Probing the Luminal Compartment of 3D Organoids via Particle Tracking Microrheology

Background

Organoids have significantly improved our ability to study tissue development and disease progression in vitro and provide a viable alternative to animal models for pre-clinical drug testing [2–4]. Gastrointestinal organoids are widely used to represent the stomach, small intestine, and colon in studies of gut health and disease [4–8]. Notably, such organoids are known to produce the protective mucus that lines the gastrointestinal tract [9–11]. Mucus is a viscoelastic material that plays an important role as the body’s first barrier against the outside world [12].

Human gastric organoids (HGOs) generally consist of the apical epithelium facing inward, separated from the outer environment [9,13]. HGOs contain both types of mucus-producing cells—MUC5AC-secreting pit mucus cells and MUC6-secreting mucus neck cells—that are present in the healthy human stomach and secrete mucus into the organoid lumen, as shown in multiple studies [5,14,15]. The topologically closed structure of HGOs makes the lumen challenging to access [12]. Investigating the suitability of HGO models for recreating in vivo gastric conditions—such as the protective mucus barrier—requires thorough characterization of their luminal composition. While fixing and sectioning organoids using histological methods can reveal a cross-section of the lumen, this process disrupts the natural state and spatial distribution of luminal contents [15]. Studying the luminal microenvironment within an organoid and the biophysical properties of its contents provides the most accurate reflection of its natural spatial complexity and minimizes experimental artifacts [16]. We and others have used micromanipulator-controlled microinjection to access the lumen of viable gastric organoids for delivering bacteria or fluorescent tracers [6,11,15,17,18]. Additionally, we have used microelectrodes to measure intraluminal oxygen content for bacterial inoculation [6] and pH [19], upon which mucus rheology is dependent. This non-destructive technique is powerful and has enabled real-time observations to be made, including that the organoid lumen maintains microaerophilic conditions and a near-neutral pH. In this study, fluorescent microspheres were microinjected into gastric organoids for passive particle tracking microrheology analysis, enabling the rheological characterization of the mucus within the organoids.

Previous work using microparticle tracking and oscillatory shear rheology has established the micro- and bulk rheological properties of mucus isolated from the stomach and other organs, as well as of several purified mucins from the lung, salivary, gastric, and reproductive systems [20–23]. These studies concluded that gastric mucins and mucus exhibit both viscous and elastic properties, with certain types having the ability to form elastic gels at low pH [24]. Many soft materials exhibit a viscoelastic response to applied shear forces that depends on how liquid-like (viscous) or solid-like (elastic) the material behaves [25]. More specifically, viscosity refers to a material’s ability to dissipate energy and resist flow, while elasticity refers to a material’s ability to store energy and return to its original shape following deformation [26,27]. The extent of each can depend upon the various forces acting on the material. Understanding the rheological behavior of gastric mucus may have physiological relevance to questions such as the response of mucus to shear forces associated with digestion [28] and the transport of nutrients and microorganisms across the mucus barrier [29].

In this protocol, gastric organoid luminal mucus is characterized by visualization of microinjected fluorescent microspheres into the lumen for particle tracking microrheology (PTM) using microscopy. We employ analytical methods to interrogate the thermally driven, Brownian motion of the injected microspheres to extract information about the mechanical properties of the luminal material [30]. We confirm that the mucus secreted by the organoids is viscoelastic, and that the viscoelasticity varies temporally, suggesting that gastric organoids can serve as a physiologically relevant model system for investigating the influence of other factors on mucus structure and dynamics [20,27,31,32]. Note that, throughout this protocol, we utilize the term microspheres to refer to the fluorescent polystyrene probes (e.g., 1.0 μm Fluoresbrite) injected into the gastric organoid lumen. However, it should be noted that in the broader microrheology literature, these probes can be referred to interchangeably as beads, particles, or microparticles [33,34].