Technological tools and strategies for culturing human gut microbiota in engineered in vitro models

Abstract

The gut microbiota directly impacts the pathophysiology of different human body districts. Consequently, microbiota investigation is an hot topic of research and its in vitro culture has gained extreme interest in different fields. However, the high sensitivity of microbiota to external stimuli, such as sampling procedure, and the physicochemical complexity of the gut environment make its in vitro culture a challenging task. New engineered microfluidic gut-on-a-chip devices have the potential to model some important features of the intestinal structure, but they are usually unable to sustain culture of microbiota over an extended period of time. The integration of gut-on-a-chip devices with bioreactors for continuous bacterial culture would lead to fast advances in the study of microbiota-host crosstalk. In this review, we summarize the main technologies for the continuous culture of microbiota as upstream systems to be coupled with microfluidic devices to study bacteria-host cells communication. The engineering of integrated microfluidic platforms, capable of sustaining both anaerobic and aerobic cultures, would be the starting point to unveil complex biological phenomena proper of the microbiota-host crosstalks, paving to way to multiple research and technological applications.

Keywords: anaerobiosis; bioreactors; gut-brain axis; microfluidic systems; organ-on-a-chip.

Figure 1

The gastrointestinal tract is a complex system both from a physicochemical and biological point of view. Moving from the upper to the lower GI tract, there are different parameters that vary accordingly to the site, such as oxygen partial pressure, pH and intensity of the dynamic stimuli, as well as an increased number and diversity of bacteria shaping the microbiota. Regardless from the tract considered, the distribution of bacterial strains is influenced by the oxygen gradient. Figure made with biorender (https://biorender.com) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

(a) The typical growth curves of batch and fed‐batch cultures are compared to the continuous culture performed by a bioreactor, such as a chemostat, where the presence of a medium inflow and outflow allow for a potentially infinite stationary phase. (b) The different approaches for the continuous culture in a bioreactor with reference to the chosen method to control D. In particular, the increasing or decreasing of the dilution rate corresponds to the accelerostat or decelerostat condition (red and blu line respectively). Differently, a constant D defines the chemostat approach (in the figure, two different D are represented by the green and orange line). Figure made with biorender [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Schematic representation of the microbiota culture methods with increasing level of complexity, including batch (a), fed‐batch (b), and continuous culture (d). The bioreactors for continuous culture of microbiota were classified accordingly to the number of vessels: single‐, double‐ and triple‐stage bioreactors (d, e and f, respectively). Figure made with biorender (https://biorender.com) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

The different approaches used to develop in vitro AOI in a crescendo of complexity. The gradient of oxygen was statically engineered by the presence of O₂ permeable/impermeable environments (a) or agar‐based gels (b). Differently, the aerobic/anaerobic medium flows allowed modeling AOI in dynamic gut‐on‐a‐chip systems (c). Figure made with biorender (https://biorender.com) [Color figure can be viewed at wileyonlinelibrary.com]