Microcontact printing | EPFL Graph Search

Microcontact printing (or μCP) is a form of soft lithography that uses the relief patterns on a master polydimethylsiloxane (PDMS) stamp or Urethane rubber micro stamp to form patterns of self-assembled monolayers (SAMs) of ink on the surface of a substrate through conformal contact as in the case of nanotransfer printing (nTP). Its applications are wide-ranging including microelectronics, surface chemistry and cell biology. History Both lithography and stamp printing have been around for centuries. However, the combination of the two gave rise to the method of microcontact printing. The method was first introduced by George M. Whitesides and Amit Kumar at Harvard University. Since its inception many methods of soft lithography have been explored. Procedure Preparing the master Creation of the master, or template, is done using traditional photolithography techniques. The master is typically created on silicon, but can be done on any solid patterned surf

Muscle stem cell's in vitro bipolar niche

Adrien de Tonnac

In the domain of regenerative medicine research there are many fronts aimed at increasing our understanding of induced pluripotent, embryonic and adult stem cells. Recently a new front has appeared, stem cell niches, the microenvironment that is a crucial component in the regulation of stem cells. New in vitro platforms are continually being created to answer the multitude of biological questions that surround stem cell niches. The transition from in vitro to in vivo is a heavy setback in cellular research due to a huge gap between the two conditions, which can only be reduced if the in vitro conditions better mimic those found in vivo. This project has reduced the gap and has the potential to continue minimizing the differences between the two conditions in the field of muscle stem cell research. Through a combination of the forefront techniques in biological research, we were able to create a new innovative bioengineered platform, a bipolar hydrogel microenvironment. First, we took advantage of polyethylene glycol (PEG) hydrogels, which are highly hydrophilic polymer networks that have properties that mimic physiologic tissue, including high water content and low Young's Modulus. Microfabrication techniques allowed us to topographically pattern the hydrogel at micro-dimensions resembling the native cell environment. Utilizing microfluidics and micro-printing techniques, the three-dimensional hydrogel surface was also patterned with tethered niche proteins found in the in vivo muscle stem cell niche. The swelling properties of the PEG hydrogel were considered as an advantage and controlled to form niche gaps with physical properties that were tuned to correspond to those found in vivo. This new complex and controlled surface for cells to be cultured on is a tool, which will allow biologists to probe new questions and gain insight into the regulatory networks that control cell fate

2009

Calcium Dynamics and Intercellular Communication in Arterial Smooth Muscle Cells in Vitro

Nadia Halidi

Tissue blood flow is controlled by changes in the diameter of the arteries and arterioles through coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contraction of SMCs is primarily regulated by the intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i, in response to stimuli, can propagate from cell to cell, as an intercellular Ca2+ wave along the vessel wall and can activate the process of contraction. The aim of this thesis is to elucidate the mechanisms underlying intercellular Ca2+ wave propagation between SMCs. In the first part of this thesis, we used A7r5 cells, a rat aortic SMC line, loaded with the fluorescent Ca2+ dye Fluo-4 to study intercellular Ca2+ wave propagation. Local mechanical stimulation evoked a Ca2+ wave in the stimulated cell that failed to propagate to neighboring cells. Using primary cultured rat mesenteric smooth muscle cells (pSMCs) instead, intercellular Ca2+ wave propagation was observed. To understand the difference in junctional communication between A7r5 and pSMCs, we investigated the expression of connexin37 (Cx37), Cx40, Cx43 and Cx45. RNA and protein analysis demonstrated that Cx40 – in contrast to A7r5 cells – is not expressed in pSMCs. To confirm that coexpression of Cx40 and Cx43 interfered with junctional communication, we used 6B5N cells, a clone of A7r5 cells with a higher Cx43:Cx40 expression ratio. Junctional communication, assessed by transfer of Lucifer Yellow and propagation of Ca2+ waves, was comparable between 6B5N cells and pSMCs. In addition, Ca2+ wave propagation was inhibited with the connexin-mimetic peptide 43Gap 26, that targets Cx43. Our results demonstrate that Cx43 gap junctions are primarily involved in mediating intercellular Ca2+ waves between pSMCs, and the coexpression of Cx43 with Cx40 may interfere with Cx43 gap junction formation, affecting cell-cell communication. In the second part of this thesis, we applied the microcontact printing technique to culture pSMCs on collagen lines. The aligned arrangement of the cells facilitates the observation of Ca2+ wave progression from one cell to another. To induce a Ca2+ wave, a single pSMC was locally stimulated with a micropipette (transientmechanical stimulation) or by microejection of KCl. Mechanical stimulation evoked two distinct Ca2+ waves: 1) a fast wave (∼2 mm/s) that propagated to all observed neighbouring cells, and 2) a slow wave (∼20 µm/s), that was most often limited in propagation to the first cell. KCl induced only fast Ca2+ waves of the same velocity as the mechanically-induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors, showed that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca2+ influx, whereas, the slow wave was due to Ca2+ release primarily through IP3 receptors. Together, these results suggest a mechanism by which intercellular Ca2+ waves can propagate between SMCs of the arterial wall.

EPFL2011

Metal and metaloxide nanostructures on and in plant viruses

Sinan Balci

Tobacco Mosaic Virus (TMV) is composed of 2130 identical coat protein subunits following the right-handed helix of an accompanying RNA strand to produce a 300 nm hollow cylinder with an outer diameter of 18 nm and a 4 nm wide central channel. In this work, TMV is used as a bionanotemplate for the synthesis of interesting nanomaterials. When the TMV is activated with a catalyst (Pd (II)), nickel, copper, and cobalt nanowires can be grown within the central channel of the virion. In the same way alloy nanowires (cobalt-iron) are produced inside the central channel. Due to the non-galvanic protocol, the deposition method is called electroless deposition (ELD). Apart from the deposition of metals (Ni, Co, Cu, CoFe alloy), zinc oxide, a large band gap semiconductor, is deposited on the exterior surface of the virus. The chemical compositions of the nanomaterials are revealed at the nanoscale by energy-filtering transmission electron microscopy. Various physical properties of some of the synthesized nanomaterials are measured: Electrical transport measurements on the TMV rods, contacted by gold electrodes, indicate that the TMV rods show insulating behavior at room temperature. High-resolution transmission electron microscopy images of individual nickel nanowires show the crystalline bulk phase, confined to a 3 nm wide wire, with (111) planes perpendicular to the wire axis. Cathodoluminescence measurements from individual zinc oxide rods exhibit ultraviolet emission with a photon energy of around ∼3.4 eV, which is equal to the band gap of the bulk material. Furthermore, 6 nm diameter gold nanoparticles (Au-nps) are selectively attached to the ends of the TMV particles, and they are enlarged by electroless deposition of gold. The RNA strand plays an important role in the selective attachment of the Au-nps. It is possible to print and align TMV particles on a variety of surfaces by the microcontact printing method. Several micrometer long end-to-end assembled TMV rods can be placed close to the edges within the recessed regions of polydimethysiloxane stamp structures. The reason for the selective assembly of the virions is the dewetting of the inking solvent (water) on the stamp surface. By printing, the virion pattern is transferred to solid surfaces. Alternatively, TMV particles can be selectively assembled on flat rectangular submicrometer patches with amine groups, which are generated by electron beam lithography. The experimental results show that TMV can be a model system for new bionanotemplates with huge potential in nanoscale science and possibly technology.