A calcium flux assay has been developed for the LabChip 3000 microfluidic system for use in drug screening against GPCR targets. The miniaturized platform results in low cellular and reagent consumption, high data quality and a high degree of automation.
The LabChip microfluidic assay accesses test compounds from a microplate and mixes the samples with suspended cells continuously flowing through the chip. Flow through the microchannel network results from a vacuum applied to the waste wells of the chip. The system measures the fluorescence properties of individual cells flowing past a fluorescence detector. The LabChip system measures as few as 50 cells and uses less than 50 nL of sample and 15 nL of agonist per measurement.
The steps involved in developing and validating an assay to conduct screens for GPCR agonists and antagonists will be discussed here. CHO-m1 is used as a model cell line.
CHO-m1 Assay Summary
Typical cell usage as low as 600-1000 cells/well
Z' = 0.86 for Agonist, Z' = 0.72 for Antagonist
Unattended run time of between 4 and 8 hr
Throughput of up to 8,000 samples in 8 hr
The general steps followed in the development of a calcium flux assay are outlined in Figure 1. A preliminary choice of cell line, growth conditions, and cell assay buffer is made (Step 1). A protocol for labeling, washing and suspending cells is then developed (Step 2), after which confirmation is made that the labeled cells can be detected (Step 3). Once feasibility has been established, the kinetics and magnitude of response to agonist exposure is measured (step 4) and, in the case of an antagonist assay, the concentration of agonist to be placed on-chip is determined (Step 5).
The dilution factor used to calculate the appropriate microplate and control concentration is measured (Step 6). Assay stability can then be assessed by examining the stability and robustness of the response over time (Step 7).
If the assay is deemed stable, then validation will continue with optimization of throughput and cell usage and confirmation of assay stability over multiple runs (Steps 8 and 9). If the assay is not stable, then optimization of buffer conditions and cell growth conditions will be required prior to validation.
III. Assay Development Steps
1. Choose Cell Line, Cell Growth and Cell Buffer Conditions
A microfluidic assay using the CHO-m1 cell line was developed. Cells were grown in Ham's F12 medium adjusted to contain 1.5 g/L sodium bicarbonate with 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM L-glutamine, 0.1 mg/mL Geneticin G418, 1 mM sodium pyruvate, 10 mM HEPES and 10% Fetal Bovine Serum.
Cells were maintained at 37 oC and 5% CO2 and subcultured in monolayers in T75 flasks using standard cell culture methodologies. Cells were split at 80-90% confluency. The day before each assay cells were seeded at 3 x 106 cells into T75 flasks and incubated overnight at 37 oC and 5% CO2.
2. Label, Suspend and Wash Cells
Cells are labeled at room temperature with two calcium sensitive dyes , Fluo4 and Fura Red, while attached to the plate to minimize cell stress due to multiple pelleting and suspension steps. The minimum concentration of dye that yields bright fluorescently-labeled cells should be used. Typically, 6 x 106 cells are loaded with 6 mL of physiological buffer containing 1 μM Fluo-4 and 1 μM Fura Red. In addition, anion transport inhibitors such as probenecid should be used for cell lines such as CHO-m1 that rapidly export Ca2+ indicators. After loading of dye, the cells are suspended and washed to remove unincorporated dye. It is not critical to wash cells extensively as the LabChip assay is relatively insensitive to background fluorescence. Suspensions of adherent cell lines can be formed by a variety of methods such as trypsinizing or EDTA treatment. After lifting and washing, cells are suspended in cell assay buffer prior to loading on the microfluidic chip.
Media was aspirated from the T75 flask and cells were washed once with 10 mL HBSS with 20 mM HEPES. The HBSS/HEPES solution was aspirated and 6 mL dye loading solution was added to the flask (See Section IV). The flask was covered with aluminum foil and incubated at room temperature for 45 min. After the dye loading solution was aspirated, the cells were washed once with 10 mL of PBS without Ca2+ or Mg2+ and then with 5 mL of EDTA. The EDTA was aspirated and 2 mL of Trypsin-EDTA solution was added. After ~ 5 minutes, it was confirmed that the cells were suspended and 1 mL of Trypsin inhibitor solution was added to stop the reaction.
9 mL of cell preparation buffer (See Section IV) was added to the flask and mixed vigorously by pipette. The contents of the flask were then transferred to a 15mL centrifuge tube and centrifuged for 3 min at 200 g. The supernatant was aspirated and the pellet was loosened by flicking. 1 mL of cell preparation buffer was added and pipetted vigorously. An additional 2 mL of cell preparation buffer was added and pipetted vigorously (note: care was taken to avoid introduction of air bubbles; if introduced these can be aspirated from liquid surface). The cell suspension was incubated at room temperature for 5 minutes and an additional 4 mL of cell preparation buffer was then added and gently mixed by pipette. Cell counts were performed and the suspension was then centrifuged for 3 min at 200 x g. The supernatant was aspirated and the pellet was loosened by flicking prior to resuspension in 3 mL of cell assay buffer at 3-5 x 106 cells/mL (note cell assay buffer may require optimization, but an initial buffer composition given in Section IV should suffice for early phase feasibility).
3. Confirm Cell Detection
Typically, once cells have been loaded with dye and suspended in solution, the cells are added to the cell wells on the microfluidic chip. The cells are then carried to the detection point in the chip by applying a vacuum to the waste wells. Cell fluorescence excited by a 488 nm laser is detected at both 530 (Fluo-4) and 685 (Fura Red) nm. A spike or peak in fluorescence is observed as each cell passes the detector. Robust signals display peak heights greater than ten times the baseline noise.
4. Determine Kinetics of Response
The LabChip 3000 calcium flux chip is designed such that calcium response for each cell is measured at a fixed incubation time. The incubation time is determined by the cell velocity and the length of the incubation channel. Cell velocity is a function of pressure applied to the chip, the chip temperature and the solution viscosity, while incubation channel length can be varied by selecting detection points at three locations (or zones) along the cell path. Table 1 lists the dependence of incubation time on pressure and detection zone. Typically pressures of -1.2, -2 and -3 psi are sufficient to gather statistics on optimum incubation time. In this experiment, a candidate agonist is sipped up onto the chip and contacted with cells. The ratio of fluorescence emission for each cell at 530 and 685 nm is measured and plotted as a function of incubation time. From this experiment 15 s (pressure = -2 psi at Zone 1) was determined to be the optimum incubation time for CHO-m1 cells stimulated by carbachol, since detecting at a point after the peak calcium response yields maximum sensitivity to low agonist doses while maintaining high responses to high agonist doses (Figure 3).
5. Determine On-Chip Agonist Concentration
For antagonist assays, the concentration of agonist to be placed on the chip is determined. The CHO-m1 assay was run with on-chip agonist concentration equal to the compound EC80.
EC80 is determined by placing increasing concentrations of agonist in the wells of a microplate and determining cell response. This response is compared to a control response such as ionomycin and a % activity is calculated. Analysis of the dose-response curve (Figure 4) yielded an EC50 of ~ 0.07 μM and an EC80 of ~ 0.6 μM. The concentration of agonist in the agonist well on the chip was then set at 6 μM to yield 0.6 μM in the reaction channel of the chip.
6. Determine Compound Plate and 100% Control Concentrations
Compound concentrations in microplates and control troughs must be chosen to account for on-chip dilution from cell sidearms and dispersion in the main channel. Sidearm dilutions for agonist and antagonist chips is ~50%. Sample dilution due to dispersion depends on assay parameters such as sample sip time, detection zone and applied pressure. For agonist and antagonist chips, typical operating conditions of -2 psi, (zone 1), and sample sip times > 5 s result in effectively no dispersion-related dilution of compound from the sipper. On-plate and 100% control compound concentrations were therefore doubled to account for on-chip dilutions.
7. Assay Stability
Once reaction conditions have been determined, the stability of the assay over the anticipated run time can be assessed. Typical run times for cell-based assays range from 4 to 8 hr. Stability of the assay can be assessed by examining the cell count rate and the cell viability (e.g. EC50) over time.
CHO-m1 cells are loaded in the chip wells at a density of between 3-5 x 106 cells/mL. The cell count over time is monitored to ensure that sufficient statistics are gathered on the last plate of the run. Figure 5 shows that cell counts are > 100 cells per well after 5 hr. Figure 6 shows the stability of the cell response over a typical run time.
8. Optimize Throughput and Cell Usage
Optimization of throughput is achieved by gathering data on a sufficient number of cells for each sample well visited on all assay plates. Figure 7 shows the dependence of data quality (Z') on cell count per sample. Typically more than 50 cells per sample are required for sufficient statistics to develop high Z' assays.
As cell count numbers are dependent on sample sip time, throughput and data quality can be optimized as needed. In this assay, sample sip times of 12 and 5 s were chosen as low- and high-throughput versions of both agonist and antagonist calcium flux assays. The sip time of degassed buffer between samples should also be chosen to maximize throughput while still maintaining a low dissolved gas content on the chip. Buffer sip times of 5 and 3 s were chosen for 12 and 5 s sample sip times, respectively.
The number of wells sampled during an assay run will affect cell usage. Higher-throughput assays will have lower Z' but reduced cell consumption on a per-well basis. Table 2 summarizes throughput and cell usage for CHO-m1 agonist and antagonist assays.
9. Confirm Z': % Activity
Stability vs. Time over URT. Once assay conditions had been set, the assay was validated. Typical validation criteria include data quality (Z') assessment and assay stability over multiple runs. Figure 8 shows the calculated Z' values for the agonist assay with both a 12 and 5 s sample sip time. Assay stability was measured over several runs and was determined to be adequate over the intended run time of between 4 and 8 hr. Average Z' values were calculated at 0.86 and 0.75 over 8 hr for the 12 and 5 s sample sip times, respectively.
Figure 9 shows the Z' values for the associated antagonist assay, with stability and reproducibility within expected limits. Average Z' values were calculated at 0.72 and 0.57 over 5 hr for the 12 and 5 s sample sip times, respectively. Note that the shorter sip time does result in slightly lower Z' values, as discussed in section 8.
In addition to Z' analysis , the stability of both the agonist and antagonist response was measured over several runs for both 12 and 5 s sample sip times. Figures 10 and 11 show the stability of the agonist and antagonist response relative to an ionomycin and atropine control, respectively, over several hours for a 12 s sample sip time. The 5 s sample sip response is similar over an extended run (data not shown).
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