Hierarchical encapsulation of bacteria in functional hydrogel beads for inter- and intra- species communication

To sequester prokaryotic cells in a biofilm-like niche, the creation of a pertinent and reliable microenvironment that reflects the heterogeneous nature of biological systems is vital for sustenance. Design of a microenvironment that is conducive for growth and survival of organisms, should account for factors such as mass transport, porosity, stability, elasticity, size, functionality, and biochemical characteristics of the organisms in the confined architecture. In this work we present an artificial long-term confinement model fabricated by natural alginate hydrogels that are structurally stable and can host organisms for over 10 days in physiologically relevant conditions. A unique feature of the confinement platform is the development of stratified habitats wherein bacterial cells can be entrapped in the core as well as in the shell layers, wherein the thickness and the number of shell layers are tunable at fabrication. We show that the hydrogel microenvironment in the beads can host complex subpopulations of organisms similar to that in a biofilm. Dynamic interaction of bacterial colonies encapsulated in different beads or within the core and stratified layers of single beads was demonstrated to show intra- species communication. Inter- species communication between probiotic bacteria and human colorectal carcinoma cells was also demonstrated to highlight a possible bidirectional communication between the organisms in the beads and the environment. Statement of significance: Bacteria confinement in a natural soft hydrogel structure has always been a challenge due to the collapse of hydrogel architectures. Alternative methods have been attempted to encapsulate microorganisms by employing various processes to avoid/minimize rupturing of hydrogel structures. However, most of the past approaches have been unfavorable in balancing cell proliferation and functionality upon confinement. Our study addresses the fundamental gap in knowledge necessary to create favorable and complex 3D biofilm mimics utilizing natural hydrogel for microbial colonization for long-term studies. Our approach represents a cornerstone in the development of 3D functional architectures not only to advance studies in microbial communication, host-microbe interaction but also to address basic and fundamental questions in biology.