posted on 2021-03-22, 22:13authored byNingwei Li, Feiyu Yang, Subiksha Parthasarathy, Sarah St. Pierre, Kelly Hong, Narciso Pavon, ChangHui Pak, Yubing Sun
Recent
advances in human pluripotent stem cells (hPSCs)-derived in
vitro models open a new avenue for studying early stage
human development. While current approaches leverage the self-organizing
capability of hPSCs, it remains unclear whether extrinsic morphogen
gradients are sufficient to pattern neuroectoderm tissues in vitro. While microfluidics or hydrogel-based approaches
to generate chemical gradients are well-established, these systems
either require continuous pumping or encapsulating cells in gels,
making it difficult for adaptation in standard biology laboratories
and downstream analysis. In this work, we report a new device design
that leverages localized passive diffusion, or LPaD for short, to
generate a stable chemical gradient in an open environment. As LPaD
is operated simply by media changing, common issues for microfluidic
systems such as leakage, bubble formation, and contamination can be
avoided. The device contains a slit carved in a film filled with solid
gelatin and connected to a static aqueous morphogen reservoir. Concentration
gradients generated by the device were visualized via DAPI fluorescent intensity and were found to be stable for up to
168 h. Using this device, we successfully induced cellular response
of Madin–Darby canine kidney (MDCK) cells to the concentration
gradient of a small-molecule drug, cytochalasin D. Furthermore, we
efficiently patterned the dorsal–ventral axis of hPSC-derived
forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal
gradient generated by the LPaD devices. Together, LPaD devices are
powerful tools to control the local chemical microenvironment for
engineering organotypic structures in vitro.