Hepatic C9 cells switch their behaviour in short or long exposure to soft substrates.

BACKGROUND INFORMATION: There have been several studies to understand the influence of stiffness of the culture substrates for different types of adherent cells. It is generally accepted that cell proliferation, spreading and focal adhesions increase with higher matrix stiffness. However, what remains unclear is whether this kind of cell behaviour may be reverted by culturing on soft substrates those cell lines that were originally selected or primed on stiff surfaces. RESULTS: Here, we studied the influence of substrate softness on proliferation, adhesion and morphology of the highly proliferative hepatic C9 cell line. We cultured C9 cells on soft and stiff polydimethylsiloxane (PDMS) substrates prepared with two different elastic moduli in the range of 10 and 200 kPa, respectively. Lower cell proliferation was observed on substrates with lower stiffness without affecting cell viability. The proliferation rate of C9 cell line along with extracellular growth-regulated kinase (ERK) phosphorylation was decreased on soft PDMS. Despite this cell line has been described as a hepatic epithelial cell, our characterisation demonstrated that the origin of C9 cells is uncertain, although clearly epithelial, with the expression of markers of several hepatic cells. Surprisingly, consecutive passages of C9 cells on soft PDMS did not alter this mesenchymal phenotype, vimentin expression did not decrease when culturing cells in soft substrates, even though the ERK phosphorylation levels eventually were increased after several passages on soft PDMS, triggering again an increase of cell proliferation. CONCLUSIONS AND SIGNIFICANCE: This study shows that the exposure of C9 cells to soft substrates promoted a decrease of cell proliferation rate, as reported for other types of cells on PDMS, whereas a much longer term exposure caused cells to adapt to softness after trained for several passages, reactivating proliferation. During this phenomenon, the morphology and phenotype of trained cells was modified accompanying the increase of cell proliferation rate contrary to the effect observed in short periods of cell culture. In contrast to previous reports, cell death was not observed during these experiments, discarding a cell selection mechanism and suggesting soft cell adaptation may be limited in time in C9 cells.

Micro-Macro: Selective Integration of Microfeatures Inside Low-Cost Macromolds for PDMS Microfluidics Fabrication.

Microfluidics has become a very promising technology in recent years, due to its great potential to revolutionize life-science solutions. Generic microfabrication processes have been progressively made available to academic laboratories thanks to cost-effective soft-lithography techniques and enabled important progress in applications like lab-on-chip platforms using rapid- prototyping. However, micron-sized features are required in most designs, especially in biomimetic cell culture platforms, imposing elevated costs of production associated with lithography and limiting the use of such devices. In most cases, however, only a small portion of the structures require high-resolution and cost may be decreased. In this work, we present a replica-molding method separating the fabrication steps of low (macro) and high (micro) resolutions and then merging the two scales in a single chip. The method consists of fabricating the largest possible area in inexpensive macromolds using simple techniques such as plastics micromilling, laser microfabrication, or even by shrinking printed polystyrene sheets. The microfeatures were made on a separated mold or onto existing macromolds using photolithography or 2-photon lithography. By limiting the expensive area to the essential, the time and cost of fabrication can be reduced. Polydimethylsiloxane (PDMS) microfluidic chips were successfully fabricated from the constructed molds and tested to validate our micro-macro method.

Progress on the Use of Commercial Digital Optical Disc Units for Low-Power Laser Micromachining in Biomedical Applications.

The development of organ-on-chip and biological scaffolds is currently requiring simpler methods for microstructure biocompatible materials in three dimensions, to fabricate structural and functional elements in biomaterials, or modify the physicochemical properties of desired substrates. Aiming at addressing this need, a low-power CD-DVD-Blu-ray laser pickup head was mounted on a programmable three-axis micro-displacement system in order to modify the surface of polymeric materials in a local fashion. Thanks to a specially-designed method using a strongly absorbing additive coating the materials of interest, it has been possible to establish and precisely control processes useful in microtechnology for biomedical applications. The system was upgraded with Blu-ray laser for additive manufacturing and ablation on a single platform. In this work, we present the application of these fabrication techniques to the development of biomimetic cellular culture platforms thanks to the simple integration of several features typically achieved with traditional, less cost-effective microtechnology methods in one step or through replica-molding. Our straightforward approach indeed enables great control of local laser microablation or polymerization for true on-demand biomimetic micropatterned designs in transparent polymers and hydrogels and is allowing integration of microfluidics, microelectronics, surface microstructuring, and transfer of superficial protein micropatterns on a variety of biocompatible materials.

Low-cost formation of bulk and localized polymer-derived carbon nanodomains from polydimethylsiloxane.

We present two simple alternative methods to form polymer-derived carbon nanodomains in a controlled fashion and at low cost, using custom-made chemical vapour deposition and selective laser ablation with a commercial CD-DVD platform. Both processes presented shiny and dark residual materials after the polymer combustion and according to micro-Raman spectroscopy of the domains, graphitic nanocrystals and carbon nanotubes have successfully been produced by the combustion of polydimethylsiloxane layers. The fabrication processes and characterization of the byproduct materials are reported. We demonstrate that CVD led to bulk production of graphitic nanocrystals and single-walled carbon nanotubes while direct laser ablation may be employed for the formation of localized fluorescent nanodots. In the latter case, graphitic nanodomains and multi-wall carbon nanotubes are left inside microchannels and preliminary results seem to indicate that laser ablation could offer a tuning control of the nature and optical properties of the nanodomains that are left inside micropatterns with on-demand geometries. These low-cost methods look particularly promising for the formation of carbon nanoresidues with controlled properties and in applications where high integration is desired.

Rapid fabrication of on-demand high-resolution optical masks with a CD-DVD pickup unit.

A low-cost, direct fabrication technique with a micrometer range resolution has been implemented for rapid prototyping of optical masks for photolithography and structured light and diffraction optics applications. Using a setup based on the optical unit of a compact disc-digital versatile disc burner, a low-energy infrared laser beam was focused on a thin polymeric layer with embedded absorbing carbon nanopowder coated on a transparent glass substrate. This allowed for the generation of a custom-made transparent pattern in a computer numerical control fashion. In addition to its great simplicity and repeatability, the method also enables grayscale contrasts for each pixel individually, and fabricated masks proved to resist high intensities.