Characterization of a Human In Vitro Intestinal Model for the Hazard Assessment of Nanomaterials Used in Cancer Immunotherapy  
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1 Institute of Biomedical Studies, Baylor University, One Bear Place #97266, Waco, TX 76798-7266, USA; [EMAIL_ADDRESS]  
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3 Viro Advisors, LLC, Boston, MA 02130-4323, USA; [EMAIL_ADDRESS] (J.D.E. ); [EMAIL_ADDRESS] (J.A.S.) 4  
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3 Viro Advisors, LLC, Boston, MA 02130-4323, USA; [EMAIL_ADDRESS] (J.D.E. ); [EMAIL_ADDRESS] (J.A.S.) 4  
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1 Institute of Biomedical Studies, Baylor University, One Bear Place #97266, Waco, TX 76798-7266, USA; [EMAIL_ADDRESS]  
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in assessing the toxicity of therapeutics is in vitro human intestinal models. To avoid the pitfalls associated with interspecies variations, complex human cell cultures have received attention from many different local, national, and international agencies as a viable alternative-to-animal strategies that can provide valuable insights in infectious disease, immunology, drug discovery, and toxicology studies [5; 6]. The most recognized in vitro cell-type used for intestinal permeability studies is enterocytes (e.g., Caco-2 cell line). The structure and function of this cell is similar to enterocytes of the human gut, as the cells were harvested from a human and subsequently cultured in vitro. However, the use of enterocytes as a monolayer in in vitro studies is limited as Caco-2 cells are derived from the colon rather than the small intestine. Cells that reside in the small intestine have fewer tight junctions to allow for absorption of nutrients and water while cells in the colon have more tight junctions that result in lowered absorption [7; 8; 9; 10]. However, Caco-2 cells can be differentiated into a phenotype with elevated absorption abilities (i.e., Caco-2 to M-cells) via the addition of B-cells (such as Raji B) which allows for considerations of the non-specific absorption of digested materials in the lumen [11]. The morphological, physiological, and biochemical differences among cells lining the colon versus small intestine should be taken into consideration when interpreting results from monocultures of precursor cells (Caco-2) to differentiated cultures (those that include M-cells) in order to develop a more sophisticated model. Cell model choice strongly depends on the biological function of interest because the structure-function relationship that exists at the molecular level is dependent upon biochemical processes inherent to specific cell-types. For example, phagocytic cells, such as M-cells, are responsible for active transport mechanisms, where they maintain homeostasis through the trafficking of antigens from the lumen to immune cells creating either a tolerogenic or inflammatory response [12; 13; 14]. Disruptions in homeostasis can lead to exacerbated immune responses and subsequent disease. Because immune cells, such as T-cells or B-cells, are responsible for most of the interferon and interleukin cytokine production, incorporating M-cells into intestinal co-culture models is critically important to translate from in vitro responses to potential in vivo outcomes [15; 16; 17; 18]. The move towards co-cultured intestinal models allows for the integration of critical cell-types at the luminal interface. Because the intestinal tract consists of a compounded network of cells, communications, and involuntary biokinetics, the utilization of triple cell cultures which mimic the intestinal cellular architecture provides a better toxicological and pharmacological model than single monolayers. Within the human intestinal tract, there are approximately six different types of mammalian cells as well as the mucosa and microbiome [19; 20; 21]. Therefore, an in vitro intestinal model should include mucus-producing cells. Most commonly, goblet cells (e.g., HT29-MTX cell line) serve in this role [22; 23; 24].