Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation (pdf)

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Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

ARTICLE https://doi.org/10.1038/s41467-022-31898-w OPEN Building programmable multicompartment artiﬁcial cells incorporating remotely activated protein channels using microﬂuidics and acoustic levitation 1234567890():,; Jin Li 1 ✉, William D. Jamieson 2, Pantelitsa Dimitriou 1, Wen Xu 3, Paul Rohde 4, Boris Martinac Matthew Baker 6, Bruce W. Drinkwater 7 ✉, Oliver K. Castell 2 ✉ & David A. Barrow1 ✉ 4,5, Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artiﬁcial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artiﬁcial cells, using microﬂuidics and acoustic levitation. Such levitated models provide free-standing, dynamic, deﬁnable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of largeconductance. The assembly of droplet networks is three-dimensionally patterned with ﬂuidic input conﬁgurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconﬁgure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artiﬁcial cell by the application of acoustic and magnetic ﬁelds to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artiﬁcial cells as life-like materials. 1 School of Engineering, Cardiff University, The Parade, Cardiff CF24 3AA, UK. 2 School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Ave, Cardiff CF10 3NB, UK. 3 Cardiff Business School, Cardiff University, Aberconway Building, Colum Dr, Cardiff CF10 3EU, UK. 4 Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool St, Darlinhurst, NSW 2010, Australia. 5 School of Clinical Medicine, UNSW, Sydney, NSW 2052, Australia. 6 School of Biotechnology and Biomolecular Science, UNSW, Sydney, NSW 2052, Australia. 7 Department of Mechanical Engineering, University of Bristol, University Walk, Bristol BS8 1TR, UK. ✉email: ; ; ; NATURE COMMUNICATIONS | (2022)13:4125 | https://doi.org/10.1038/s41467-022-31898-w | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31898-w hrough evolution, eukaryotic cells have exploited intracellular compartmentalisation to increase the efﬁciency and range of functional complexity in cellular metabolism1. There is wide interest in harnessing such principles in the creation of life-like synthetic materials. The bottom-up construction of artiﬁcial cells aims to harness and imitate some of these key cellular functions, with de novo structures2,3. Protocells mimic possible primitive cells, and have been routinely fabricated by forming small, singularly compartmentalised droplets or vesicles with relatively simple infrastructure4–6, usually built through the macromolecular assembly of lipids or other amphiphiles7,8. Such basic models can encapsulate different reagents, representing minimal ‘cellular’ systems for cell-free studies, including those of membrane properties9, chemically mediated communication10–12, and the manipulation of genetic information13. State-of-art protocell materials can interact with living cells14 for potential biotechnological applications, such as immunogenicity enhancement15, organoid formation16 or blood vessel vasodilation17. To fabricate increasingly complex artiﬁcial cells, a key objective is to develop functionalities that are underpinned by intracellular compartmentalised architectures. Such structures can be formed by the encapsulation of lipid-bounded aqueous droplets and biomolecule complexes, within a host (envelope) droplet18,19, where each compartment may contain different biochemical species. Recent work has demonstrated that organelle-like components can work as functional units to process molecular signals20,21, regulate sequential reactions22–24, and may be used for energy harvesting25–28 within artiﬁcial cells. Furthermore, compartmentalisation has been harnessed in similar ways in the packing and patterning of individual protocells to construct tissue-like materials, incorporating protein channels29, DNA sequences30, and functional hydrogels31. These works indicate that structural complexity can display a range of emergent properties deﬁned by the contents and connectivity of constituent components in such soft, biomimetic materials. However, few efforts have focused on the permutation of compartments within artiﬁcial cells, and the subsequent processing and functionality of these architectures. This is, in part, because high-order emulsiﬁcation processes for the creation of multi-compartmental structures with controllable (bio) chemical reagent distribution, remain a signiﬁcant challenge. Most work to date has relied largely on the manual juxtaposition, or 3D printing, of multiple droplets to create tissue-like materials32–34. Droplet microﬂuidics provides the ability to control emulsion formation and has become an invaluable tool in the fabrication of vesicles and protocells35–37. Meanwhile, acoustic manipulation is a contactless, and non-invasive tool38 that has been applied to protocell formation and patterning in microﬂuidic environments39. However, to date, the full range of opportunities for artiﬁcial cell construction and control provided by the combination of these techniques has yet to be fully realised. Current technological bottlenecks in complex emulsion processing have limited the architectural complexity and functionality development of multicompartment artiﬁcial cell models. Here, we report the development of a droplet laboratory platform for the creation, manipulation, control, and measurement of artiﬁcial cells with distributed cores (ACDC droplet), using precision droplet microﬂuidics and acoustic levitation (Fig. 1a-1, a-2, Fig. S1). Membrane proteins can be incorporated in the levitated ACDC droplets, and their functions are remotely controlled in situ by the acoustic levitator. The application of programmable microﬂuidics and acoustics, with the incorporation of responsive bio-elements, represents a possible route to affording increased control over the compartmental organisation, connectivity, and communication, alongside opportunities for spatial and temporal control of protein f (...truncated)