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Pompeo Mixon
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We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.
The ability to direct cell behavior and tissue formation in vitro and in vivo is a central design feature for the development of a range of biomaterials, cell biotechnologies and tissue engineering based therapies for improving human health1,2,3,4,5. Traditional molecular biology methods have significantly advanced cell function understanding and provided a range of tools for manipulating cell behavior. Recently, cell surface engineering strategies that use bottom-up chemical approaches have gained increasing attention due to their ability to affect cell surface interactions but not require genomic manipulations6,7,8. Several chemical strategies have been used to tailor cell surfaces including metabolite analogues, cationic polymer adhesion and polymersome attachment9,10,11,12. An alternate chemical approach has used the addition of synthetic lipids delivered directly to cells in culture in order to add new functions to cell membranes13,14. New methods that rewire cell surfaces with the capability to control cell interconnectivity in space and time would allow for further exploration of a range of fundamental cell behavior studies and provide new ways to install imaging probes, advance cell based biotechnologies and accelerate regenerative medicine and tissue engineering based therapies.
Herein, we develop a general strategy that delivers photo-active and bio-orthogonal chemistry via liposome fusion to cell surfaces for subsequent in-situ tailoring for on-demand microtissue assembly and disassembly. We demonstrate this photo-active cell surface engineering system by conjugating and tracking cell surface ligands and applying a photo-cleavable click (oxime) type ligation between cells for the spatial and temporal control of multilayer tissue assembly and disassembly for generating multicellular tissues as well as manipulating stem cell differentiation. This methodology allows for real-time manipulation of tissue dynamics and may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.
To generate tissue assemblies containing multiple cell types for a range of fundamental cell behavior, cell imaging and tissue engineering applications, we used a bottom-up synthetic approach to rewire cell surfaces. Cell surface tailoring was achieved by a straightforward new liposome fusion method to incorporate complementary bio-orthogonal molecules capable of an intercellular click chemical reaction upon physical cell-cell contact13,14. The external cell surface click conjugation between cells proceeds at physiological conditions in the presence of serum and allows for stable cell interconnectivity. The limited suite of bio-orthogonal click reactions is increasingly becoming important tools in chemical biology and cell biological research15,16. To access spatial and temporal control of cell-cell interactions, the synthetic ligation tether between cells was engineered to contain a photochemical cleavage site17. Remote controlled tissue disassembly proceeds by a programmed photo-initiated cleavage of the intercellular ligation tether (Fig. 1 top). The key features are the delivery of synthetic chemical groups to cell surfaces (via liposome fusion)13, the intercellular oxime click ligation bond16 (bio-orthogonal) and a photo-cleavage site contained within the oxyamine lipid tether (Fig. 1 bottom)17.
(top) Two or more different cell types are engineered via liposome fusion to present complementary and bio-orthogonal molecules on their cell surfaces. Upon interaction, a stable and covalent click (oxime) type reaction occurs and tissue like assemblies are formed. After a defined period of time, tissue disassembly can be remotely controlled by photo-cleavage of the intercellular ligation tether. (bottom) A schematic showing the interfacial molecular lock and cleave sequence for conjugating and releasing cell associations. The lipid lock molecule contains both a photocleavable group and an aminooxy group for bio-orthogonal oxime ligation to ketone presenting cells. Disassociation of cell assemblies via photo-cleavage occurs upon UV illumination.
(A) A photolabile oxyamine molecule is mixed with POPC and positively charged DOTAP to generate photo-active liposomes that are delivered to cell surfaces via liposome fusion. (B) Dodecanone is mixed with POPC and DOTAP molecules to generate ketone presenting liposomes that are delivered to cell surfaces via liposome fusion. (C) O-dodecyloxyamine is mixed with POPC and DOTAP molecules to generate oxyamine presenting liposomes that are delivered to cell surfaces via liposome fusion. (D) No cell assembly occurred when control Jurkat cells were mixed with ketone presenting Jurkat cells (6). (E) Programmed microtissue assembly when Jurkat-ONH2 (10) cells were mixed with jurkat-ketone cells (6). No disassembly occurred upon uv light illumination. (F) Cell Assembly proceeded upon mixing of jurkat-photo-ONH2 (5) with jurkat-ketone (6) cells. Microtissue disassembly to single cells proceeded upon cleavage of the intercellular linkage through uv light illumination.
(A) Fibroblasts presenting the photo-active oxyamine chemoselectively adhere to materials presenting aldehyde groups (B, C) through an interfacial oxime linkage. The rewired cells could then be selectively released from the material upon UV illumination (D).
Two different types of cells (fibroblasts and hMSCs), tailored with photo-oxyamine (A) and ketone (B) respectively, were peeled from tissue culture plates and assembled via the oxime ligation (C, D) to form a stable multi-tissue co-culture system. (E) Phase contrast microscopy of the tissue showing regions of multilayer and monolayer. (F) Monolayer of mixed RFP and GFP cells with no ketone or oxyamine chemistry. (G) Multilayer of mixed RFP and GFP cells via oxime ligation. (H) Oriented RFP and GFP cells via sequential addition of cells presenting ketone and oxyamine groups. The cell layers only adhered if the complementary chemistry was present on their cell surfaces.
We used flow cytometry to quantify and characterize the amount of photo-oxyamine lipid delivered to cells via liposome fusion, for subsequent photo-oxime ligation induced microtissue assembly (Fig. 5). The photo-oxyamine was designed to have 3 components that are essential for spatial and temporal control of cell interconnectivity (Fig. 5A). A lipid hydrophobic component to insert into membranes, a photo-cleavable center and a bio-orthogonal oxyamine group for cell surface ligation of a range of ligands or other cells presenting ketone groups.
As further characterization of cell surface presenting photo-oxyamine groups, we developed a novel mass spectrometry method (Fig. 5F). Lipid transfected cells were added to SAM surfaces presenting aldehyde groups, which allow cells to attach to the surface via the interfacial oxime ligation16,21. Cells were then removed from the surface (lysed) with vigorous agitation through a stream of PBS solution and H2O. Due to the covalent nature of the interfacial oxime bond between the cells and SAM surface, the lipid was essentially pulled out of the membrane and remained conjugated to the substrate. Because the substrate is conductive, MALDI mass spectrometry could be performed directly on the substrate to characterize the interfacial reaction. Fig. 5G shows MALDI analysis and clearly shows the presence of the oxime molecule on the substrate. This strategy may be used to characterize a range of lipid presenting molecules on cell surfaces and as a new pull-down method of proteins if the lipid molecule spans the plasma membrane and covalently associates with cytoplasmic proteins.
We have developed a novel liposome fusion system to deliver bio-orthogonal photo-active lipid and ketone-lipid like molecules to cell membranes. Upon mixing these cells in different formats, co-culture spheroids and multilayers could be generated due to an intercellular oxime (click) ligation. Furthermore, we also showed the feasibility of producing macroscopic-scale tissue and achieving multiple tissue-tissue assembly, which could lead to a series of tissue engineering, especially organ printing technologies. Since the ligation tether contains a photo-cleavable group, remote control of disassembly could be achieved upon UV light illumination. We demonstrated this system in several cell lines to generate switchable co-culture spheroids and multi-layers. Flow cytometry and mass spectrometry analysis quantified and characterized the interfacial cell surface reaction. The ability to engineer cell surfaces with a straightforward and inexpensive liposome fusion strategy will find wide use in fundamental studies of membrane biophysics, paracrine signaling and adapt to generate new biomaterials and as a biotechnology platform for screening complex cell behaviors in tissue microarrays. Several other bio-orthogonal chemical ligation strategies including diels-alder, huisgen, oxime, hydrazone, thiol-ene, etc. may be used to tailor cell surfaces with nanoparticles, redox groups and a range of other molecules for targeted delivery and as cell tracking and imaging beacons. The spatial and temporal control of cell interactions between multiple different cell types will lead to new studies of dynamic cellular communication. Furthermore, the combination of bioreactor technologies with intercellular ligation methods may provide new ways to generate large-scale complex multi-cell type tissues. When combined with traditional polymer scaffolds, molds or printing technologies, a range of complex 3D tissues and organs may be possible for an array of biomedical diagnostic and transplantation applications28,29,30.
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