The Stiffness-Sensitive Transcriptome of Human Tendon Stromal Cells.

Extracellular matrix stiffness is a major regulator of cellular states. Stiffness-sensing investigations are typically performed using cells that have acquired "mechanical memory" through prolonged conditioning in rigid environments, e.g., tissue culture plastic (TCP). This potentially masks the full extent of the matrix stiffness-driven mechanosensing programs. Here, a biomaterial composed of 2D mechanovariant silicone substrates with simplified and scalable surface biofunctionalization chemistry is developed to facilitate large-scale cell culture expansion processes. Using RNA sequencing, stiffness-mediated mechano-responses of human tendon-derived stromal cells are broadly mapped. Matrix elasticity (E.) approximating tendon microscale stiffness range (E. ≈ 35 kPa) distinctly favors transcriptional programs related to chromatin remodeling and Hippo signaling; whereas compliant stiffnesses (E. ≈ 2 kPa) are enriched in processes related to cell stemness, synapse assembly, and angiogenesis. While tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, mechanovariant substrates abrogate this activation with tenogenic stiffnesses inducing a transcriptional program that strongly correlates with established tendon tissue-specific expression signature. Computational inference predicts that AKT1 and ERK1/2 are major stiffness-sensing signaling hubs. Together, these findings highlight how matrix biophysical cues may dictate the transcriptional identity of tendon cells, and how matrix mechano-reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon fibroblasts.

The relationship between metastatic potential and in vitro mechanical properties of osteosarcoma cells.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.

Easy and Accurate Mechano-profiling on Micropost Arrays.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow "mechano-profiling" of cell phenotypes. A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Connecting µ-fluidics to electron microscopy.

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.

High-density micro-arrays for mass spectrometry.

Functional high-density micro-arrays for mass spectrometry enable rapid picolitre-volume aliquoting and ultrasensitive analysis of microscale samples, for example, single cells.

Microdevice DNA forensics by the simple tandem repeat method.

We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures.

A high-performance multilane microdevice system designed for the DNA forensics laboratory.

We report preliminary testing of "GeneTrack", an instrument designed for the specific application of multiplexed short tandem repeat (STR) DNA analysis. The system supports a glass microdevice with 16 lanes of 20 cm effective length and double-T cross injectors. A high-speed galvanometer-scanned four-color detector was specially designed to accommodate the high elution rates on the microdevice. All aspects of the system were carefully matched to practical crime lab requirements for rapid reproducible analysis of crime-scene DNA evidence in conjunction with the United States DNA database (CODIS). Statistically significant studies demonstrate that an absolute, three-sigma, peak accuracy of 0.4-0.9 base pair (bp) can be achieved for the CODIS 13-locus multiplex, utilizing a single channel per sample. Only 0.5 microL of PCR product is needed per lane, a significant reduction in the consumption of costly chemicals in comparison to commercial capillary machines. The instrument is also designed to address problems in temperature-dependent decalibration and environmental sensitivity, which are weaknesses of the commercial capillary machines for the forensics application.

Evaporation driven pumping for chromatography application.

A continuous transport process for liquids in micro-channels is reported. Flow was generated by evaporation at the channel end plus capillary forces. The micro-channels integrated into a two-glass-layer device were 110 microm wide, 28 microm deep and 4 or 10 cm long. A continuous liquid transport velocity of up to 2.25 mm s(-1) was observed for aqueous solutions. The flow velocity is shown to increase when an air stream is guided over the evaporation zone.