The nanoscale IFC consists of a plastic carrier and a polydimethylsiloxane (PDMS) core (Figure 2A) or fluidic circuit. The carrier contains reservoir wells for input and output of reagents and circuit control. It provides a platform to facilitate interfacing with the fluidic circuit. The fluidic circuit with the desired microfluidic control components was fabricated using multilayer soft lithography (MSL^®) process (Unger et al., 2000). Fluidic circuit components include flow and control channels, valves, multiplexors, and logic devices [such as serial-to-parallel shift register (SR)]. Fabrication and operational details of the fluidic logic circuits and devices were reported earlier (Devaraju and Unger, 2012). The IFC is designed to have the capability to actively select single cells based on fluorescent markers, isolate them to a desired holding location (cell capture site), apply individual conditions (feed medium and dose reagents to cells), and finally study the functional response. Execution of all these complex functions in a routine fashion requires flexible, programmable operational control, which in turn requires many controls in a parallel manner. Traditionally, in microfluidics, a dedicated external control line is required to independently control a set of valves. This imposes a limitation on the number of practical on-chip control operations and poses a challenge for scalability by requiring more external hardware. On-chip control architecture capable of receiving and processing data by elementary computation and decision-making can integrate programmability of controls on-chip and allows an increase in the number of on-chip control lines for the same number of external chip connections (Figure 2B). We developed such a microfluidic fluidic logic and implemented it on our Polaris IFC.

The state-based microfluidic fluidic logic devices and circuits utilize static gain and normally closed valves (NCVs). NCVs are fabricated by filling specialized control channels with a flash curable prepolymer and curing while the valve is closed. The resulting closed valve exerts certain force against fluidic pressure to keep the valve closed. The valves are characterized by breakthrough pressure: the threshold pressure in the flow channel required to push open the valve and restore the continuity of the flow. Breakthrough pressure for an NCV can be tailored by controlling the pressure at which they are cured. Using these NCVs, we have developed static gain valves (SGV) that have the ability to control higher (or equal) fluidic pressure using a lower pressure. This type of valve is essential to create any logic/feedback structures (to account for signal strength losses), which can receive the output of the previous element/gate and use it as an input for decision making.

Utilizing the SGV, we next built an inverter (NOT gate), which was further used to build more complex circuits including bi-stable flip flops, clocked flip flops (latches), delay flip flops (D-FF, one bit of the SR), and complex microprocessors (SR). A SR that is capable of processing n + 1 bits of data is formed by combining n D flip flops (bits of SR). The SR presented here uses air as the medium and receives three active high-pressure inputs: source, clock, and data (Figure 3). The pneumatic output of the SR cannot be used to control the flow of liquids in microchannels directly, due to risk of introducing bubbles. In order to address this issue, the signal medium is converted from air to liquid using an inverter.

The Polaris IFC microprocessor receives 28 external signals serially and processes them into 28 parallel independent controls capable of controlling individual valves or a set of valves. Five dedicated high-pressure external active signals are required for a SR. The CAD drawing of the various microfluidic components on a Polaris IFC is shown in Figure 3. The IFC can accept up to 20 independent reagents. The fluorescently labeled cells are loaded in a serpentine partition channel. Based on a desired combination of up to three fluorescent markers (refer to Section “Polaris Instrument Design” for excitation and emission details), single cells are selected and sequentially isolated to the cell capture sites through a multiplexer. Up to 48 single cells can be sequestered on a single Polaris IFC. Subsequently, these 48 cells are processed through template-switching chemistry for full-length cDNA generation for mRNA-seq. In brief, the cells are lysed and reverse-transcribed, and full-length cDNA is preamplified by long and accurate PCR.

The Fluidigm Polaris system (Figure 4A) consists of four major modules: (1) thermal control module; (2) imaging module; (3) pneumatic control module; and (4) environmental control (EC) module. The thermal module consists of a Peltier-based thermoelectric couple (TEC) device for heating/cooling. The TEC module can provide temperature in the range of 4–99°C. Vacuum grooves on the thermal module are designed to enable tight contact with the glass-based integrated heat spreader (IHS) on the Polaris IFC. This ensures thermal uniformity across the fluidic circuit. The imaging module contains a five-color LED light engine for excitation (Ex wavelengths: 438, 475, 530, 575, and 632 nm). The light source from the engine is collected and projected onto the fluidic circuit using fiber optics. The emitted signal from the fluidic circuit passes through an emission filter (five Em wavelengths: 488, 525, 570, 630, and 700 nm) and is collected by CCD camera with 6-μm pixel resolution through a custom-designed collimator lens.

The pneumatic control module generates and stores air with volume up to 1 L. The system can achieve a maximum pressure of 100 psi. The pneumatic controller generates a vacuum on the thermal chuck, clamps the Polaris IFC against the EC interface plate (IP) to enable a closed environment around the IFC, and loads reagents from the inlets on the IFC carrier to the microchannels and reagent chambers of the fluidic circuit. The system contains segregated zones to regulate four different pressures simultaneously. The EC module provides an environment suitable for cell culture using user-desired gas composition. Environmental parameters such as temperature, relative humidity (RH), and mix gas flow rate across the fluidic circuit are monitored and controlled. The gas inside the closed chamber is heated by two heater coils. The gas inlet on the EC IP is used to regulate the flow of gas across the fluidic circuit. The EC IP (Figure 4B) contains an indium tin oxide (ITO) coated glass on the top to maintain thermal control in the EC while allowing imaging through the EC IP. During cell culture operation, the ITO glass is heated to prevent condensation. During cell culture, the environment around the fluidic circuit is maintained by blood gas (5% CO₂, 5% oxygen, and 90% nitrogen) or premixed gas of choice (for example, 5% CO₂, 20% oxygen, and 75% nitrogen). Before on-IFC cell culture, a rectangular sponge saturated with water is installed inside the closed chamber to provide the desired humidity through heating. The EC IP is equipped with two sensors (T/RH) to measure and maintain temperature at 37°C and RH at 90%.

K562 cells (ATCC^® CCL-243) are cultured in T25 flasks in a volume ranging from 10 to 15 mL in an incubator (37°C, 5% CO₂). The culture medium contains IMDM + GlutaMAX™-I + 25 mM HEPES + 3.024 g/L sodium bicarbonate (Gibco, 31980-030) and is supplemented with 10% FBS. The cells were fed every 2–3 days by dilution to 200,000 cells/mL. The K562 cells were stained with CellTracker™ Orange (CTO) CMRA Dye (Thermo Fisher Scientific, C34551) as universal marker and Alexa Fluor^® 647 conjugated CD59 antibody. The recommended dyes and corresponding excitation and emission filters on the Polaris system are shown in Table 1. Immediately before use, the cell staining solution was prepared by adding 0.6 μL of 1 mM CTO to 2 mL of HBSS without calcium or magnesium (−/−) at a final concentration of 0.3 μM. The cell staining solution was protected from light until use within 30 min. A total of ~1.5 × 10⁶ cells was aliquoted in a 15 mL non-pyrogenic conical tube. The cell suspension was centrifuged at 300 × g for 3 min. Following this, the medium was aspirated without disturbing the pellet, and 2 mL of cell staining solution was added to the pellet and gently suspended by pipetting up and down three times. The cells were then incubated in the dark at 37°C for 20 min with occasional inverting and flicking. Following this, the cells were washed by adding 12 mL of HBSS to the cells in the 2 mL of staining buffer and then centrifuged at 300 × g for 5 min. Supernatant was aspirated and discarded without disturbing the pellet. The pellet was then resuspended in 200 μL of HBSS. The CTO-stained K562 cells were split into two tubes of 100 μL each. One tube was used as negative surface-stained cell population, and the other tube was processed further to stain CD59 epitope. In order to stain the surface CD59 epitope, 10 μL of CD59 biotinylated antibody (BD Biosciences, 555762, 100 tests, 2.0 mL) was added to 100 μL of CTO-stained cells. For negative surface-stained cell control, 10 μL of HBSS was added. Both the tubes were incubated at room temperature for 20 min with occasional inverting and flicking. Subsequently, 13 mL of HBSS was added to each tube and centrifuged at 300 × g for 5 min. The pellet was resuspended in 100 μL of HBSS. To this, 0.5 μL of Streptavidin Alexa Fluor^® 647 (Thermo Fisher Scientific, S32357, 2 mg/mL stock) was added to positive-stain tube with CD59 biotinylated antibody in 100-μL cell suspension. This solution was mixed gently by pipetting up and down five times. Following this, the stain solution was incubated at room temperature for 15 min with occasional flicking. Again, 13 mL of HBSS was added to each tube, mixed by gently pipetting up and down, and centrifuged at 300 × g for 5 min. The supernatant was removed, and the pellet was resuspended in ~100–150 μL culture medium with FBS, but without phenol red, to prevent high background fluorescence during cell selection on the Polaris system. The resuspension volume of culture medium accounts for cell losses during the staining procedure and was chosen to yield a cell concentration greater than the target concentration of 550 cells/μL. Typically, 10 μL of cell mix is loaded into a C-Chip™ Disposable Hemocytometer (INCYTO, DHC-N01) and imaged on the Polaris system to estimate the staining intensity and purity. In order to achieve optimal buoyancy, cells in the range of 333–550 cells/μL are mixed with suspension reagent (Fluidigm, 101-0434). Typically, the ratio of cells to cell suspension reagent is 3:2. However, this ratio might need optimization depending on the cell type.

The Polaris IFC is first primed to fill the control lines on the fluidic circuit, load cell capture beads, and the inside of PDMS channels is blocked to prevent non-specific absorption/adsorption of proteins. In order to capture and maintain the single cells in the sites, the capture sites (48 sites) are preloaded with beads that are linked on-IFC to fabricate a tightly packed bead column during the IFC prime step. In the case of adherent cells, ECM is coated inside the cell capture chambers during prime step. After completion of the prime step, the cell mix (cells with suspension reagent) is loaded on the Polaris IFC and single CTO⁺/CD59⁺ cells are selected to capture sites. We extensively tested the performance of the Polaris IFC and system at three different cell purities (3, 10, and 50%). The cell purity is defined as the ratio of CTO⁺/CD59⁺ cells to CTO⁺/CD59⁻ cells. During the cell selection step, the suspended cells are loaded into the serpentine partition channel (Figure 3). Subsequently, the flow inside the partition channel is stopped (Figure 5A), and the cells are imaged in the partition channel for different fluorescent markers, as selected by the user. Based on automated image analyses by the system’s software, only single cells with the desired combination of fluorescent markers are selected and isolated to the cell capture site. Any doublets or single cells with undesired fluorescent combinations are not selected by the software for further experimentation (Figure 5B). The selected single cells are moved to capture sites through a multiplexer (Figure 5C). The system takes images of the capture sites to confirm the arrival of single cells from a particular position in the partition to a particular capture site number. Figure 5C shows a typical image from the Polaris system showing K562 single cells captured inside sites packed with a column of beads. The system will then select and isolate all available single cells from a partition fill as per desired fluorescent marker combination. Once it completes selection of candidate cells, the system refills the serpentine partition to look for more candidate single cells. The system repeats this process to select and isolate single cells until it fills all 48 capture sites.

If desired, the single cells can then be cultured in the capture sites. It is possible to culture either suspension (e.g., K562) or adherent (e.g., BJ fibroblast) cells. For adherent cells, extracellular matrix can be coated inside the capture site during the IFC priming step. Figure 5D shows a Polaris image of a cultured BJ fibroblast (adhered). Based on the experimental design, it is possible to dose these single cells and on-IFC-cultured single cells with drugs or other cell stimuli. Finally, the single cells are processed through template-switching mRNA-seq chemistry for full-length cDNA generation and preamplification on-IFC.

Preamplified full-length cDNA of selected single cells are generated on-IFC, and the amplicons are harvested through 48 different outlets. We used the SMARTer Ultra^® Low RNA Kit for Illumina Sequencing (Clontech^®, 634936) to generate preamplified cDNA. The selected and sequestered single cells were lysed using Polaris cell lysis mixture. The 28-μL cell lysis mix consists of 8.0 μL of Polaris Lysis Reagent (Fluidigm, 101-1637), 9.6 μL of Polaris Lysis Plus Reagent (Fluidigm, 101-1635), 9.0 μL of 3′ SMART™ CDS Primer II A (12 μM, Clontech, 634936), and 1.4 μL of Loading Reagent (20X, Fluidigm, 101-1004). Synthetic RNA spikes can be optionally used with cell lysis mix. We typically use ArrayControl™ RNA spikes 1, 4, and 7 (Thermo Fisher Scientific, AM1780) to establish the functionality of RT and PCR on-IFC. We also use ERCC spikes at 1:50,000 dilution (final in lysis mix) for efficiency and quantification estimations. In order to implement synthetic RNA spikes, we thoroughly mix 96.5 μL of loading reagent with 2.5 μL of SMARTer Kit RNase Inhibitor (40 U/μL; Clontech, 634936) and subsequently add 1 μL of synthetic RNA spike to this spike mix. If RNA spike is used, then 1.4 μL of the loading reagent is replaced with the spike mix. The thermal profile for single-cell lysis is 37°C for 5 min, 72°C for 3 min, 25°C for 1 min, and hold at 4°C.

The 48-μL preparation volume for RT contains 1X SMARTer Kit 5X First-Strand Buffer (5X; Clontech, 634936), 2.5-mM SMARTer Kit Dithiothreitol (100 mM; Clontech, 634936), 1-mM SMARTer Kit dNTP Mix (10 mM each; Clontech, 634936), 1.2-μM SMARTer Kit SMARTer II A Oligonucleotide (12 μM; Clontech, 634936), 1-U/μL SMARTer Kit RNase Inhibitor (40 U/μL; Clontech, 634936), 10-U/μL SMARTScribe™ Reverse Transcriptase (100 U/μL; Clontech, 634936), and 3.2 μL of Polaris RT Plus Reagent (Fluidigm, 101-1366). All the concentrations correspond to those found in the RT chambers inside the Polaris IFC. The thermal protocol for RT is 42°C for 90 min (RT), 70°C for 10 min (enzyme inactivation), and a final hold at 4°C.

The 90-μL preparation volume for PCR contains 1X Advantage 2 PCR Buffer [not short amplicon (SA)] (10X, Clontech, 639206, Advantage^® 2 PCR Kit), 0.4-mM dNTP Mix (50X/10 mM, Clontech, 639206), 0.48-μM IS PCR Primer (12 μM, Clontech, 639206), 2X Advantage 2 Polymerase Mix (50X, Clontech, 639206), and 1X Loading Reagent (20X, Fluidigm, 101-1004). All the concentrations correspond to those found in the PCR chambers inside the Polaris IFC. The thermal protocol for preamplification consists of 95°C for 1 min (enzyme activation), five cycles (95°C for 20 s, 58°C for 4 min, and 68°C for 6 min), nine cycles (95°C for 20 s, 64°C for 30 s, and 68°C for 6 min), seven cycles (95°C for 30 s, 64°C for 30 s, and 68°C for 7 min), and final extension at 72°C for 10 min. The preamplified cDNAs are harvested into 48 separate outlets on the Polaris IFC carrier.

Harvested samples from Polaris IFCs were analyzed by qPCR using 96.96 Dynamic Array™ IFCs and the Biomark™ HD system from Fluidigm. Processing of the IFCs and operation of the instruments were performed according to the manufacturer’s procedures. For detection using the RNA expression and splice variant assays, a Master Mix was prepared consisting of 360-μL SsoFast™ EvaGreen^® Supermix with Low ROX (BioRad 172-5211) and 36-μL 20 × DNA Binding Dye Sample Loading Reagent (Fluidigm 100-5360), and 3.3 μL of this mix was dispensed to each well of a 96-well assay plate. Diluted harvest product (2.7 μL) was added to each well, and the plate was briefly vortexed and centrifuged. Following priming of the IFC in the IFC Controller HX, 5 μL of the sample + Master Mix were dispensed to each sample inlet of the 96.96 IFC. Five microliters of each 10 × assay [5 μM each primer, 1 × assay Loading Reagent (Fluidigm 100-5359)] were dispensed to each Detector Inlet of the 96.96 IFC. After loading the assays and samples into the IFC in the IFC Controller HX, the IFC was transferred to the Biomark HD, and PCR was performed using the thermal protocol GE Fast 96 × 96 PCR + Melt v2.pcl. This protocol consists of a thermal mix of 70°C, 40 min; 60°C, 30 s, hot start at 95°C, 1 min, PCR cycle of 30 cycles of 96°C, 5 s; 60°C, 20 s, and melting using a ramp from 60 to 95°C at 1°C/3 s. Data were analyzed using Fluidigm Real-Time PCR Analysis software using the Linear (Derivative) Baseline Correction Method and the Auto (Global) Ct Threshold Method. The data are exported as a.csv file into an Excel^® macro to compile and compare the data against in-house specifications.

In order to statistically evaluate the performance of the Polaris IFC, we designed and developed two performance tests: (1) total-RNA-based performance test (RNA PT) and (2) single-cell-based key performance test (KPT). Since single cells are heterogeneous, it would be difficult to evaluate the performance uniformity across 48 capture sites using a cell-based test method. Hence, we developed a 20-cell-equivalent total-RNA PT to evaluate and improve the performance of the Polaris IFC during the initial phase of the IFC development process.

An alternative way to evaluate performance of single-cell mRNA-seq on the Polaris system is to implement use of the ERCC RNA Spike-In Mix 1 in the lysis mix. The ERCC control mix consists of 92 polyadenylated transcripts with a size range of 273–2,022 bases and six orders of magnitude range in concentration. We tested both qPCR- and sequencing-based methods for detection of ERCC spikes. Ninety-two primer pairs were designed to target the corresponding transcripts for qPCR testing. qPCR was performed on a Fluidigm 96.96 Dynamic Array IFC. Stochastic distribution of transcripts was observed when the input concentration was 25 copies per reaction or less on the Polaris IFC followed by qPCR detection on the 96.96 Dynamic Array (Figure 14A). Single-copy RNA detection is demonstrated, although intermittently, likely due to sampling at the reaction site. Transcripts at 1.6 copies per reaction were intermittently detected by qPCR on the 96.96 Dynamic Array IFC. We also evaluated the detection rate of ERCC spikes (>1.6 copies/reaction) using an approach based on massive parallel sequencing. There were 7 ERCC spikes (ERCC-00170; ERCC-00148; ERCC-00126; ERCC-00099; ERCC-00054; ERCC-00163; ERCC-00059), which were at a concentration of 1.6 copies per Polaris reaction chamber. One of the 7 ERCC spikes (ERCC-00054) was not detected in any of the 19 single-cell samples. If we remove this datapoint as an outlier, the average detection rate of ~1.6 copies is 28%. Based on Poisson estimates, single-copy detection rate should be ~33% (67% should be a failure event). The single-copy detection rate (28%) from the Polaris system is very close to expected theoretical estimates based on Poisson statistics (Figure 14B).

In order to demonstrate dosing and functional response analyses, we transfected single K562 cells with nuclear green fluorescent protein (nGFP) mRNA and cultured the transfected single K562 for 16 h. During this culture duration, the cells translated the nGFP mRNA and expressed the GFP inside the cell. The imaging capability of Polaris enables monitoring of GFP expression. Subsequently, the cells were processed for mRNA-seq chemistry on-IFC and sequenced on MiSeq to quantify the reads mapped to GFP. K562 cells stained with CTO were selected on Polaris IFC. To carry out the single-cell transfection, 10 μL of Stemfect RNA transfection reagent was mixed with 240-μL Stemfect transfection buffer (Stemgent^® Stemfect™ RNA Transfection Kit, 00-0069) (Mix A). The stock nGFP mRNA (Stemgent, 05-0019) at 100 ng/μL was diluted with Stemfect transfection buffer first and then further diluted with Mix A to make mRNA transfection complex. This complex was incubated at room temperature for 15 min and further diluted with K562 cell culture medium (refer to Section “K562 Cell Culture and CD59 Staining”) to achieve different final concentrations (0.5 and 1 ng/μL) of nGFP mRNA. Selected single K562 cells were cultured with the nGFP mRNA transfection complex with culture medium at 37°C with 5% CO₂ on the Polaris IFC. During this cell culture incubation time, images were taken every hour to monitor the onset of GFP expression. Image analyses (Figure 15) showed that single cells picked up nGFP mRNA at 0.5 and 1 ng/μL concentrations and expressed the green fluorescent proteins, thereby reinforcing the fact that single cells on Polaris IFC are healthy and are able to uptake naked mRNA and translate it to protein capable of being transfected. Figure 15A shows typical time-series images, which can be obtained from the Polaris system. It should be noted that for this particular experiment, the imaging interval was set to 1 h, but the Polaris system is capable of taking successive images in a rapid mode. We noted onset of GFP gene expression around the 3-h time frame at single-cell resolution. The cDNA pool showed a length range from 0.3 to 9.2 kb, with an average length ~2 kb. It is also noted that >85% of the total cDNA pool lies between 0.5 and 9.2 kb (Figure 15B). Sequencing data show that the cells transfected with nGFP mRNA harbored the extracellular mRNA even after 16 h of culture. As expected, the control cells without nGFP transfection did not show any mapping to GFP sequence. The transfection of nGFP did not alter the mapping to genome and transcriptome when compared to the control cells (Figure 15C). The nGFP-transfected cells showed percentage average reads mapping of 0.57, 87.99, and 47.82% to GFP, genome, and transcriptome, respectively (n = 7), while the control K562 showed percentage average mapping of 0, 88.03, and 49.22 (n = 7).