## 4 Discussion  
Globally, reducing animals used in pharmacological and toxicological testing is a priority. Alternative methods, such as in vitro co-culture models, are fundamental in addressing this reduction and for expediting the process of testing, validating, and mass-producing novel therapeutics. The United States EPA has vowed to reduce requests and funding of mammal studies by 30% by 2025; the agency plans to eliminate all mammal study requests by 2035. Utilizing alternative methods, such as AOP development or in silico approaches as well as in vitro culture models not only allows for a better recapitulation of in vivo readouts but can aid in the immediate need to improve testing methods for the ever-increasing amount of chemicals in production and use. The use of monolayers is still dominant today despite poorly imitating in vivo conditions. Monoculures do not properly represent an in vivo model system in terms of tissue architectures or biochemical signaling [49; 50; 51]. While current literature acknowledges the former (i.e., tissue architecture), only a few in vitro based papers discuss the shortcomings of the latter (i.e., biochemical signaling) [52; 53]. Correlations among in vitro to in vivo model systems are largely dependent upon the cell-type, organ system, and animal selected for the study. Due to the diversity of the intestinal tract, it is apparent that single monolayers of gut epithelial cells are not sophisticated enough to assess biological responses accurately. The triple cell culture model can better assess immunomodulatory effects after ingestion of nanomaterials [54]. To get a better understanding of inflammation and subsequent biochemical responses, this study focused on the proinflammatory cytokine interleukin-6 (IL-6) [55]. It has been shown that IL-6 activates STAT 3, a transcription factor which leads to tissue repair, differentiation, cell survival, migration, and cell death [56; 57]. Importantly, IL-6 is necessary for the differentiation of Caco-2 cells to enterocyte-like phenotype as well as to inflammation after tissue injury [58; 59]. In the case of pathophysiology, IL-6 plays an important role in the ability of intestinal cancers to progress and should be considered a fundamental cytokine when testing for initial inflammatory responses [56]. Inflammatory response measured by IL-6 concentration shows that there are differential measurements in bio  
Figure 5: Monolayer integrity, as measured by transendothelial electrical resistance (TEER), of the three individual cell types, as well as the tri-culture, used in the study. All cells were seeded at the same density (13,000 cells/cm\({}^{2}\)) and were maintained in standard cell culture conditions (5% CO\({}_{2}\), 10% humidity) for 8 days. Experiments were conducted with n = 3 in triplicate (n = 9). chemical signaling among monocultures versus triple cell cultures of the same cell-types. The monocultures consistently produce different baseline expression, by either up- or down-regulating depending on the cell-type, of key signature biomarkers relative to the triple cell culture system. When comparing results measured from in vitro model systems to those collected from in vivo models, there are considerable differences. In vivo models have clearance mechanisms, innate and adaptive immune systems, and three-dimensionality. While in vitro systems do not have these features, they can recapitulate relevant aspects of in vivo models using specialized cell culture scaffolds and multiple human cell-types. Human cells can express cytokines and chemokines at a level concordant with humans if cell seeding density and cell-type ratios are purposefully applied and proper cell differentiation has occurred [60]. For this study, scanning electron microscopy proved to be useful in observing how cells interact with each other as well as qualifying individual cell-type differentiation and effects of seeding densities. SEM was able to validate Caco-2 enterocyte differentiation to Caco-2 M-cell phenotype [11].