## 3 Results
### Incentive for Using FITC Surrogate to Track Actual Compound Distributions across Device Wells 3-11  
To ascertain actual study compound concentrations across the wells, i.e., spatial and time distributions, 1 uM FITC surrogate was added into a source well in a row parallel to the compound dosing row, simulating an actual compound dose of volume and frequency (drug study). The FITC relative fluorescence units (RFU) are used as a surrogate evaluator in drug dissemination. The fluorescence FITC was measured to estimate drug concentrations after 1 day following a single bolus dose in well 1 (Figure 5a) and after 7 days with daily dosing in well 1 (Figure 5b). In kinetic displays, data curves show the % FITC on the \(y\)-axis (i.e., % of 1 uM FITC signal) versus device well-sites on the \(x\)-axis. The relationship aligns time-resolved exposure gradients with % FITC surrogate, used to approximate % drug/well [23]. At 1 Day (24 h), culture well 3 approaches 79.6% of initial dose, well 5 (20.2%), well 7 (3.7%), and well 11 (0.5%). Similarly, at 7 days (168 h), well 3 approaches 131.4% of initial dose, well 5 (107.1%), well 7 (88.9%), and well 11 (39.9%). The dose values > 100% (Figure 5b) are a function of repeat dosing and residual FITC in wells that accumulate in value. These data show how the platform can be used to generate trackable and repeatable FITC gradient concentrations across the ufluidic system. In this, it  
Figure 4: Operational insights. (**a**) Top view simulation of one microfluidic channel signifying dual exposure gradients of test compound (dosed into well 1) and cell generated metabolite. (**b**) Modulating experimental exposures of test compound throughout one device row having ten (10) re-doses of 100 uL over 3 days. Values 0\(\rightarrow\)1 equate to 0\(\rightarrow\)100% of dose. Dose “0” is culture media without compound (i.e., no-dose baseline). (**b1**) Linescans displaying a timeline of aggregate exposure kinetics, per well, with compound exposure traits ranging from asymptotic (wells 3, 4), approximately linear (wells 7, 8), and delayed increase (lag of exposures in wells 10, 11). Subsequently, fully developed linescans progress into natural logarithm, linear, and delayed exponential growth [23]. (**b2**) Well-specific, data centric, cumulative exposures over time (i.e., area under curve); similar to a radiation model. The final equilibrium drug concentration is very similar across the entire plate (dose 10; wells 3-11; 67.4\(\rightarrow\)62.4%). (**b3**) Total “Cumulative Exposure” is very different in each well, 6.21\(\rightarrow\)2.69.  
is acknowledged that molecular weight differences between FITC and other drug categories will shift gradients of flowing exposure dynamics; an awareness that trending line-shifts can be calibrated for predictive accuracy [23]. Validation of Tempered DMSO Compound as an Effective Control Vehicle Using High-Content Cell Imaging across the Sequential Assembly-Line of \(\mu\)Fluidic Culture Sites  
To investigate effects of the DMSO control vehicle on the viability of cells, HepaRGs were seeded in the \(\mu\)fluidic platform and exposed to the DMSO vehicle for 7 days. HepaRG morphology was evaluated for cells experiencing fluid flow and for cells in no-flow static protocols (Figure 6(a1,a3)). Cell structures were morphologically unchanged with areas of tightly/loosely packed cell densities achieving 100% culture confluency. Yet, given that upstream culture wells feed into downstream well sites, as shown in Figure 6(a2), a variance in cell viability could occur amid adjacent multiwells. Adjacent wells might show  
Figure 5: Percentage and quantification of FITC-flow and drug-concentrations in multiwell device. (**a**) FITC concentrations after 1 day, i.e., 24 h, and a single FITC bolus dose into well 1. (**b**) FITC concentrations after 7 days, i.e., 168 h, and daily FITC dosing into well 1. (**TABLE: case study using drug diclofenac):** Comparison of CYP3A4 activity after diclofenac treatment in HepaRG cells.