Fig. 1: An in vitro 3D growth matrix with tunable viscoelastic properties.

a The physical properties of the mucosal layer are diverse and are altered by factors such as a change in diet, infection, inflammation, and enzymatic degradation of fibres by microbes. b Different gut-derived microbial strains from red flour beetles, were imaged in brightfield (pseudo-coloured for enhanced contrast). Schematic of the red flour beetle is created in BioRender (M. S. (2024) BioRender.com/y37r627). c By packing highly swollen polymeric hydrogel granules beyond jamming concentrations, we design an in vitro 3D growth medium, which provides a granular and internally porous microenvironment for bacterial growth under confinement. d–f The physical properties of the microgel growth medium are highly tunable based on the mass percentage of hydrogel granules relative to liquid LB (wt%/volume). Here, we show both soft and low confinement (0.50%), as well as stiff and high confinement (0.85%) matrices, the viscoelastic properties of which approximately match mucosal samples from natural sources²⁴. Rheological measurements shown here either apply d a unidirectional shear at different rates to measure the viscosity, or, e a small amplitude (1%) of oscillatory strain at different frequencies to measure the shear moduli of the 3D growth media. The storage modulus (G’) is a measure of the elastic solid-like nature, while the loss modulus (G”) signifies the viscous properties of the material. f The porosity of the medium, shown here as a complementary cumulative distribution function (1-CDF) of all the inter-particle pore spaces, determines the degree of confinement, and can be tuned by altering the packing fraction of the hydrogel granules.