## 3 Results
### Use-Model, Drug Exposure, and CYP3A4 Comparisons between _u_Fluidic Systems and Static Cultures
#### 3.4.2 Extrapolation of Drug Exposures in _u_Fluidic System, Diclofenac (Days 1 and 7)  
Day 1: We compared CYP3A4 activities after HepaRG cells were exposed to different concentrations of diclofenac for 24 h (Figure 5 Case Study; a1-a5). The measured FITC concentration per well, column a1, is the % of FITC dispersed across the multiwell system and correlates with Figure 5a. The calculated uM diclofenac concentration, column a2, is column a1 \(\times\) 125 ulM dose (i.e., well 3 is 79.6% \(\times\) 125 ulM = 99.5 ulM diclofenac). The actual uM diclofenac drug concentration, not determined, is line-shifted as FITC (389.3 g/mol) and diclofenac (296.1 g/mol) have different molecular weights, but trending flow patterns remain analogous [23]. Column a3 illustrates CYP3A4 outcomes from static culture protocols (no flow) using equivalent drug exposure concentrations. Column a4 shows CYP3A4 outcomes with cultures experiencing kinetic nutrient movements (_u_fluidic device). At 24 h, we did not observe any significant difference in CYP3A4 activity between the static and ulfluidic systems (column a5, statistical _\"p\"_, ns). Day 7: We then compared the effect of different concentrations of diclofenac on CYP3A4 activity after 7 days of exposure (Figure 5 case study; b1-b5), with daily drug re-dosing. Notably, the well-1 dose % (column b1) is listed as >1\(\times\) concentration and is a function of repeat dosing with rising concentrations above the initial/starting well-1 dilutions to correlate with Figure 5b. Paraphrased, residual compound in well 1 with repeat dosing accumulates values. The % FITC concentrations are increased across all wells ranging from upstream 131.4% (well 3) to downstream 39.9% (well 11), column b1. The calculated diclofenac uM drug concentration, column b2, is column b1 \(\times\) 125 ulM dose (i.e., well 3 is 131.4% \(\times\) 125 \(\upmu\)M = 164.3 \(\upmu\)M diclofenac). CYP3A4 activity in HepaRG cells was decreased in the \(\upmu\)fluidic device wells 3-9 (column b4) as compared with the static culture (column b3), indicating a significant difference noted in column b5, statistical \"\(p\)\". These data infer that flow in upstream well (well 3), or cascading flows across \(\upmu\)fluidic wells 4-11, create cell-signaling concentration gradients of either dose-compound or endogenous cellular byproducts that factor into cell variances of culture responses. Figure 6: Morphology and viability of HepaRG cells in device multiwell culture locations, post 0.1% DMSO vehicle compound treatment. **(a2)** Profile of device displaying two adjacent wells, two flat culture surfaces, and \(\upmu\)channels entering wells. **(a1)** Kinetic (fluid flow) cultures with cell images at days 1 and 7, phase contrast brightfield 10\(\times\). **(a3)** Comparative cell images at days 1 and 7 in static (no-flow) cultures, 10\(\times\). **(b1–b3)** Fluorescent image captures of cell nuclei stains (DAPI; blue) and non-viable stains (CellTox Green; green) for adjacent device wells, 4\(\times\) pictorials. **(b1)** Equivalent high cell viability **(d1,d2)**, or equivalent lower cell viability **(e1,e2)**. **(b2)** Upstream device well displaying lower cell viability vs. downstream well. **(b3)** Upstream device well displaying higher cell viability vs. downstream well. **(c)** Fluorescent image captures of cell nuclei stains (DAPI; blue), viable cells (Calcein AM; green), and non-viable cells (Ethidium Homodimer; red) to assess variance of cell viability across device wells 3–11. (Day 1) Auto-DMSO concentration gradients after a single 0.1% DMSO bolus dose (0.1 _v/v_) into well 1. (Day 3) After three 0.1% DMSO re-doses into well 1; daily re-dosing. (Day 7) After seven 0.1% DMSO re-doses into well 1. Overall, cell viability remains effectively unchanged, 95–99%.