Microfabricated Polyacrylamide Devices for the Controlled Culture of Growing Cells and Developing Organisms

<div><p>The ability to spatially confine living cells or small organisms while dynamically controlling their aqueous environment is important for a host of microscopy applications. Here, we show how polyacrylamide layers can be patterned to construct simple microfluidic devices for this purpose. We find that polyacrylamide gels can be molded like PDMS into micron-scale structures that can enclose organisms, while being permeable to liquids, and transparent to allow for microscopic observation. We present a range of chemostat-like devices to observe bacterial and yeast growth, and <i>C. elegans</i> nematode development. The devices can integrate PDMS layers and allow for temporal control of nutrient conditions and the presence of drugs on a minute timescale. We show how spatial confinement of motile <i>C. elegans</i> enables for time-lapse microscopy in a parallel fashion.</p> </div

Results of the stochastic model.

<p>(A) Example time-series of cell growth rate for a cell with fast (green), slow (blue) and very slow (red) response. (B) Fluorescence time-series for the cells shown in (A). Inset: The same data on a logarithmic scale, showing that cells with higher expression levels at the time of shift of medium tend to be induced more rapidly. (C) Histograms of growth (red) and fluorescence (green) recovery times, ΔTμ₂ and ΔT_F. In panels (C) and (D), cells at ΔTμ₂ = 0 showed a decrease in growth rate of less than 20%. (D) <i>Lac</i> expression of each lineage at t_shift plotted against growth recovery time. Cells which did not reach the induction threshold in the time of the simulations are placed at ΔTμ₂ = 500 min. Cells with initial concentrations above ∼10 nM typically have a rapid recovery of growth rate. Note that the plot range does not represent the full range of initial expression levels.</p

Diffusion in unstructured and structured polyacrylamide hydrogel membranes

<p>(A) Sketch of the flow cell device. An unstructured acryl gel (height 500 µm) is sandwiched between a PDMS layer comprising a channel (height 113 µm), and a glass coverslip, similar to the design in Figure 1A. (B) Fluorescence of the small dye 2-NBDG is proportional to its concentration in the flowing solution. (C) Fluorescence signal after infusion (squares) or depletion (circles) of the dye 2-NBDG was measured at the gel-glass interface (black cross in A). Lines show fits to the 1D diffusion equation. Open symbols and dashed lines correspond to flow rates of 50 µl.min^-1, closed symbols and solid lines to flow rates of 20 µl.min^-1. (D) Sketch of the linear gradient generator device. A structured polyacrylamide hydrogel (height 1 mm) is sandwiched between a PDMS layer and a glass slide. Water containing 3.5 <a href="http://%C2%B5g.ml" target="_blank">µg.ml</a>^-1 fluorescein is flown through the left channel, while pure water is flown through the right channel, thereby creating a linear concentration gradient within the gel. (E) Image of the fluorescence intensity profile at mid-channel height (black line in D) taken 85 min after the flows were established. Red lines indicate channel walls. (F) Fluorescence intensity profile (blue crosses) plotted versus distance (along the blue line in E). The fluorescence intensity in the acryl gel in between the channels is linear (red line).</p

Dynamics at the population level and in single cells.

<p>(A) Growth curve for a typical microcolony, indicating the sum of all cell lengths within the colony. (B) Mean fluorescence intensity (per unit area) within cells, averaged over a microcolony. (C) Single-cell length over time for three different lineages, representing cases with no growth rate decrease (green), a lag phase (blue) and a longer lag phase (red). Arrows indicate cell division events. The curves are vertically shifted for clarity. (D) Elongation rates obtained by exponential fits to the length data at sub-cell cycle resolution. Drawn lines are fitted parameterized functions. ΔTµ₂ is the time difference between the time of shift and the half maximum to growth recovery after shift. (E) Fluorescence levels for the three lineages in (C) and (D). Drawn lines are fitted parameterized functions. ΔT_F is the time difference between the time of shift and the half maximum to induction after shift. Black bar: 120 min before the shift, over which data was averaged to determine the expression level prior to the shift.</p

Single-Cell Dynamics Reveals Sustained Growth during Diauxic Shifts

<div><p>Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of <i>E. coli</i> cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring <i>lac</i> expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic <i>lac</i> expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between <i>lac</i> expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, <i>lac</i> expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.</p></div

Switching synchrony of sister cells.

<p>The growth recovery delays ΔTµ₂ are plotted for pairs of sister cells. (A) Data obtained from experiments. N = 75, r² = 0.52, p-value <0.001. (B) Data resulting from simulations. N = 660, r²≈0.13 and p<0.001. Note that in both cases lineages in which one cell switches but its sister or its progeny does not are not plotted (in total: 22 pairs for the experimental data, 146 pairs for the numerical data).</p

Schematics of devices for cells or organisms culture in polyacrylamide membranes.

<p>In all devices presented here, the different layers are held together by mechanical clamping, and the cells or organisms (represented as black circles) grow at the interface between the polyacrylamide gel and a glass coverslip through which microscopy is performed. (A) Side view of a microfabricated membrane comprising culture chambers, mechanically clamped between a glass slide and a glass coverslip. The device is sealed with a glass or metal contour. Sealing can be enhanced by adding vacuum silicon grease between surfaces. (B) Top view of panel A showing the array of microchambers surrounded by the glass or metal contour. (C) Flow cell using a PDMS control channel in contact with a polyacrylamide monolayer, which allows transfer of the flowing medium to the cells. In this design, cells are compressed underneath a flat polyacrylamide monolayer. (D) A more complex device combining the microchambers of the design in panel A and with the PDMS control channel in panel C.</p

Monitoring bacterial growth by time-lapse microscopy.

<p>(A) Phase contrast images of <i>E</i>. <i>coli</i> cells growing in minimal medium supplemented with lactose. (B) Typical cell detection performed on a phase contrast image. (C) Sum of cells length for microcolonies growing on minimal medium with lactose (green), maltose (red), lactate (yellow) and limiting lactose (blue) as sole carbon source. (D) Sum of cells length during a shift from lactose to glucose and (E) corresponding mean fluorescence intensity of the colony. For comparison, fluorescence intensity for a colony growing only on lactose is shown in grey.</p

Stochastic model.

<p>(A) Within each cell the concentrations of lactose, LacYZ and LacI are simulated, as well as the operator state. Lactose imported from the environment or glucose lead to cell growth. (B) Each cell is simulated until it reaches a specified length, at which point it divides to produce two daughter cells. The proteins of the parent cell are partitioned randomly between the two daughters. The daughters are then simulated until their subsequent division. Growth and fluorescence recovery times (T_F and T_µ) are extracted from the reconstructed cell lineages.</p

<i>C. elegans</i> growth in microchambers.

<p>(A) Growth of a single <i>C</i>. <i>elegans</i> animal through the L1 larval stage constrained in a 200 x 200 x 18 µm polyacrylamide microchamber filled with OP50 as source of food. Time is shown in hours after hatching. At 12 h after hatching the animal has entered the lethargus at the end of the L1 larval stage. (B) Worm length as a function of time after hatching. Different colors indicate animals grown in parallel on the same device. Horizontal bars show the duration of lethargus, ending with the molt at the start of the L2 larval stage. The markers indicated by the arrow correspond to the time points shown in (A).</p