Abstract
Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.
Highlights
Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge
Fibrin can be remodelled and digested by cells over time providing a system to potentially escape compression associated with cell division,[17] while its stability can be increased by covalent modification.[18]
In this study we present a versatile platform for integrating live imaging with individual cell retrieval
Summary
Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining live imaging with cell retrieval for functional and molecular analysis could greatly help us better understand dynamic cellular process in controlled conditions For this purpose, we have designed a highly flexible cell encapsulation system and microfluidic platform, and validated it using embryonic stem (ES) cell differentiation as a model system. Paper heterogeneity.[11,12,13] at any given point in time during exit from the ES cell state, cells can be in different stages of the differentiation process and respond very differently to differentiation cues.[14] This asynchrony has confounded our understanding of how ES cells change state and initiate multilineage differentiation With these challenges in mind, we set out to design a platform that would enable: (i) live imaging a dynamic and asynchronous process using reporter systems; (ii) isolating individual events for downstream molecular and functional characterisation at specific timepoints. The platform had to fulfil two requirements: cells had to be cultured in contact with extracellular matrix (ECM), without attaching to the microfluidic device directly; and channels had to be controlled individually so that specific cells or cell aggregates of interest could be extracted independently
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