Alveolar epithelium protects macrophages from quorum sensing-induced cytotoxicity in a three-dimensional co-culture model.

The quorum sensing signal N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12) HSL), produced by Pseudomonas aeruginosa, exerts cytotoxic effects in macrophages in vitro, which is believed to affect host innate immunity in vivo. However, the medical significance of this finding to pulmonary disease remains unclear since the multicellular complexity of the lung was not considered in the assessment of macrophage responses to 3-oxo-C(12) HSL. We developed a novel three-dimensional co-culture model of alveolar epithelium and macrophages using the rotating wall vessel (RWV) bioreactor, by adding undifferentiated monocytes to RWV-derived alveolar epithelium. Our three-dimensional model expressed important architectural/phenotypic hallmarks of the parental tissue, as evidenced by highly differentiated epithelium, spontaneous differentiation of monocytes to functional macrophage-like cells, localization of these cells on the alveolar surface and a macrophage-to-epithelial cell ratio relevant to the in vivo situation. Co-cultivation of macrophages with alveolar epithelium counteracted 3-oxo-C(12) HSL-induced cytotoxicity via removal of quorum sensing molecules by alveolar cells. Furthermore, 3-oxo-C(12) HSL induced the intercellular adhesion molecule ICAM-1 in both alveolar epithelium and macrophages. These data stress the importance of multicellular organotypic models to integrate the role of different cell types in overall lung homeostasis and disease development in response to external factors.

Evaluating the effect of spaceflight on the host-pathogen interaction between human intestinal epithelial cells and Salmonella Typhimurium.

Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host-pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Recellularization of decellularized lung scaffolds is enhanced by dynamic suspension culture.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

The generation of 3-D tissue models based on hyaluronan hydrogel-coated microcarriers within a rotating wall vessel bioreactor.

With the increasing necessity for functional tissue- and organ equivalents in the clinic, the optimization of techniques for the in vitro generation of organotypic structures that closely resemble the native tissue is of paramount importance. The engineering of a variety of highly differentiated tissues has been achieved using the rotating wall vessel (RWV) bioreactor technology, which is an optimized suspension culture allowing cells to grow in three-dimensions (3-D). However, certain cell types require the use of scaffolds, such as collagen-coated microcarrier beads, for optimal growth and differentiation in the RWV. Removal of the 3-D structures from the microcarriers involves enzymatic treatment, which disrupts the delicate 3-D architecture and makes it inapplicable for potential implantation. Therefore, we designed a microcarrier bead coated with a synthetic extracellular matrix (ECM) composed of a disulfide-crosslinked hyaluronan and gelatin hydrogel for 3-D tissue engineering, that allows for enzyme-free cell detachment under mild reductive conditions (i.e. by a thiol-disulfide exchange reaction). The ECM-coated beads (ECB) served as scaffold to culture human intestinal epithelial cells (Int-407) in the RWV, which formed viable multi-layered cell aggregates and expressed epithelial differentiation markers. The cell aggregates remained viable following dissociation from the microcarriers, and could be returned to the RWV bioreactor for further culturing into bead-free tissue assemblies. The developed ECBs thus offer the potential to generate scaffold-free 3-D tissue assemblies, which could further be explored for tissue replacement and remodeling.

Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions.

Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.