2. Materials and
Methods
2.1 Fabrication of microfluidic
devices
The T-junction microfluidic device used in this study consists of two
inlets for infusing the disperse (aqueous) phase and continuous (oil)
phase, respectively, one outlet for transporting microdroplets into the
collection tube and one rectangular chamber for observation of
cell-laden microdroplets. A corona-shaped filter was designed at the
inlets to prevent any possible dust entry. The connecting microchannels
have an aspect ratio of height/width = 4:5 (height: ~40
µm; width: ~50 µm), and the rectangular observation
chamber dimensions of 1.4 × 0.65 cm (Fig. S1 & Fig. S2).
This droplet-based microfluidic device was fabricated from a silicon
wafer patterned with SU-8 mould (SU-8 2035, MicroChem, Newton, MA, USA)
using standard soft-lithography techniques (Xia and Whitesides, 1998).
Degassed poly(dimethyl siloxane) (PDMS, Sylgard 184, Dow Corning,
Midland, MI, USA) in liquid form prepared by mixing the base and curing
agent at a ratio of 10:1 was poured onto the SU-8 mould and cured in an
oven at 80 °C for two hours. Then the PDMS slab with microchannels was
peeled off from the mould and the fluidic access holes were created
using PDMS biopsy puncher with an outer diameter of 1.5 mm. After
cleaning the channel side of PDMS with scotch tape, isopropanol and DI
water in order, the PDMS slab and a standard glass slide was treated
using an oxygen plasma cleaner to increase the surface energy and
immediately pressured to each other for an irreversible bonding. Lastly,
the channel was rendered hydrophobic by infusing with 0.02 %
Trichloro(octadecyl)silane (OTS, 104817, Sigma-Aldrich, St. Louis, MO,
USA) in isopropanol for 5 mins and drying with nitrogen gas followed by
drying in an oven at 100 °C for 10 mins.
2.2 Cell
preparation
We used a total of four yeast strains, the haploid S. cerevisiaestrain (CEN.PK113-7D), the PA evolved mutant S. cerevisiae strain
(PA-3), GFP-tagged S. cerevisiae strain (CEN.PK2-1C-GFP) and
GFP-tagged P. pastoris strain (CBS7435-GFP) in the single-cell
growth assays (Table S1).
CEN.PK113-7D and PA-3 were grown overnight at 30 °C, 200 rpm in 5 mL
buffered minimal medium (Xu et al., 2019). The overnight culture was
washed twice and re-inoculated into 5 mL buffered minimal medium at
three different concentrations of PA: 15 mM, 25 mM and 35 mM. To
investigate the effect of K-ions on the tolerance to PA in yeast,
CEN.PK2-1C-GFP was pre-cultured overnight at 30 °C, 200 rpm in synthetic
drop-out medium, without uracil
(contains 1× yeast nitrogen base (YNB) without amino acids mix (Y0626,
Sigma-Aldrich, St.Louis, MO, USA), 1% glucose and Yeast Synthetic
Drop-out Medium Supplements without uracil (Y1501, Sigma-Aldrich,
St.Louis, MO, USA)). Cells were washed twice and re-inoculated into 5 mL
uracil drop-out medium containing defined concentrations of potassium
(1× translucent K+ free YNB, 1% glucose, and 0, 1, 10
and 50 mM potassium chloride), supplemented with or without 25 mM PA.
CBS7435-GFP was pre-cultured overnight in the same drop-out medium as
the one used for CEN.PK2-1C-GFP at 25 °C, 200 rpm, and the culture was
washed twice and re-inoculated into 5 mL uracil drop-out medium. All the
cultures were reinoculated at an initial OD600 of 0.2
before being encapsulated into microdroplets.
2.3 Generation and storage of
microdroplets
In the T-shaped droplet generator of 50 × 50 µm (width × depth),
continuous phase flowed to the observation chamber and the disperse
phase flowed and sheared at the interface to generate monodisperse
microdroplets with a diameter of
~65 µm. Continuous
phase used here was Novec™ 7500 Engineered Fluid (3M, St. Paul, MN, USA)
containing 2% Pico-Surf™ 1 (Sphere Fluidics, Cambridge, UK). Disperse
phase used here was culture medium added with 20%
OptiPrepTM (D1556, Sigma-Aldrich, St. Louis, MO, USA).
Cells were diluted to an OD600 of 0.1 for the disperse
phase. Two syringe pumps (Fusion 100, Chemyx, Stafford, TX, USA) were
used to inject the two phases, respectively. When the ratio of the flow
rates of the two phases reached 4:1 (continuous phase: 16 µL/min vs
disperse phase: 4 µL/min), ~ 144 pL monodisperse
microdroplets were created in a high-throughput fashion
(~116 droplets per second). By Poisson distribution,
this size of droplet can ensure substantial droplets containing single
cells (28.0%) and maintain relatively low ratio of droplets containing
two cells (6.0%) and less than 1.0% droplets containing more than two
cells, and the rest 65.0% droplets are empty. A fluorinated ethylene
propylene (FEP) tubing (IDEX, Lake Forest, IL, USA) with an inner
diameter of 0.5 mm was used to transfer microdroplets into a 2 mL
EppendorfTM safe-lock tube (Hamburg, Germany)
pre-filled with 100 µl continuous (Fig. S3).
2.4 Image acquisition and
analysis
An initial observation of microdroplets containing single cells was
performed at a rectangular chamber of the microdevice before
microdroplets were transported into the collection tube. Bright-field
snapshots of the generated microdroplets were captured by a digital
camera (DS-Qi1Mc, Nikon, Tokyo, Japan) installed on an inverted
microscope (Eclipse Ti-U, Nikon, Tokyo, Japan). After images were taken,
the number of cells per droplet was counted using
ImageJ® (National Institutes of Health (NIH),
Bethesda, MD, USA).
To quantify growth of single yeast cells, the values for the specific
growth rate μ were determined based on the number of cells per droplet
at hourly time points up to 10 hours. The number of cells per droplet
(N) was converted into the logarithmic scale as ln(N), and the
estimation of the biokinetic constant, μ, over time of culture, t, was
obtained by the equation below:
\begin{equation}
\begin{matrix}\mu=\frac{\text{dln}\left(N\right)}{\text{dt}}\#\left(1\right)\\
\end{matrix}\nonumber \\
\end{equation}Fluorescence images of GFP-tagged cells were obtained at fluorescein
isothiocyanate (FITC) channel by a confocal microscope FV3000 (Olympus,
Tokyo, Japan). The number of yeast cells in a complex cluster were more
easily counted from the fluorescence images compared to bright-field
images. Regarding uncountable
agglomeration, images of eight
slices were stacked up and the corresponding fluorescence intensity was
measured by ImageJ®. The image processing includes the
following four main steps: 1) the bit depth of images was reduced to 8
bits; 2) area of each droplet was recognized; 3) The threshold was set
by Yen’s algorithm to remove noise; and 4) “limit to threshold” was
chosen and the mean fluorescence intensity within the area of each
droplet was measured.
2.5 Cell viability
test
To measure the viability of yeast cells in microdroplets, live/dead
staining tests were performed at three selected time points: before
encapsulation, at 24 hours of culture and 48 hours of culture. At each
time point, 50 µl emulsion microdroplets in oil were collected into a
centrifuge tube, and 2 μL Pico-Break™ (Sphere Fluidics, Cambridge, UK)
was added subsequently to release yeast cells from the microdroplets.
After a short centrifugation at 2000 rpm for 30 s, the oil phase was
kept at the bottom and the supernatant was transferred into a new
centrifuge tube. 30 µl staining solution consisting of 2 µM SYTO 9
(Thermo Fisher Scientific, Waltham, MA, USA) and 4 µM EthD-III (Biotium,
Hayward, Ca, USA) was added into the suspension of released yeast and
co-cultured for 20 mins. Fluorescence images were taken when excited at
a wavelength of ~495 nm and ~530 nm,
respectively: live cells showed green fluorescence in 515 nm channel
while dead cells showed red fluorescence in ~635 nm
channel and yellow colour in merged images. The cell viability over time
was tested based on three random frames for each measurement, and a
total of 100 cells were tested for each measurement. The viability test
results were analysed by one-way repeated measures analysis of variance
(ANOVA) to determine whether a significant difference was existed among
different time points.