Presentation Information
[P03-429]Dual-photoresponsive substrates enabling image-based single-cell recovery of nonadherent mammalian cells
○Ryogo Nakashima1, Xueyang Li2, Satoshi Yamaguchi1 (1. The University of Osaka (Japan), 2. The University of Tokyo (Japan))
Keywords:
Single-cell array,Photoresponsive cell patterning,Photoclickable materials,Cell sorting,Mammalian cells
[Purpose]
Single-cell arrays are essential tools for visualizing cellular heterogeneity and linking cellular phenotypes observed by imaging to intracellular molecular systems. This is achieved through molecular analysis of cells retrieved after imaging. However, most conventional single-cell array techniques have limited in applicability due to the narrow microstructures required for trapping single cells. To overcome this limitation, we developed photoresponsive surfaces that enable the simple formation of single-cell arrays without the use of microstructures [1-4]. Using these surfaces, we achieved single-cell analysis of dynamic morphological changes [2], as well as cell-cell communication without spatial constraints [3,4]. Unlike microstructure-based single-cell arrays, however, cells of interest could not be retrieved from the substrate after observation due to strong interactions between the cell surface and the cell-trapping substrate. To address these challenges, this study presents a dual-photoresponsive surface that enables single-cell photopatterning without microstructures and the selective, light-induced release of individual cells.
[Method]
We used a material containing a photoactivatable precursor of dibenzocyclooctyne (pDBCO) to coat the surface [3]. UV irradiation activates the pDBCO moiety to generate DBCO, which subsequently reacts with the azide groups of the cell-trapping molecule containing the photocleavable moiety. Upon cell seeding, cells become anchored at the sites exposed to light. After observation for identification of the cells of interest on the array, a second UV light is applied to the selected cells to cleave the photocleavable moiety, resulting in their selective detachment from the substrate surface, followed by cell recovery using a glass capillary.
[Results]
We demonstrated light-induced patterning of mammalian immune cells on the present dual-photoresponsive surface via binding to patterned cell-anchoring materials. This process enabled the formation of microstructure-free single-cell arrays on a flat glass substrate. Furthermore, we selectively released photopatterned individual cells via localized light irradiation and subsequently retrieved them at the single-cell level using cell pickers. These results represent the first demonstration of selective cell isolation from photoclickable surfaces following cell patterning and phenotypic observation.
[Consideration]
[Conclusion]
We reported a dual-photoresponsive substrate based on a photoclickable material coating and photocleavable cell anchoring molecules. This surface is expected to be applicable to a wide range of single-cell analyses because it allows cell-anchoring molecules, designed cell patterns, and downstream molecular analyses to be tailored as needed.
[1] M. Tan, et al., Lab Chip, 17, 1933 (2017); [2] S. Yamahira, et al., Macromol. Biosci., 14, 1670 (2014); [3] S. Yamahira, et al., J. Am. Chem. Soc., 144, 13154 (2022); [4] T. Kosaka, et al., J. Am. Chem. Soc., 144, 17980 (2022).
Single-cell arrays are essential tools for visualizing cellular heterogeneity and linking cellular phenotypes observed by imaging to intracellular molecular systems. This is achieved through molecular analysis of cells retrieved after imaging. However, most conventional single-cell array techniques have limited in applicability due to the narrow microstructures required for trapping single cells. To overcome this limitation, we developed photoresponsive surfaces that enable the simple formation of single-cell arrays without the use of microstructures [1-4]. Using these surfaces, we achieved single-cell analysis of dynamic morphological changes [2], as well as cell-cell communication without spatial constraints [3,4]. Unlike microstructure-based single-cell arrays, however, cells of interest could not be retrieved from the substrate after observation due to strong interactions between the cell surface and the cell-trapping substrate. To address these challenges, this study presents a dual-photoresponsive surface that enables single-cell photopatterning without microstructures and the selective, light-induced release of individual cells.
[Method]
We used a material containing a photoactivatable precursor of dibenzocyclooctyne (pDBCO) to coat the surface [3]. UV irradiation activates the pDBCO moiety to generate DBCO, which subsequently reacts with the azide groups of the cell-trapping molecule containing the photocleavable moiety. Upon cell seeding, cells become anchored at the sites exposed to light. After observation for identification of the cells of interest on the array, a second UV light is applied to the selected cells to cleave the photocleavable moiety, resulting in their selective detachment from the substrate surface, followed by cell recovery using a glass capillary.
[Results]
We demonstrated light-induced patterning of mammalian immune cells on the present dual-photoresponsive surface via binding to patterned cell-anchoring materials. This process enabled the formation of microstructure-free single-cell arrays on a flat glass substrate. Furthermore, we selectively released photopatterned individual cells via localized light irradiation and subsequently retrieved them at the single-cell level using cell pickers. These results represent the first demonstration of selective cell isolation from photoclickable surfaces following cell patterning and phenotypic observation.
[Consideration]
[Conclusion]
We reported a dual-photoresponsive substrate based on a photoclickable material coating and photocleavable cell anchoring molecules. This surface is expected to be applicable to a wide range of single-cell analyses because it allows cell-anchoring molecules, designed cell patterns, and downstream molecular analyses to be tailored as needed.
[1] M. Tan, et al., Lab Chip, 17, 1933 (2017); [2] S. Yamahira, et al., Macromol. Biosci., 14, 1670 (2014); [3] S. Yamahira, et al., J. Am. Chem. Soc., 144, 13154 (2022); [4] T. Kosaka, et al., J. Am. Chem. Soc., 144, 17980 (2022).
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