Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Abstract

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues [1][2][3][4][5][6] . The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a highaspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads ( Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor ( Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions ( Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial celltype specific properties.The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type 1,[7][8][9][10][11][12][13] . Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability ( Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/d...

Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae

“…To construct the human 3-D endometrial epithelial cell (EEC) model, we used the rotating wall vessel (RWV) bioreactor technology (28) and the previously established endometrial epithelial cell line HEC-1A. The HEC-1A cell line was isolated in 1968 by H. Kuramoto from a patient with stage IA endometrial cancer (29).…”

Optimal 3D Culture of Primary Articular Chondrocytes for Use in the Rotating Wall Vessel Bioreactor

“…During rotation of the vessel containing media and cells seeded on appropriate 3D carriers, the cells are exposed to a constant rotation, producing vector-averaged forces comparable with that of near-earth free fall orbit (9). The RWV system has been used by several investigators to study many different cell types including epithelial cells (19), Schwann cells (26), cells of the intervertebral disc (27), and trophoblast cells (28), as well as for tissue engineering studies (2) specifically for liver cells (8), bone cells (16,23), and elastic cartilage (25). To use the RWV bioreactor, cells must be in suspension.…”

<i>In Vitro</i> Evaluation of Anticancer Drugs with Kinetic and Static Alternating Cell Culture System

“…To mimic these two physical forms in which cancer cells exist in the body, an ideal bioreactor must allow cells to grow well in both static and kinetic states, and these two states should ideally be controlled and regulated in a dynamic fashion by automated control systems. Unfortunately, current commercially-available 3D bioreactors can only maintain cells in either a kinetic state (K bioreactor) [1] [2] [5] [6] [7] or a static state (S bioreactor) [1] [2] [3] [4]. When cells are cultured in a K bioreactor [5] [6] [7], they are kept in motion by agitation or similar mechanism, accumulating at fixed points within the bioreactor when agitation ceases.…”

“…Unfortunately, current commercially-available 3D bioreactors can only maintain cells in either a kinetic state (K bioreactor) [1] [2] [5] [6] [7] or a static state (S bioreactor) [1] [2] [3] [4]. When cells are cultured in a K bioreactor [5] [6] [7], they are kept in motion by agitation or similar mechanism, accumulating at fixed points within the bioreactor when agitation ceases. Cell accumulation adversely affects the growth of cells in culture as cells in the center of the accumulation experience hypoxic conditions and lower concentrations of anti-cancer drugs in the medium.…”