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. 2017 Jul 11;114(28):7283-7288.
doi: 10.1073/pnas.1705059114. Epub 2017 Jun 26.

Microfluidic Guillotine for Single-Cell Wound Repair Studies

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Free PMC article

Microfluidic Guillotine for Single-Cell Wound Repair Studies

Lucas R Blauch et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute-more than 200 times faster than current methods-is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Keywords: Stentor coeruleus; microfluidics; microguillotine; single cell; wound healing.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Design and validation of the microfluidic guillotine device and regeneration of cell fragments. (A) Scheme of the microfluidic guillotine device. The red box shows details of the knife region. The dimensions are listed in Table S1. (B) Micrographs showing the time sequence of the flow of a Stentor cell in the microchannel, the bisection of the cell at the knife, and the subsequent splitting of the two cell fragments into two separate outlets downstream. (C) Micrographs of the regeneration process (at time t = 0, 4, 24, and 48 h after the cut) of two cell fragments generated from cutting one cell using the microfluidic guillotine. (D) Micrographs of the regeneration process (at time t = 0, 4, 24, and 48 h after the cut) of two cell fragments generated from cutting one cell using a glass needle by hand. The two fragments (indicated by red arrow at t = 0) were kept in two separate PDMS wells. By t = 24–48 h all cell fragments have regenerated their trumpet shapes. The blue arrows indicate air bubbles that dissolved eventually. Videos of regeneration for A and B are shown in Movies S1 and S2, respectively.
Fig. 2.
Fig. 2.
Characterization of the effect of applied flow rate on the cutting process and cell survival. (A) Schemes and micrographs showing the morphology of the cell during the cutting process at low velocity (regime I) and high velocity (regime II). For each case, the cells were suspended in a methylcellulose solution at a concentration of 0.2% wt/vol or 0.5% wt/vol. (B) The normalized extension length of the cell during cutting and (C) cell survival rates, as a function of cell velocity measured immediately upstream of the knife. The blue and red symbols correspond to experiments performed in 0.2% and 0.5% methylcellulose solutions, respectively. In B, the markers indicate the mean from 7 cells. In C, the markers indicate the mean from four to seven cells from each of the three separate experiments (or total of >15 cells from all experiments). The height of the error bar represents one SD from the mean. The width of the error bar represents one SD from the mean cell velocity at a fixed flow rate as applied from the syringe pump.
Fig. S1.
Fig. S1.
Effect of the blade angle. Snapshots of the cells cut by a knife with a blade angle of (A) θ = 6°, (B) θ = 40°, and (C) θ = 122°, respectively. (D) The normalized maximum extension length εmax of the cell fragment tended to increase with increasing blade angle. The marker represents the mean of at least three cells cut, and the height of the error bar represents one SD from the mean. (E) Cell survival rate decreased with increasing blade angle. Each marker represents the mean of survival rate of 12 cells cut, and the height of the error bar represents one SD from the mean. The dashed lines in both D and E are guides to the eye only.
Fig. S2.
Fig. S2.
Wound size within 1 min after cutting the cell in (A) regime I at cell velocity = 0.70 cm/s and (B) regime II at cell velocity = 8.38 cm/s. To perform these experiments, we incubated the cells in a DiBAC4(3) solution in PSW (5 μM) for 5 min before cutting the cells [see the main text and SI Materials and Methods for other details on the DiBAC4(3) assay]. The cells were then cut in the microfluidic guillotine directly in this solution. We stopped the flow after the cell was cut and imaged the cut cells within 1 min of the cut in the same channel that consisted of a large imaging chamber downstream of the knife. Compared with the assay performed in Fig. 3, incubating cells in DiBAC4(3) before they were cut allowed the DiBAC4(3) molecules to diffuse into cell immediately after the cells were cut. The resulting DiBAC4(3) fluorescence could better reflect the extent of membrane rupture during the cutting process. We note, however, that the absolute fluorescence intensity distribution depends also on the local diffusivity of the DiBAC4(3) molecules, as well as the local concentration of intracellular proteins that DiBAC4(3) associates with. It is thus difficult to quantify the actual size of the membrane rupture during the cut based on the absolute fluorescence intensity distribution. Nevertheless, in regime I, if the knife had penetrated the cell membrane and split the cell that way we would expect to see a large wound that would expose half of the cytoplasm. DiBAC4(3) could then diffuse into the cell easily. The resulting DiBAC4(3) fluorescence should then spread throughout the majority of the cut cell. As shown in the images, however, we observed that the size of the wound was relatively small and localized. In some cases, we did not observe any fluorescence. In addition, during the cutting process, we did not observe any spilling of the cytoplasm until almost the entire cell length had entered the two outlet channels. In contrast, for cells cut in regime II, the fluorescence spread over almost the entire cut cell. Such results are consistent with the extended degree of membrane rupture in regime II.
Fig. S3.
Fig. S3.
Effect of flow rate on a shear-thinning viscoelastic droplet. The drop consisted of methylcellulose solution (2% wt/vol), and the continuous phase consisted of HFE-7500 containing EA surfactant (2% wt/wt). The infinite rate viscosity of the drop solution was measured as described in SI Materials and Methods to be 900 cP. (A) Snapshots of the drop in regime I and II. The definition of the two regimes was the same as that in the text for a cell. (B) Extension length of the drop as a function of the velocity of the drop. We note that similar transition in droplet shape occurred with increasing flow rate in a Newtonian droplet consisting of water or a glycerol solution (20% wt/wt), except that in regime II the rear end of the drop often broke into smaller drops instead of forming a long thread hanging at the knife.
Fig. 3.
Fig. 3.
Imaging the degree of membrane rupture. (A) Bright-field (Left) and DiBAC4(3) fluorescence (Right) images of uncut Stentor cell, a cell fragment cut in regime I, and a cell fragment cut in regime II, respectively. These cells were cut at the flow conditions indicated by blue arrows in Fig. 2 B and C. (B) DiBAC4(3) fluorescence intensities from cells cut in regime I and in regime II, where DiBAC4(3) was added immediate after the cut (“0h”) or 24 h after the cut (“24h”). The fluorescence intensity of uncut cells is shown as a benchmark. Each marker represents the data for one cell.
Fig. 4.
Fig. 4.
Self-cleaning of the knife using droplets and parallelization of microfluidic guillotine device. (A) Micrographs of the knife after cutting 1, 5, and 10 cells, respectively, for the cases when the cells are not encapsulated inside droplets (“− droplets”), and when the cells are encapsulated inside droplets containing 0.2% wt/vol methylcellulose “MC” solution (“+ droplets”). (B) Micrographs showing the time sequence of the removal of cell residue by droplets. (C) Scheme showing the design of the parallel microfluidic guillotine device, containing eight parallel T-junction droplet generators where the cells are encapsulated into droplets and eight sets of knives downstream. (C, Inset) Micrographs showing the cut cell inside a droplet collected at the outlet of the device. The drops can be merged subsequently and the cell fragments can be collected and imaged out of the drops. (D) Comparison of the cutting performance using a glass needle by hand, a single microfluidic guillotine channel, and eight parallel channels where cells are also encapsulated in droplets.
Fig. S4.
Fig. S4.
Droplet velocity in the eight channels in the parallel microfluidic guillotine device. 1* represents a second measurement of the velocity of drops in channel 1 ∼30 min after the first measurement. The horizontal line represents the mean velocity of droplets in the eight channels. Droplet velocities were calculated using custom MATLAB scripts to track the velocities of drops in videos taken of each channel.

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