## 4 Discussion
As previously demonstrated by [PERSON] et al., utilizing SEM to observe loss of microvilli and cell invagination is sufficient to identify M-cell differentiated cells [28]. Along with cytokines and chemokines, other immunoregulatory factors, such as reactive oxygen species (ROS), are important indicators of cell stress and potential intestinal pathologies (e.g., irritable bowel disease). Importantly, ROS is strongly associated with tumorigenesis via increased survivability, genetic instability and damage, and loss of tumor-suppressor gene functionality [61]. Therefore, quantifying basal rates of ROS production prior to incorporating any exposure is paramount to fully understanding in vitro intestinal models and their ability to recognize perturbations in ROS levels [62]. We demonstrate that seeding density affects the ROS production. Lower densities showed elevated levels of ROS when compared to higher densities. This result indicates that on a per-cell basis, cells showed higher quantities of ROS. In other words, lower seeding densities induce high cell stress. HT29-MTX cells express higher basal levels of IL-6 at early timepoints compared to Caco-2 or Raji B cells. Previous work has shown HT29 cells have increased sensitivity to local stimulation, which could account for the marked increase in early timepoint secretion of IL-6 [63]. Activation, maturation, survival, and proliferation of B cells requires binding to the B cell receptor (BCR) [64]. As such, Raji B cells are inactive in the described model system translating to an insignificant amount of secreted cytokines. If Raji B cells are activated, then a significant amount of cytokines would be readily produced [65; 66]. As B cells showed no increase in cytokine expression in this study, the triple cell culture proved to be a suitable model as it considers the expressions of multiple cell-types simultaneously. Cellular communication is related to increased formation of tight junctions which in turn increases resistance over time. Studies using Caco-2 and HT29-MTX co-cultures, without incorporating Raji B cells, are unable to show the significance of M-cell differentiation from Caco-2 [10; 67; 68]. M-cells allow for better representation of intestinal barrier integrity while simultaneously incorporating a crucial cell-type associated with absorption and antigen retrieval and subsequent contact with immune cells. It is the interaction among a triple cell culture that can serve as a representative model for in vivo systems. These results are in concordance with [PERSON] et al. which showed that the monocultures and triple cell culture increased in confluency over time; however, the triple cell culture increased in resistance at a faster rate when compared to the individual monocultures [69]. Further research in TEER endpoint analyses and subsequent data interpretation is needed to fully exploit the utility of this functional parameter [28; 34]. [PERSON] et al. showed that the co-culture of Caco-2 and HT29-MTX offers a suitable option for testing drug permeability properties over traditional in vivo testing regimes [34]. Co-cultures are more robust in terms of barrier integrity as compared to simple monocultures of either cell-type cultured by itself. The authors state that while the co-culture model can give a preliminary qualitative indication of permeability, it falls short of quantifyingthe amount or rate of material transfer. Because the model lacks immune cells, probing for pro-inflammatory biomarkers is not a suitable auxiliary to measuring barrier integrity. As no mammalian system consists of a single cell-type, it is becoming increasing evident that to properly mimic the complexity of in vivo cellular interactions, in vitro models must include multiple cell types and proper cellular architecture in order to better translate from in vitro to in vivo outcomes. Models for the varying compartments of the lungs [70; 71], brain [72], liver [73], bone [74], and heart [75] have already been investigated and determined to be better mimics of in vivo responses.