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.

Microfluidic positioning of pollen grains in lab-on-a-chip for single cell analysis.

A lab-on-a-chip device with a knot shaped microfluidic network is presented to enable trapping of single pollen grains at the entrances of a series of microchannels. This set-up serves to create identical growth conditions for serially arranged tip growing plant cells such as pollen tubes. The design consists of an inlet to introduce the pollen suspension into the chip, three outlets to evacuate excess medium or cells, a distribution chamber to guide the pollen grains toward the growth microchannels and a serial arrangement of microchannels with different geometries connected to the distribution chamber. These microchannels are to harbor the individual pollen tubes. Two different criteria were established to assess the efficiency and optimize the device: trapping probability and uniformity of fluid flow conditions within the microchannels. The performance of different geometries of the microfluidic network was numerically analyzed and experimentally tested.

Lab-on-a-chip for studying growing pollen tubes.

A major limitation in the study of pollen tube growth has been the difficulty in providing an in vitro testing microenvironment that physically resembles the in vivo conditions. Here we describe the development of a lab-on-a-chip (LOC) for the manipulation and experimental testing of individual pollen tubes. The design was specifically tailored to pollen tubes from Camellia japonica, but it can be easily adapted for any other species. The platform is fabricated from polydimethylsiloxane (PDMS) using a silicon/SU-8 mold and makes use of microfluidics to distribute pollen grains to serially arranged microchannels. The tubes are guided into these channels where they can be tested individually. The microfluidic platform allows for specific testing of a variety of growth behavioral features as demonstrated with a simple mechanical obstacle test, and it permits the straightforward integration of further single-cell test assays.

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.