ABSTRACT
During fetal skin development, over thirty distinct cell types emerge to form a solid barrier capable of immune
surveillance and sensory detection. Due to its complexity, the skin has been challenging to reproduce in
cultures. Maintenance of excised skin has only been possible for short durations. Yet, a longer-term skin
culture system could greatly benefit efforts to model congenital diseases, investigate the mechanisms of
cancer initiation, or mimic the site of infection, inflammation, and wounds. A critical obstacle to progress has
been our inability to identify culture conditions that satisfy the metabolic needs of cells found in every skin
subdomain, including skin appendages, such as hair follicles and sweat glands, and accessory structures,
such as vasculature and nerves. Incorporating diverse immune cell populations has evaded previous
engineering attempts. Recently, we invented a novel culture system that uses human pluripotent cells (hPSCs)
to generate full-thickness skin with many of the cellular components of normal skin, including epidermis,
dermis, hair follicles, and sensory nerves—collectively known as skin organoids. Although an improvement
over previous skin models in terms of completeness, the resulting skin arises as a massive floating tissue that
is challenging to monitor over time in culture and is devoid of immune cells. Here, we will build on preliminary
data showing that skin organoids can reformat onto easily imaged microfluidic chips that we have maintained
for over 100 days without apparent tissue degradation. For Goal 1, we will optimize a manufacturing process
for creating hair-bearing skin organoids-on-a-chip by iteratively evaluating chip geometry, matrix composition,
mechanical properties, and chemical treatments supplied to the developing tissue. We will use quantitative
imaging and single-cell and spatial transcriptomics to assess the quality of our chip designs. For Goal 2, we will
evaluate a novel chip design that better integrates neuro-vascular structures into the skin organoid chips. For
Goal 3, we will build on exciting preliminary data to integrate myeloid and lymphoid lineage cells into
developing skin organoid chips. We will define the proper timing and medium requirements for immune cell
seeding and assess the fidelity of immuno-competent skin organoid chips through direct comparison to fetal
histological specimens. We will test the system by simulating immune reactions due to hPSC donor
incompatibility and bacterial infection. These experiments will yield chips with stereotyped neural inputs and
vascular networks. Our collaborative team has strong bioengineering, material science, neuroscience, and
immunology expertise and is uniquely suited to execute the project goals. Our research strategy considers how
others could adopt our methodology, and we include plans for beta-testing tissue chip production in less
equipped laboratories. We anticipate that our skin organoid tissue chips will provide researchers with a
powerful new tool to watch and learn as human skin develops under normal and diseased conditions.