Temporary Nanoencapsulation of Mammalian Cells With Silk Ionomers Using Electrostatic Layer-By-Layer Deposition...

Nanoencapsulation of mammalian cells is a novel approach offering a protective microenvironment for cells, enabling their sustained viability and function in hostile or non-physiological conditions. This protection is particularly valuable in applications, such as cell therapy, tissue engineering, and immunotherapy, where maintaining cell functionality is critical for therapeutic success. The encapsulation process involves surrounding cells with a biomaterial barrier that acts as an artificial cell wall, shielding them from immune system attacks, toxic substances, and mechanical stress, while still allowing the exchange of essential nutrients, gases, and waste; thus, preserving cell viability and function during biochemical processing and applications. Utilizing THP-1 immune cells and human intestinal organoids (HIOs) as examples, this article provides a comprehensive guide for encapsulating single cells and stem cell-derived large cell aggregates with silk ionomers derived from the silkworm Bombyx mori, applying an electrostatic layer-by-layer deposition technique. We provide a detailed protocol for preparing silk fibroin (SF) from silk cocoons, synthesizing silk ionomers using the prepared SF, and encapsulating immune cells and organoids through electrostatic layer-by-layer deposition. This article also outlines the characterization methods, such as confocal microscopy, scanning electron microscopy (SEM), quartz crystal microbalance with dissipation monitoring (QCM-D) for coating thickness, and atomic force microscopy (AFM) for stiffness measurement. Guidelines for assessing cellular function post-encapsulation are also provided, enabling researchers to build on these methods and advance silk-based encapsulation in biomedical applications. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of aminated and carboxylated silk ionomers Basic Protocol 2: THP-1 cell and human intestinal organoid culture Basic Protocol 3: Nanoencapsulation of THP-1 cells and organoids through electrostatic layer-by-layer deposition Basic Protocol 4: Imaging: Scanning electron microscopy and confocal microscopy Basic Protocol 5: Quartz crystal microbalance with dissipation monitoring analysis: Determining coating thickness Basic Protocol 6: Atomic force microscopy: Measuring cell stiffness Basic Protocol 7: Cell viability and propagation post-encapsulation.