Dynamic, high precision targeting of growth modulating agents is able to trigger pollen
tube growth reorientation
Amir Sanati Nezhad1, Muthukumaran Packirisamy1,* Anja Geitmann2,*
Supplementary Material
Design considerations for the microfluidic network
The depth of the entire microfluidic network was set to 80 µm, slightly larger than the diameter of the
pollen grains to let the pollen grains pass freely along the main chamber and reach the traps, but to
prevent vertical stacking. The main chamber also has an outlet to evacuate excess pollen in order to
prevent their accumulation at the microchannel entrance or at the exit of the main chamber.
The pollen grain/tube is a non-adherent cell and must be trapped to not be carried away by the flow.
Pollen grain trapping is ensured by the narrow size of the initial portion of the microchannel. With 30 µm it
is narrower than the diameter of 50-60 µm of the Camellia pollen grain, but wider than the typical diameter
of 12-19 µm of the pollen tube. This size constraint traps the pollen grains at the microchannel entrance
and allows the pollen tube to grow into the microchannel. At a distance of 100 µm from the microchannel
entrance, the width of the microchannel widens to 60 µm to allow for significant displacements of the
pollen tube tip in lateral direction (x-direction) while the 80 µm depth of channel results in the pollen tube
occupying only about 4-5% of the channel cross section. Consequently the presence of the tube was not
expected to significantly influence the fluid distribution and flow pattern within the channel. Conventional
soft lithography methods were used to fabricate the microfluidic device as detailed in earlier works
(Agudelo et al. 2013a, Agudelo et al. 2012, Agudelo et al. 2013b, Sanati Nezhad et al. 2013). The pollenmedium inlet (PMI) and two side inlets (SI1) and (SI2) for medium were connected to syringe pumps.
Pollen tube growth is monitored using optical microscopy and quantified by image analysis.
Design modification for truly local agent administration
The above proposed experimental setup is able to administer two different solutions to the two sides of an
elongating cell. Given that in tip growing cells only the apex expands and responds to directional cues, this
approach is sufficient to be designated "local application" for the case of a tip growing cell and it permits
continuous readjustment of the fluid interface. However, for certain cell types or certain biological
questions to be addressed, it may be necessary to only expose one side of the growing tip, but not any
parts of the basal region of the cell. We therefore designed a modification to the set-up that allows
administering one of the agents through a side channel entering the microchannel half way along its
length (or at any other required position) (Fig. 1h,i). This allows for application of an agent to half of the tip
of the growing cell only while the tip grows past this entry point. Several side channels can be arranged in
series to repeatedly target the growing tip. The following simulations and experimental validation focus on
the longitudinal gradient described in the previous section, but the same principles apply to the design
modification.
Finite element model of the behavior at the fluid interface
The model consists of two side inlets for the two different media, one main chamber, the growth
microchannel and one medium outlet. The second microchannel is neglected in the model as it is blocked
during experimentation. The flow within the microfluidic network is governed by Navier-Stokes and the
continuity equations (Wang et al. 2008). The media are assumed to behave like water with the density of
1000 kg/m3 and dynamic viscosity of 0.001 Pa.s. The inlet velocity is defined as the boundary condition for
the medium inlets. The pressure at the main chamber outlet is set to atmospheric pressure. At all other
boundaries, no-slip condition is defined. COMSOL Multiphysics 3.5 software is used to solve the
governing equation of fluid flow.
Unit concentration (C=1) is set for the medium applied through side inlet 1 and zero concentration (C=0)
for side inlet 2. No-flux conditions are imposed at all channel walls. The outlet of the growth microchannel
has the boundary condition of convective flux, as there is flow outward. Diffusion analysis is carried out
using a diffusion coefficient of D=10-10 m2 s−1 for calcium ions (Donahue and Abercrombie 1987, Kamholz
and Yager 2001). Finite element analysis was used to solve the convection-diffusion transport equation
using COMSOL software 3.5. The simulation predicts that in the case of 1 mm/s inlet velocities imposed at
both side inlets, the steepness and width of the gradient present at the beginning of the microchannel is
maintained without significant change over the entire length of the microchannel. At the flow velocities
used here, the calcium ion gradient across the microchannel is sufficiently steep and narrow to expose the
advancing pollen tube apex to different concentrations of the ion even at a distance of 500 µm from the
trapped grain. The simulations show that for a diffusion coefficient of D=10 -10 m2s−1, the effective width of
the gradient (distance between C=0 and C=1) is 14% of the microchannel width at a distance of 500 µm
from the trapped grain. At the current microchannel width of 60 µm this corresponds to 8.4 µm which is
significantly smaller than the width of the Camellia pollen tube. If the interface (C=0.5) is targeted to the
pole of the cell, the flanks of the cell are thus exposed to C=0 and C=1, respectively. Even for agents with
higher diffusion coefficient, the width of the gradient does not change substantially. Simulations predict
that for a diffusion coefficient of D=10-9 m2 s−1, the effective width increases to 18% of the microchannel
width (or 10.8 µm in a 60 µm wide microchannel), which remains below the pollen tube diameter.
Chip fabrication
A micromold is fabricated on SU-8, a negative photoresist (MicroChem Corp.) using photolithographic
technique. SU-8 2035 is spin coated on silicon wafer to a thickness of 80 μm. The SU-8 is baked for 3 min
at 65ºC and for 7 min at 95ºC in a hotplate. After soft-baking, the resist is cooled down at room
temperature and exposed to UV light using a photo mask for 30 s. Post-exposure bake step is then carried
out, baking the resist for 3 min at 65ºC and for 6 min at 95ºC in order to cross-link the SU-8. The SU-8
layer is then developed to obtain the SU-8 mold. The microfluidic device is then fabricated pouring a layer
(4 mm) of polydimethylsiloxane (PDMS) prepolymer mixture with curing agent with a mass ratio of 10:1 on
the SU-8 mold. This PDMS layer is cured at 80ºC for 30 min, peeled off from the master mold and bonded
to a glass layer to seal the fluidic network. The inlet and outlet holes are then punched and tubing is
attached for injection purposes.
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