The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells

Cell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells. We review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (~30 nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment. As confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique. SLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity. •Cell resealing technique enables intracellular delivery of membrane-impermeable molecules by using pore-forming toxins.•Streptolysin O-type resealing is optimal for creating disease model cells, especially lifestyle-related diseases.•Listeriolysin O, which forms smaller pores in the plasma membranes, can be specifically used for screening mid-sized biological drugs and their intracellular functions.•Semi-intact cell system is suitable for investigating the biochemical requirements for the specific processes, in which the concerted biological reactions occur in cells.