Optimization of flow assisted entrapment of pollen grains in a microfluidic platform for tip growth analysis.

A biocompatible polydimethylsiloxane (PDMS) biomicrofluidic platform is designed, fabricated and tested to study protuberance growth of single plant cells in a micro-vitro environment. The design consists of an inlet to introduce the cell suspension into the chip, three outlets to conduct the medium or cells out of the chip, a main distribution chamber and eight microchannels connected to the main chamber to guide the growth of tip growing plant cells. The test cells used here were pollen grains which produce cylindrical protrusions called pollen tubes. The goal was to adjust the design of the microfluidic network with the aim to enhance the uniformly distributed positioning of pollen grains at the entrances of the microchannels and to provide identical fluid flow conditions for growing pollen tubes along each microchannel. Computational fluid analysis and experimental testing were carried out to estimate the trapping efficiencies of the different designs.

Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling.

Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants.

Quantification of the Young's modulus of the primary plant cell wall using Bending-Lab-On-Chip (BLOC).

Biomechanical and mathematical modeling of plant developmental processes requires quantitative information about the structural and mechanical properties of living cells, tissues and cellular components. A crucial mechanical property of plant cells is the mechanical stiffness or Young's modulus of its cell wall. Measuring this property in situ at single cell wall level is technically challenging. Here, a bending test is implemented in a chip, called Bending-Lab-On-a-Chip (BLOC), to quantify this biomechanical property for a widely investigated cellular model system, the pollen tube. Pollen along with culture medium is introduced into a microfluidic chip and the growing pollen tube is exposed to a bending force created through fluid loading. The flexural rigidity of the pollen tube and the Young's modulus of the cell wall are estimated through finite element modeling of the observed fluid-structure interaction. An average value of 350 MPa was experimentally estimated for the Young's modulus in longitudinal direction of the cell wall of Camellia pollen tubes. This value is in agreement with the result of an independent method based on cellular shrinkage after plasmolysis and with the mechanical properties of in vitro reconstituted cellulose-callose material.

TipChip: a modular, MEMS-based platform for experimentation and phenotyping of tip-growing cells.

Large-scale phenotyping of tip-growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high-throughput execution of more sophisticated assays, an experimental platform, the TipChip, was developed based on microfluidic and microelectromechanical systems (MEMS) technology. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels equipped with microscopic experimental set-ups. The tip-growing cells (pollen tubes, filamentous yeast or fungal hyphae) may be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform, we were able to answer several outstanding questions on pollen tube growth. We established that, unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air, raising the question of how and where water is taken up by the cell. The platform opens new avenues for more efficient experimentation and large-scale phenotyping of tip-growing cells under precisely controlled, reproducible conditions.

Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers.

Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)-agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS-agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS-agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS-agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.