As noted earlier, the combination of PK-PD models and a microfluidic co-culture system could work as a novel platform for reproducing the action of drugs in the body. Several initial attempts have been made and reported, some of which have been already mentioned. Here we describe a few other studies that have specifically focused on creating a physical realization of a PK-PD model using a microfluidic co-culture system. The Shuler group [49
] described a PK-PD model-based μCCA device, which allowed for the evaluation of drug metabolism and prediction of the inter-organ interactions between the liver and other organs in the human body. Subsequently, the design, fabrication, and operation of a three-chamber microscale cell culture analog (μCCA) system were attempted; the chambers represented the lung, liver, and other tissues. The system consisted of a silicon substrate that contained fluidic networks and chambers, confined within Plexiglass housing. Mammalian cells (L2 and H4IIE) were cultured for over 24 h and the cell viability was then examined. The oxygen exchange rate for cell cultures was measured using an integrated fluorescent-based oxygen sensor [50
]. The μCCA system was validated using naphthalene as a model toxic chemical [51
]. The system contained four compartments (representing the lung, liver, other tissues, and fat). The lung and liver cells were cultured in two separate chambers. The tissue and fat compartments did not contain cells, but had a controlled fluid flow that was induced by channel geometry to mimic in vivo residence times. This was intended so that the drug distribution in the chip would mimic that of the human body. After the medium passed through the inlet, the fluid moved through the lung compartment and was distributed to the liver, fat, and other tissue compartments. The fluid was then combined and recirculated from the outlet to the inlet for recirculation. Naphthalene, added to the circulating culture medium, was metabolized and generated reactive metabolites (Naphthoquinone and naphthalene diol) by a P450 enzyme in the liver compartment; the metabolite circulated and induced GSH depletion in the lung and liver cells (L2 and C3A cells, respectively), resulting in cell death. L2 cells were more sensitive to GSH depletion, as the GSH re-synthesis rate of C3A cells was faster than that of L2 cells. Cell cultures performed in a 2D monolayer failed to replicate the physiological response of native tissue in vivo, mainly because 2D monolayer culture systems cannot imitate cell-to-cell and cell-to-ECM interactions, which are important for maintaining the physiological function of cells. Therefore, a 3D hydrogel-based cell culture system is required for tissue-specific functions and differentiated states of cells [52
]. Colon cancer cells (HCT-116), hepatoma cells (HepG2/C3A), and myeloblasts (Kasumi-1) were embedded and cultured in the tumor, liver, and marrow chamber, respectively. The chambers were connected by the microfluidic network, mimicking the blood flow around the target organs. The cytotoxic effect of Tegafur, an oral prodrug of 5-fluorouracil (5-FU), was compared in a 96-well microtiter plate and the µCCA. The hypothesis set up by the authors was that the presence of liver metabolism in the µCCA would produce the active drug, which would kill tumor cells. As predicted, in the µCCA, Tegafur was converted to 5-FU by the P450 enzyme in the liver, and consequently induced the death of cancer cells. The μCCA system was used to mimic in vivo pharmacokinetic and pharmacodynamic drug profiles. In addition, a gravity-induced fluidic system was developed and the toxicity of 5-FU was analyzed by using a PK-PD model (Figure 4
]. Cells showed a higher sensitivity to 5-FU using this system compared to the results obtained using the static system. In addition, the difference in responses based on cell types was distinct using the dynamic system compared to responses generated using the static system. A corresponding PK-PD model was constructed to quantify the action of the drug in the chip, providing insight into the in vivo drug mechanism.