Alexandra M. Greiner

Adding spatial control to click chemistry: phototriggered Diels-Alder surface (bio)functionalization at ambient temperature.

A photoconjugation strategy based on light-triggered Diels–Alder addition of o-quinodimethanes is compatible with biomolecules and proceeds rapidly at ambient temperature without the need of a catalyst. Spatial control was confirmed by photopatterning of a small-molecule ATRP initiator, a polymer, and a peptide in a time-of-flight secondary-ion mass spectrometry investigation.

Micro-engineered 3D scaffolds for cell culture studies.

Cells in physiological 3D environments differ considerably in morphology and differentiation from those in 2D tissue culture. Naturally derived polymer systems are frequently used to study cells in 3D. These 3D matrices are complex with respect to their chemical composition, mechanical properties, and geometry. Therefore, there is a demand for well-defined 3D scaffolds to systematically investigate cell behavior in 3D. Here, fabrication techniques, materials, architectures, biochemical functionalizations, and mechanical properties of 3D scaffolds are discussed. In particular, work focusing on single cells and small cell assemblies grown in tailored synthetic 3D scaffolds fabricated by computer-based techniques are reviewed and the influence of these environments on cell behavior is evaluated.

Multifunctional polymer scaffolds with adjustable pore size and chemoattractant gradients for studying cell matrix invasion

Transmigrating cells often need to deform cell body and nucleus to pass through micrometer-sized pores in extracellular matrix scaffolds. Furthermore, chemoattractive signals typically guide transmigration, but the precise interplay between mechanical constraints and signaling mechanisms during 3D matrix invasion is incompletely understood and may differ between cell types. Here, we used Direct Laser Writing to fabricate 3D cell culture scaffolds with adjustable pore sizes (2-10 μm) on a microporous carrier membrane for applying diffusible chemical gradients. Mouse embryonic fibroblasts invade 10 μm pore scaffolds even in absence of chemoattractant, but invasion is significantly enhanced by knockout of lamin A/C, a known regulator of cell nucleus stiffness. Nuclear stiffness thus constitutes a major obstacle to matrix invasion for fibroblasts, but chemotaxis signals are not essential. In contrast, epithelial A549 cells do not enter 10 μm pores even when lamin A/C levels are reduced, but readily enter scaffolds with pores down to 7 μm in presence of chemoattractant (serum). Nuclear stiffness is therefore not a prime regulator of matrix invasion in epithelial cells, which instead require chemoattractive signals. Microstructured scaffolds with adjustable pore size and diffusible chemical gradients are thus a valuable tool to dissect cell-type specific mechanical and signaling aspects during matrix invasion.

Three‐Dimensional Microscaffolds Exhibiting Spatially Resolved Surface Chemistry

Spatial control over the surface chemistry of 3D organic-inorganic hybrid microscaffolds is achieved by a two-photon-triggered cycloaddition. Following a coating step with photoactivatable dienes via silanization, surface irradiation with a femtosecond-pulsed laser in the presence of functional dienophiles enables a site-selective alteration of the surface chemistry. Bioconjugation with fluorescent protein tags is employed to reveal the 3D molecular patterns.

Interdigitated multicolored bioink micropatterns by multiplexed polymer pen lithography.

Multiplexing, i.e., the application and integration of more than one ink in an interdigitated microscale pattern, is still a challenge for microcontact printing (μCP) and similar techniques. On the other hand there is a strong demand for interdigitated patterns of more than one protein on subcellular to cellular length scales in the lower micrometer range in biological experiments. Here, a new integrative approach is presented for the fabrication of bioactive microarrays and complex multi-ink patterns by polymer pen lithography (PPL). By taking advantage of the strength of microcontact printing (μCP) combined with the spatial control and capability of precise repetition of PPL in an innovative way, a new inking and writing strategy is introduced for PPL that enables true multiplexing within each repetitive subpattern. Furthermore, a specific ink/substrate platform is demonstrated that can be used to immobilize functional proteins and other bioactive compounds over a biotin-streptavidin approach. This patterning strategy aims specifically at application by cell biologists and biochemists addressing a wide range of relevant pattern sizes, easy pattern generation and adjustment, the use of only biofriendly, nontoxic chemicals, and mild processing conditions during the patterning steps. The retained bioactivity of the fabricated cm(2) area filling multiprotein patterns is demonstrated by showing the interaction of fibroblasts and neurons with multiplexed structures of fibronectin and laminin or laminin and ephrin, respectively.

Cyclic tensile strain controls cell shape and directs actin stress fiber formation and focal adhesion alignment in spreading cells

The actin cytoskeleton plays a crucial role for the spreading of cells, but is also a key element for the structural integrity and internal tension in cells. In fact, adhesive cells and their actin stress fiber–adhesion system show a remarkable reorganization and adaptation when subjected to external mechanical forces. Less is known about how mechanical forces alter the spreading of cells and the development of the actin–cell-matrix adhesion apparatus. We investigated these processes in fibroblasts, exposed to uniaxial cyclic tensile strain (CTS) and demonstrate that initial cell spreading is stretch-independent while it is directed by the mechanical signals in a later phase. The total temporal spreading characteristic was not changed and cell protrusions are initially formed uniformly around the cells. Analyzing the actin network, we observed that during the first phase the cells developed a circumferential arc-like actin network, not affected by the CTS. In the following orientation phase the cells elongated perpendicular to the stretch direction. This occurred simultaneously with the de novo formation of perpendicular mainly ventral actin stress fibers and concurrent realignment of cell-matrix adhesions during their maturation. The stretch-induced perpendicular cell elongation is microtubule-independent but myosin II-dependent. In summary, a CTS-induced cell orientation of spreading cells correlates temporary with the development of the acto-myosin system as well as contact to the underlying substrate by cell-matrix adhesions.

Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

Hierarchical Micro-Nano Surface Topography Promotes Long-Term Maintenance of Undifferentiated Mouse Embryonic Stem Cells.

Understanding of stem cell-surface interactions and, in particular, long-term maintenance of stem cell pluripotency on well-defined synthetic surfaces is crucial for fundamental research and biomedical applications of stem cells. Here, we show that synthetic surfaces possessing hierarchical micro-nano roughness (MN-surfaces) promote long-term self-renewal (>3 weeks) of mouse embryonic stem cells (mESCs) as monitored by the expression levels of the pluripotency markers octamer-binding transcription factor 4 (Oct4), Nanog, and alkaline phosphatase. On the contrary, culturing of mESCs on either smooth (S-) or nanorough polymer surfaces (N-surfaces) leads to their fast differentiation. Moreover, we show that regular passaging of mESCs on the hierarchical MN-polymer surface leads to an increased homogeneity and percentage of Oct4-positive stem cell colonies as compared to mESCs grown on fibroblast feeder cells. Immunostaining revealed the absence of focal adhesion markers on all polymer substrates studied. However, only the MN-surfaces elicited the formation of actin-positive cell protrusions, indicating an alternative anchorage mechanism involved in the maintenance of mESC stemness.

A Facile Route to Boronic Acid Functional Polymeric Microspheres via Epoxide Ring Opening

Boronic acid-functionalized microspheres are prepared for the first time via mild epoxide ring opening based on porous cross-linked polymeric microspheres (diameter ≈ 10 μm, porosity ≈ 1000 Å). Quantitative chemical analysis by XPS and EA evidences that there is a greater functionalization with boronic acid when employing a sequential synthetic method [1.7 atom% boron (XPS); 1.12 wt% nitrogen (EA)] versus a one-pot synthetic method [0.2 atom% boron (XPS); 0.60 wt% nitrogen (EA)] yielding grafting densities ranging from approximately 2.5 molecules of boronic acid per nm(2) to 1 molecule of boronic acid per nm(2), respectively. Furthermore, the boronic acid-functionalized microspheres are conjugated with a novel fluorescent glucose molecule demonstrating a homogeneous spatial distribution of boronic acid.

Temporal responses of human endothelial and smooth muscle cells exposed to uniaxial cyclic tensile strain

The physiology of vascular cells depends on stimulating mechanical forces caused by pulsatile flow. Thus, mechano-transduction processes and responses of primary human endothelial cells (ECs) and smooth muscle cells (SMCs) have been studied to reveal cell-type specific differences which may contribute to vascular tissue integrity. Here, we investigate the dynamic reorientation response of ECs and SMCs cultured on elastic membranes over a range of stretch frequencies from 0.01 to 1 Hz. ECs and SMCs show different cell shape adaptation responses (reorientation) dependent on the frequency. ECs reveal a specific threshold frequency (0.01 Hz) below which no responses is detectable while the threshold frequency for SMCs could not be determined and is speculated to be above 1 Hz. Interestingly, the reorganization of the actin cytoskeleton and focal adhesions system, as well as changes in the focal adhesion area, can be observed for both cell types and is dependent on the frequency. RhoA and Rac1 activities are increased for ECs but not for SMCs upon application of a uniaxial cyclic tensile strain. Analysis of membrane protrusions revealed that the spatial protrusion activity of ECs and SMCs is independent of the application of a uniaxial cyclic tensile strain of 1 Hz while the total number of protrusions is increased for ECs only. Our study indicates differences in the reorientation response and the reaction times of the two cell types in dependence of the stretching frequency, with matching data for actin cytoskeleton, focal adhesion realignment, RhoA/Rac1 activities, and membrane protrusion activity. These are promising results which may allow cell-type specific activation of vascular cells by frequency-selective mechanical stretching. This specific activation of different vascular cell types might be helpful in improving strategies in regenerative medicine.