Increasing Fluid Viscosity Ensures Consistent Single-Cell Encapsulation

High-throughput single-cell analysis typically relies on the isolation of cells of interest in separate compartments for subsequent phenotypic or genotypic characterization. Using microfluidics, this is achieved by isolating individual cells in microdroplets or microwells. However, due to cell-to-cell variability in size, shape, and density, the cell capture efficiencies may vary significantly. This variability can negatively impact the measurements and introduce undesirable artifacts when trying to isolate and characterize heterogeneous cell populations. In this study, we show that single-cell isolation biases in microfluidics can be circumvented by increasing the viscosity of fluids in which cells are dispersed. At a viscosity of 40–50 cP (cP), the cell sedimentation is effectively reduced, resulting in a steady cell flow inside the microfluidics chip and consistent encapsulation in water-in-oil droplets over extended periods of time. This approach allows nearly all cells in a sample to be isolated with the same efficiency, irrespective of their type. Our results show that increased fluid viscosity, rather than cell-adjusted density, provides a more reliable approach to mitigate single-cell isolation biases.


Theoretical background for cell sedimentation
Cells suspended in a stationary fluid experience the forces of gravity, the Archimedes (buoyancy) force and the viscous drag force.The sum of the action of these forces defines the magnitude and the direction of the cell velocity.Under the effect of gravity,  !=  "  =  "  "  the axis of motion is directed downwards.Here, ρc represents the average density of the cell, Vc is the cell volume and g is the gravitational acceleration.The forces opposing the motion of the cell along the gravity axis (sedimentation) are the Archimedes force  # =  $  " and the viscous drag force  % = 6 " , where ρf is the fluid density, Rc is the cell radius and v is the velocity of a cell.The cell motion mc can be expressed as the sum of opposing forces: The integral of equation ( 1) can be expressed as: leading to the solution, where the cell velocity, v is expressed as a time-dependent function: For microscale objects in the order 10 -6 m (e.g.cells), the exponential function is decaying fast and thus can be omitted leading to Stokes' velocity

S3
Supplementary Tables Supplementary Table S1.Osmotic pressure of biopolymers in phosphate-buffered saline solution

Figure S1 .
Figure S1.Experiment setup.Schematics of experimental platform.The microfluidics device is connected to two syringe pumps for infusion of cell suspension (Syringe pump 1) and for infusion of immiscible carrier oil (Syringe pump 2).The syringe placed on Pump 1 is filled with mineral oil, which upon infusion, pushes the cell suspension into a microfluidics device until the entire sample is consumed.The cells passing through a microfluidics device (observation chamber) are recorded by capturing the digital images every 30 seconds using a CMOS camera (DS-Qi2).The syringe on Pump 2 is filled with HFE7500 carrier oil supplemented with droplet-stabilizing fluorosurfactant.The water-in-oil droplets are generated at a flow-focusing junction and recorded using a high-speed camera (Phantom v7 or HiSpec HS7), collected into a tube, and further inspected under the bright field microscope to estimate droplet occupancy by single cells.

Figure S2 .
Figure S2.The time trace of cell flow in 1x PBS buffer.The hybridoma (9e10) cells are being continuously injected into a microfluidics device over the course of 70 min and cell number passing the observation chamber is recorded.Note how the initial cell count passing through the microfluidics device drops down to ~1% in 3 minutes and then continues to fluctuate within the 0-10% range until the burst at 68 th minute, during which 2/3 rds of all cells pass through the device.The inset displays the same data but with Y-axis (Cell count) in a log scale.

Figure S3 .
Figure S3.The time trace of cell flow in a cell-density adjusted solution.The hybridoma (9e10) cells are being continuously injected into a microfluidics device in the presence of 20% Optiprep (ρsol = 1.053 g/ml) over the course of 70 min.Noticeably, the use of 1x PBS buffer with 20% Optiprep improves cell loading as compared to 1x PBS (FigureS2).The initial number of cells passing through the microfluidics device drops down to ~20% in 15 minutes and then slowly recovers to approx.30-40% of the initial count until the final burst during which ~2/3 rds of all cells pass through the device.The inset displays the same data but with Y-axis (Cell count) in a log scale.

Figure S4 .
Figure S4.Cell sedimentation rate in viscosity-adjusted PBS buffer.The lymphoblast cells (K562) were suspended in 1x PBS buffer supplemented with varying amounts of biopolymers and injected into a microfluidics device using the experimental setup indicated in FigureS1.The cells passing the observation chamber were counted and the sedimentation rate was extracted from the exponential decay function.Note, that the buffer in which cells are suspended undergoes continuous injection by the syringe pump, therefore sedimentation rate involves two parameters, passive cell sedimentation and injection along the gravitational axis.

Figure S5 .
Figure S5.Droplet generation with viscous fluids.Phosphate buffered saline (1x PBS) was supplemented with increasing amounts of Dextran or Xanthan gum and emulsified on an 80 µm deep microfluidics device, using flow rates at 100 µl/hr for aqueous phase and 300 µl/fr for droplet stabilization oil.The droplet generation was followed for 60 min.Droplet generation in the presence of dextran (A), xanthan gum (B), or no additive (C).Scale bars, 100 µm.

Figure S6 .
Figure S6.Cell clumping over time in a PBS buffer supplemented with dextran and Xanthan gum.Cell clumping is defined as the number of aggregated cells (n ≥ 2) over the total number of cells.The cell counting was conducted every 5 minutes as the cells traversed the microfluidics device using the experimental setup indicated in Figure S1.A) Clumping of suspension (K-562) cells over the course of 60 minutes in 1x PBS supplemented with 5, 10, or 15% dextran.B) Clumping of semi-adherent (9E10) cells over the course of 60 minutes in 1x PBS supplemented with 5, 10, or 15% dextran.C) Clumping of different types of cells in 1x PBS supplemented with 0.05% Xanthan gum.

Figure S7 .
Figure S7.Inhibition test of PCR enzymes by dextran and Xanthan gum.The DNA amplification by four different PCR enzymes, KAPA (Roche, cat.no.KK2602), DreamTaq (Thermo Fisher Scientific, cat.no.K9011), Maxima (Thermo Fisher Scientific, cat.no.K0222) and Phire Tissue Direct (Thermo Fisher Scientific, cat.no.F170S) in the presence of dextran or Xanthan gum biopolymer.A) DNA amplification by PCR in the presence of dextran.B) DNA amplification by PCR in the presence of Xanthan gum.L -ladder (ThermoFisher Scientific, SM0331), 0D, 1D, 4D and 8D indicates dextran fraction (%, [w/v]), whereas 0XG, 0.01XG and 0.05XG indicated Xanthan gum fraction (%, [w/v]).The minus sign indicates no template control.Note, at each condition tested the PCR produced a single specific amplicon band (400 bp), indicating that the presence of biopolymers does not impact reaction specificity.

Figure S8 .
Figure S8.The specificity of RT and PCR in the presence of dextran and Xanthan gum biopolymers.Each graph is represented by multiple melting curves superimposed on each other and corresponds to different biopolymer concentration and type (dextran and XG), as indicated in Figure 4.For the RT inhibition test (top row) Maxima H minus RT enzyme was used followed by qPCR (Maxima Hot Start Taq DNA Polymerase).For the PCR inhibition test (bottom row) Maxima Hot Start Taq DNA Polymerase enzyme was used.The presence of biopolymer had minimal or no impact on the melting temperature of the amplicon thereby indicating high specificity of DNA amplification.The qPCR targets were B2M, FN1, TBP, and ACTB.Further details can be found in the Material and Methods section.

Table S2 .
The Ct values of the marker genes determined by RT-

qPCR Sample The Ct values* of marker genes
* The Ct values were determined by real-time qPCR on QuantStudio 1 instrument using 40 ng/µl of total RNA extracted from K-562.The primer pairs used for qPCR are listed in Supplementary TableS4.The values represent the mean and standard deviation from three technical replicates.Supplementary TableS3.The Ct values of the marker genes determined by RT-