Abstract

The purpose of the thesis is to develop a microfluidic based lab-on-chip (LOC) platform providing an Ex-Vivotesting environment that is able to mimic certain aspects of the in vivo growth conditions of the pollen tube, a cellular protuberance formed by the male gametophyte in the flowering plants. The thesis focuses on design, fabrication, modeling and testing of various LOC devices for the study of static and dynamic behavior of pollen tubes in response to mechanical stimulation.
TipChip, an LOC platform, was developed to advance both experimentation and phenotyping in cell tip growth research. The platform enabled simultaneous testing of multiple pollen tubes. Using TipChip, we were able to answer several outstanding questions regarding pollen tube biology. We found that contrary to other types of tip growing cells such as root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, we explored the effect of geometry of the microfluidic cell culture on pollen tube growth. We found that changing the width of the microfluidic channels does not have a significant effect on the pollen tube growth rate, while the growth rate was increased by increasing microchannel depth.
We modified the original TipChip design to ascertain identical growth conditions for sequentiallyarranged pollen tubes and to ensure even distribution of entrapment probabilities for all microchannels. The effect of different dimensions of the microfluidic network on cell trapping probability was assessed using computational fluid dynamics and verified by experimental testing. The design was optimized based on trapping probability and uniformity of fluid flow conditions within the microchannels.
This thesis also presents a novel method of fabricating a high aspect ratio horizontal PDMS microcantilever-based flow sensor integrated into a microfluidic device. The performance of the flow sensor was tested by introducing various flow rates into the microfluidic device and measuring the deflection of the cantilever’s tip using an optical microscope.
The thesis addresses the quantification of cellular growth force of Camellia pollen tip growing cells using FlexChip, a flexure integrated LOC on polymer. We quantified the force that pollen tube is able to exert using a microfluidic lab-on-a-chip device integrated with flexural structure. The pollen grain is trapped in the microfluidic network and the growing tube is guided against a flexible microstructure that is monolithically integrated within the microfluidic chip. The invasive growth force of growing pollen tube was calculated from the maximal bending of microstructure modelled by Finite Element Analysis (FEA).
Furthermore, the effect of the mechanical obstacle on the pollen tube's growth dynamics was assessed by quantifying the shift in the peak frequency characterizing the oscillatory behavior of the pollen tube growth rate. Our detailed analysis of the pollen tube growth dynamic before and during the contact with microcantilever revealed that pollen tube growth rate was reduced by 44% during the contact with the microcantilever. Moreover, the peak of oscillation frequency of pollen tube growth rate was reduced more dramatically by 70-75%. This suggests that the pollen tube actively changes its growth pattern to cope with the mechanical obstacle.
Our findings in this thesis are novel in terms of pollen biology, and we believe insights from this research will lead to a better understanding of morphogenesis of a kind of tip growing cells, namely, pollen tube.