2.1 Materials

The choice of the material is highly important in the design process of organ-on-chip technologies. Biocompatible, non-toxic, optically clear for microscopic imaging are often preferred approaches. The stiffness of the material is decisive for cellular growth. One main challenge is to find the right balance between stiffer materials with more precise mechanical properties and softer materials (hydrogels) closer to physiological tissue properties, but with less opportunities to modulate mechanical stimulation.

Polymers show many advantages since they can be shaped and designed with several techniques (photolithography, multiphoton excitation leading to local degradation, electrospinning…). Polydimethylsiloxane (PDMS) is the most common polymer used to build microfluidic organ-on-chip (Figure 1a,h. Kim & Ingber, 2013), classified as elastomer for its elasticity, with a high stiffness (800 kPa ~ 10 MPa, [Seghir & Arscott, 2015]). In this context, a microengineered organ-on-chip incorporating a built-in perfusable vascular network has been described (Zhang, Lai, et al., 2018; Zhang & Radisic, 2017). This multi-material scaffolding (poly (octamethylene maleate [anhydride] citrate) and PDMS) named AngioChip is being cultivated with endothelial and parenchymal cells to model vascular systems (Figure 1d). Thanks to tunable mechanical properties and great biocompatibility, fabricated vascular networks demonstrate applications for human myocardium engineering.

Hydrogels are being investigated nowadays since they are highly biocompatible and many features like porosity, mechanical stiffness, or elasticity can be modulated (Ding et al., 2020). They are 3D polymers soft networks containing mostly water (Seliktar, 2012), with a lower but tunable stiffness (0.5 kPa ~ 100 kPa, [Zhao et al., 2019]) and can be classified into natural, synthetic and hybrid materials. Natural hydrogels exhibit closer to native tissues matrix but do not provide reproducible mechanical properties. Further considerations lead to finding the best choice between synthetic or hybrid hydrogels, guaranteeing a sufficient control on mechanical properties and a scaffold presenting interesting cell-binding sites. As an example, two hydrogel-based models mimicking intestinal crypts are shown Figure 1b (Nikolaev et al., 2020) and Figure 1c(Kim et al., 2019). Some advanced techniques like stereolithographic high-resolution printing have enabled the development of such devices achieving precise localization and patterning of cells. In light of their modular composition, hydrogels offer the possibility to create internal molecular gradients such as oxygen, which can be useful in particular for anaerobic microorganisms. In addition, hydrogels stiffness can be modulated in a reversible manner by crosslinking reactions following light stimulation (Rosales et al., 2017), thereby offering a remote spatio-temporal control useful to address mechanobiology issues.

2.2 Physical forces in microfluidic devices

Organ and tissue inside the body experience physical changes, constraints within their own microenvironment and depending on their functions. Indeed, a blood vessel or the urinary tract will experience strong shear forces due to not only pulsatile blood and urinary flow rate but also some stretching forces during diastolic and systolic cycles in cardiac tissues. Intuitively, same forces can be considered in respiratory tissues (lung, pulmonary alveoli) since air is cyclically renewed during respiration. Some tissues such as the intestine are subjected to slower flows but experience similarly a cyclic stretch during peristalsis-like motions.

Moreover, mechanical forces stimulate cell differentiation and morphological polarisation. In the gut, fluid shear stress induces for instance cell organisation into 3D crypt and villi-like structure (Figure 1g). It has been shown thanks to a kidney model that fluid shear stress not only results in a morphological polarisation triggering actin cytoskeleton reorganisation but also led to the expression of differentiated epithelial cell functions (Jang & Suh, 2010; Theobald et al., 2018). Therefore, it appears capital to reproduce mechanical forces in 3D existing models. One of the main advantages that offer organ-on-chip is that tissue grown on a porous membrane can experience multiple physical stresses at the same time (Bhatia & Ingber, 2014). Furthermore, microorganisms have been also reported to sense physical cues such as the shear stress, reinforcing the needs to recapitulate physical forces to study host-pathogen interactions (Mordue et al., 2021).

2.3 Representing specific 3D topology, interface and cellular richness

One of the main challenges of tissue engineering is to recapitulate the cellular and molecular composition of the organ. In that respect, stem cells such as induced-pluripotent stem cells (iPS) or even embryonic stem cells has been used (Wan et al., 2011). Many studies related to organ-on-chip have considered cell patterning as an efficient method to create precise cell environment with specific arrangement inside the chip. Surface treatments or even 3D printing guarantee precise patterns or complex hydrogels scaffold which can be eventually grown with cells. A recent study uses cell patterning to develop a hydrogel-based mini-gut model shaped with microcavities that recapitulate the 3D geometry and cell diversity (by loading organoids) of the native crypt and villi structure of the human gut (Figure 1b, (Nikolaev et al., 2020). Similarly, a collagen-based array of crypt-like invaginations could closely mimic the human intestinal epithelium monolayer with a gradient of proliferative to differentiated cells along crypts (Figure 1c), (Kim et al., 2019; Wang et al., 2018). These two devices enable the maintenance and spatial organisation of both stem and differentiated cells and hence represent good models to investigate gut regeneration.

A tissue exhibits specific interface and needs a precise cell organisation to be fully functional and to interact with the rest of the body. Human body displays several of them in many organs: lungs, intestine, vessels, bladder, skin, vagina are the most important. Pathogens enter the body through the airflow or through contaminated food and fluids. Organ-on-chips having two adjacent channels separated by a porous membrane offer many options in terms of cellular seeding and medium type (air, liquid or gas). Indeed, different kind of cells can be cultured on both sides of the porous membrane with their respective physiological media, allowing hence exchanges between several types of tissues/cells (Figure 1). Lung is an appropriate example of tissue interface presenting an air–liquid boundary with relevant alveolar barrier that has been recapitulated in several chips designed by Emulate, Alevolix or Epithelix (Figure 1a,e, Stucki et al., 2018). The intestinal lumen is another example of tissue liquid–liquid interface (Figure 1a–c). In addition, several devices allow at recapitulating several tissue-tissue interface as exemplified in Figure 1a,d,f.

3.1 Mechanical forces trigger pathogen virulence induction

In this part, we will briefly describe several studies reporting the induction of virulent genes by mechanical stimulation that were obtained with microfluidic devices (not truly organ-on-chip). Bacteria often experience mechanical forces when they encounter cell surfaces. They can be triggered by a fluid shear stress generated by a flow or be stimulated by the host surface stiffness and topology. It has been shown that bacteria could actually feel such signals and induce the upregulation or downregulation of their virulence factors.

Enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen that can colonise the gastrointestinal tract, was capable to adapt the expression of its virulence genes depending on the mechanical constraints of its host (Alsharif et al., 2015). More precisely, the locus of enterocyte effacement containing a range of virulence factors, notably the transcriptional activator GrlA, was shown highly expressed and localized into the cytoplasm in response to physiological relevant shear stress (Sirisaengtaksin et al., 2020).

Type IV pili are long and thin dynamic filaments based at the surface of bacteria and often involved in pathogenic processes. These surface appendages can polymerise and depolymerise from pilin molecules in a short period of time (Craig et al., 2019). Biomechanical considerations have revealed that type IV pili had an influence on not only how bacteria interact with host surfaces but also how they can be modulated by external mechanical stresses. They are actually considered by the literature as mechano-sensitive structure. The case of Neisseria meningitides, a pathogenic bacterium infecting the human nasopharynx is worth noting. When this pathogen reaches the blood–brain barrier and integrates the bloodstream, it can cause severe inflammation named meningitis. Several studies have shown that despite the strong mechanical forces generated by the blood flow, Neisseria succeed to reach the bloodstream, bind to the epithelial vessel structure and aggregate, confirming their striking abilities of adaptation (Dos Santos Souza et al., 2020). N. meningitides aggregate in microcolonies, very stable and flow-resistant which induced host cell surface reorganisation and protrusion formation enhancing the adhesion (Charles-Orszag et al., 2016). Interestingly, in vivo data showed that nesserial adhesion was mainly observed in capillaries, having heterogeneous flow, compared to normal-sized vessels and this would favour the initial adhesion of the bacterium (Mairey et al., 2006).

Biofilms are communities of bacteria held together with a specific extracellular matrix. The biofilm is created when a bacterium adheres to a surface. Interestingly, pathogens developing within a biofilm are phenotypically different form their free-living counterparts. It has been demonstrated that intracellular levels of cyclic-di-GMP participate to the initiation of Pseudomonas aeruginosa biofilms, such signals inducing a change in genes expression (Rodesney et al., 2017). In this context, the role of mechanical forces such as shear forces would trigger bacterial behaviour, enhancing the surface adhesion of P. aeruginosa, activating, via PilY1 and type IV pili, the cyclic-di-GMP pathway decisive for biofilm development (Rodesney et al., 2017).

The direct role of mechanical inputs of tissues on pathogenic gene expression has not yet been clearly investigated using organ-on-chip, but will surely be addressed in the future.

3.2 Organ-on-chip to model host-microbiota crosstalk

Microbial community is a major part of the cellular richness of some tissues, especially in the gut where the number of bacteria can reach ~10¹⁴ bacteria (Qin et al., 2010), (Turnbaugh et al., 2007), (Lozupone et al., 2012). The intestinal tract composition varies according to space and time. The interaction between microbiota and the organ participate to tissue homeostasis and function, but also turns out to be a strong protection against pathogens. Indeed, several protective mechanisms such as mucin secretion, or the triggering of bacteria sensors are controlled by certain entities of the microbiota (Ashida et al., 2012). Classical devices exhibit several drawbacks to co-cultivate eukaryotic cell monolayers with microorganisms. In fact, they fail to recapitulate the microbial diversity, the long-term microbiota interaction and the complexity of the underlying tissue structure. In this context, several studies have highlighted that organ-on-chip devices can sustain complex microbial communities growing in symbiosis with human intestinal cells, relevant to host-microorganism interaction investigations.

The lack of fluid-flow in classical devices was thought to be the main missing cue leading to bacterial overgrowth. However, peristaltic motion is another important parameter, since cyclic stretching (peristalsis-like deformations) greatly reduces bacterial overgrowth in a gut-on-chip already maintained under luminal flow (Kim et al., 2016). This suggests that modulation of mechanical deformation is decisive to prevent bacterial overgrowth (Bures et al., 2010), and that gut-on-chip are relatable models to co-culture commensal microorganisms over an extended period of time. As an example, viable bacterial microcolonies from a commercial probiotic formulation containing several strains (Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, L. bulgaricus and Streptococcus thermophiles) have been successfully maintained for several days (Kim et al., 2016).

In addition, hypoxia gradient in gut-on-chip greatly increase the microbial diversity since it increases the growth of facultative and obligate anaerobic bacteria like Bacteroides fragilis (Jalili-Firoozinezhad et al., 2019) or B. adolescentis and L. rhamnosus, (Kim et al., 2019). In particular, co-cultivating in anaerobic conditions L. rhamnosus with human colon cells in HuMiX device was able to modify the transcriptional, metabolic and immunological host responses as reported previously by in vivo studies (Shah et al., 2016).

Altogether, these studies demonstrate the huge benefit of using gut-on-chip under mechanical stimulation and oxygen gradient to provide a microbiota-intestine-on-chip relevant model.

3.3 Pathogenic mechanisms deciphered by organ-on-chip devices

Over the last few years, host-pathogen interactions have been increasingly investigated using organ-on-chip technology, some non-exhaustive examples are described below.