that direct cell behavior and cell-material dynamics. Most synthetic surfaces used for growth and differentiation of cells in the lab are impractical and cost prohibitive in clinical labs. This review focuses on the modification of low cost polymer substrates that are already widely used for cell culture so that they may be used to control and understand cell-material interactions. In addition, we discuss the ability of cells to exert dynamic control over the microenvironment leading to a more complex, less controlled surface.

Peptide Conjugation to a Polymer Coating via Native Chemical Ligation of Azlactones for Cell Culture.

Conjugation of biomolecules for stable presentation is an essential step toward reliable chemically defined platforms for cell culture studies. In this work, we describe the formation of a stable and site-specific amide bond via the coupling of a cysteine terminated peptide at low concentration to an azlactone containing copolymer coating. A copolymer of polyethylene glycol methyl ether methacrylate-ran-vinyl azlactone-ran-glycidyl methacrylate P(PEGMEMA-r-VDM-r-GMA) was used to form a thin coating (20-30 nm) on silicon and polycarbonate substrates. The formation and stability of coating-peptide bonds for peptides containing free thiols and amines were quantified by X-ray photoelectron spectroscopy (XPS) after exposure to cell culture conditions. Peptides containing a thiol as the only nucleophile coupled via a thioester bond; however, the bond was labile under cell culture conditions and almost all the bound peptides were displaced from the surface over a period of 2 days. Coupling with N-terminal primary amine peptides resulted in the formation of an amide bond with low efficiency (<20%). In contrast, peptides containing an N-terminal cysteine, which contain both nucleophiles (free thiol and amine) in close proximity, bound with 67% efficiency under neutral pH, and were stable under the same conditions for 2 weeks. Control studies confirm that the stable amide formation was a result of an intramolecular rearrangement through a N-acyl intermediate that resembles native chemical ligation. Through a combination of XPS and cell culture studies, we show that the cysteine terminated peptides undergo a native chemical ligation process at low peptide concentration in aqueous media, short reaction time, and at room temperature resulting in the stable presentation of peptides beyond 2 weeks for cell culture studies.

Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior.

Here, we have developed a novel method for forming hydrogel arrays using surfaces patterned with differential wettability. Our method for benchtop array formation is suitable for enhanced-throughput, combinatorial screening of biochemical and biophysical cues from chemically defined cell culture substrates. We demonstrated the ability to generate these arrays without the need for liquid handling systems and screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term 2-dimensional cell culture. Regardless of substrate stiffness, hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS peptide concentration. Increasing substrate stiffness also resulted in increased hMSC initial cell attachment, spreading, and proliferation; however, examination of the combinatorial effects of CRGDS peptide concentration and substrate stiffness revealed potential interplay between these distinct substrate signals. Maximal hMSC proliferation seen on substrates with either high stiffness or high CRGDS peptide concentration suggests that some baseline level of cytoskeletal tension was required for hMSC proliferation on hydrogel substrates and that multiple substrate signals could be engineered to work in synergy to promote mechanosensing and regulate cell behavior. STATEMENT OF SIGNIFICANCE: Our novel array formation method using surfaces patterned with differential wettability offers the advantages of benchtop array formation for 2-dimensional cell cultures and enhanced-throughput screening without the need for liquid handling systems. Hydrogel arrays formed via our method are suitable for screening the influence of chemical (e.g. cell adhesive ligands) and physical (stiffness, size, shape, and thickness) substrate properties on stem cell behavior. The arrays are also fully compatible with commercially available micro-array add-on systems, which allows for simultaneous control of the insoluble and soluble cell culture environment. This study used hydrogel arrays to demonstrate that synergy between cell adhesion and mechanosensing can be used to regulate hMSC behavior.

Isolation of Pristine Electronics Grade Semiconducting Carbon Nanotubes by Switching the Rigidity of the Wrapping Polymer Backbone on Demand.

Conjugated polymers are among the most selective carbon nanotube sorting agents discovered and enable the isolation of ultrahigh purity semiconducting singled-walled carbon nanotubes (s-SWCNTs) from heterogeneous mixtures that contain problematic metallic nanotubes. The strong selectivity though highly desirable for sorting, also leads to irreversible adsorption of the polymer on the s-SWCNTs, limiting their electronic and optoelectronic properties. We demonstrate how changes in polymer backbone rigidity can trigger its release from the nanotube surface. To do so, we choose a model polymer, namely poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,60-(2,20-bipyridine))] (PFO-BPy), which provides ultrahigh selectivity for s-SWCNTs, which are useful specifically for FETs, and has the chemical functionality (BPy) to alter the rigidity using mild chemistry. Upon addition of Re(CO)5Cl to the solution of PFO-BPy wrapped s-SWCNTs, selective chelation with the BPy unit in the copolymer leads to the unwrapping of PFO-BPy. UV-vis, XPS, and Raman spectroscopy studies show that binding of the metal ligand complex to BPy triggers up to 85% removal of the PFO-BPy from arc-discharge s-SWCNTs (diameter = 1.3-1.7 nm) and up to 72% from CoMoCAT s-SWCNTs (diameter = 0.7-0.8 nm). Importantly, Raman studies show that the electronic structure of the s-SWCNTs is preserved through this process. The generalizability of this method is demonstrated with two other transition metal salts. Molecular dynamics simulations support our experimental findings that the complexation of BPy with Re(CO)5Cl in the PFO-BPy backbone induces a dramatic conformational change that leads to a dynamic unwrapping of the polymer off the nanotube yielding pristine s-SWCNTs.

Polyethylene Glycol Coatings on Plastic Substrates for Chemically Defined Stem Cell Culture.

Human mesenchymal stem cells (hMSCs) are a widely available and clinically relevant cell type with a host of applications in regenerative medicine. Current clinical expansion methods can lead to selective changes in hMSC phenotype potentially resulting from relatively undefined cell culture surfaces. Chemically defined synthetic surfaces can aid in understanding the influence of cell-material interactions on stem cell behavior. Here, a thin copolymer coating for hMSC culture on plastic substrates is developed. The random copolymer is synthesized by living free radical polymerization and characterized in solution before application to the substrate, ensuring a homogeneous coating and limiting the sample-to-sample variations. The ability to coat multiple substrate types and cover large surface areas is reported. Arg-Gly-Asp-containing peptides are incorporated into the coating under aqueous conditions via their lysine or cysteine side chains, resulting in amide and thioester linkages, respectively. Stability studies show amide linkages to be stable and thioester linkages to be labile under standard serum-containing culture conditions. In addition, chemically defined passaging of hMSCs using only ethylenediaminetetraacetic acid on polystyrene dishes is shown. After passage, the hMSCs can be seeded back onto the same plate, indicating potential reusability of the coating.

Structured layer of rhenium dye on SiO2 and TiO2 surfaces by Langmuir-Blodgett technique.

We demonstrate the Langmuir-Blodgett assembly of two rhenium-bipyridine complexes containing a flexible or an aromatic bridge, and transfer of the monolayer to SiO2 and single crystal TiO2 substrates. Both of the complexes (ReEC and Re2TC) have a hydrophilic carboxylic acid group, which preferentially anchors into the water subphase, and forms stable monolayers at surface pressures up to 40 mN/m. The optimum conditions for the formation of complete monolayers of both ReEC and Re2TC were identified through characterization of the morphology by atomic force microscopy (AFM), the thickness by ellipsometry, and the surface coverage by X-ray photoelectron spectroscopy (XPS). X-ray reflectivity measurements (XRR) are consistent with the orientation of the molecules normal to the substrate, and their extension to close to their calculated maximum length. Parameters derived from XRR analysis show that there is a higher packing density for Re2TC monolayers than for ReEC monolayers, attributable to the more rigid bridge in the Re2TC molecule.

A dual functional layer for block copolymer self-assembly and the growth of nanopatterned polymer brushes.

We present a versatile method for fabricating nanopatterned polymer brushes using a cross-linked thin film made from a random copolymer consisting of an inimer (p-(2-bromoisobutyloylmethyl)styrene), styrene, and glycidyl methacrylate (GMA). The amount of inimer was held constant at 20 or 30% while the relative amount of styrene to GMA was varied to induce perpendicular domain orientation in an overlying P(S-b-MMA) block copolymer (BCP) film for lamellar and cylindrical morphologies. A cylinder forming BCP blend with PMMA homopolymer was assembled to create a perpendicular hexagonal array of cylinders, which allowed access to a nanoporous template without the loss of initiator functionality. Surface-initiated ATRP of 2-hydroxyethyl methacrylate was conducted through the pores to generate a dense array of nanopatterned brushes. Alternatively, gold was deposited into the nanopores, and brushes were grown around the dots after removal of the template. This is the first example of combining the chemistry of nonpreferential surfaces with surface-initiated growth of polymer chains.

Crosslinked PEG mats for peptide immobilization and stem cell adhesion.

We have designed a lightly crosslinked PEG based copolymer coating with compositional flexibility as well as extended stability for studying human mesenchymal stem cells (hMSCs). Copolymers contain a majority of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) as a cytophobic background with poly(ethylene glycol) methacrylate (PEGMA) for peptide coupling, and less than 10% glycidyl methacrylate (GMA) for crosslinking. Copolymer thin films were crosslinked into 30 nm thick mats by either thermal treatment or ultraviolet light and were stable for 35 days in water at 37 °C. The amount of PEGMA in the copolymer was optimized to ∼11% to minimize non-specific cell-protein interactions while maximizing the amount of total bound peptides. Following the binding of RGDSP to the mat, hMSCs were seeded. The hMSC adhesion, spreading and focal adhesion complex formation were promoted in a concentration dependent manner. Mats coupled with a non-adhesive scramble (RDGSP) maintained their cytophobicity. Competitive detachment experiments further demonstrated that cell adhesion was mediated by receptor binding to the RGDSP peptide. Cell culture experiments performed at 1 and 2 weeks show that mats can still resist cell adhesion after incubation in a serum containing medium. X-ray photoelectron spectroscopy (XPS) was effectively used to quantify the average total peptide concentration as 12.6 ± 6.14 pmol cm-2. A square 2.2 mm N (1s) element map shows an average value of 17.9 pmol cm-2 of RGDSP, which correlates well with the multipoint high resolution data. The stability of the copolymer, compositional flexibility, ease of application and the ability to precisely quantify bound peptides on the mats make these materials ideal for the study of cellular processes, where stability, functionality and topography of the biointerface are relevant.

Monodisperse porous nanodiscs with fluorescent and crystalline wall structure.

We report a facile solution process to synthesize monodisperse porous nanodiscs through confined molecular self-assembly of surfactants and ZnTPyP. The nanodiscs exhibit trimodal pores with fluorescent and crystalline wall structures, and are potentially important for sorption and separation, sensors, catalytic materials, electrode materials, etc.

Hydrogen-bonding-assisted self-assembly: monodisperse hollow nanoparticles made easy.

A facile self-assembly process for synthesizing monodisperse hollow spherical nanoparticles that are less than 50 nm in diameter has been developed. Preferential hydrogen bonding between an amphiphilic block copolymer (polystyrene-b-polyvinylpyridine, PS-PVP) and a hydrogen-bonding agent (HA) enables formation of monodisperse spherical solid polymer nanoparticles with the HA residing in the particle core surrounded by the polymer. Removal of the HA results in monodisperse hollow nanoparticles with tunable hollow cavity size and internal surface reactivity. Formation of ordered hollow nanoparticle films with controlled index of refraction for antireflective coating applications is demonstrated.

Cooperative self-assembly-assisted formation of monodisperse optically active spherical and anisotropic nanoparticles.

We report a new method in which spontaneous self-assembly is employed to synthesize monodisperse polymer nanoparticles with controlled size (<50 nm), shape, tunable functionality, and enhanced solvent and thermal stability. Cooperative noncovalent interactions, such as hydrogen bonding and aromatic pi-pi stacking, assist self-assembly of amphiphilic macromolecules (polystyrene-block-polyvinylpyridine, PS--PVP) and structure directing agents (SDAs) to form both spherical and anisotropic solid polymer nanoparticles with SDAs residing in the particle core surrounded by the polymers. Through detailed investigations by scanning electron microscopy and transmission electron microscopy (TEM), we have rationalized nanoparticle morphology evolution and dependence on factors such as SDA concentration and PVP size. By keeping the PS chain size constant, the particle morphology progresses from continuous films to spherical particles, and on to cylindrical nanowires or rods with increasing the PVP chain size. The final nanoparticles are very stable and can be redispersed in common solvents to form homogenous solutions and thin films of ordered nanoparticle arrays through solvent evaporation processes. These nanoparticles exhibit tunable fluorescent colors (or emissions) depending on the choices of the central SDAs. Our method is simple and general without requiring complicated synthetic chemistry, stabilizing surfactants, or annealing procedures (e.g., temperature or solvent annealing), making scalable synthesis feasible.