π-SACS: pH Induced Self-Assembled Cell Sheets Without the Need for Modified Surfaces

The ability to form tissue-like constructs that have high cell density with proper cell-cell and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration. Newly emerging bioprinting methods sometimes lack the high cellular density needed to provide biophysical cues to orchestrate cellular behavior to recreate tissue architecture and function. Alternate methods using self-assembly can be used to create tissue-like constructs with high cellular density and well-defined microstructure in the form of spheroids, organoids, or cell sheets. Cell sheets have a particularly interesting architecture in the context of tissue regeneration and repair as they can be applied as patches to integrate with surrounding tissues. Until now, the preparation of these sheets has involved culturing on specialized substrates that can be triggered by temperature or phase change (hydrophobic to hydrophilic) to release cells growing on them and form sheets. Here a new technique is proposed that allows delamination of cells and secreted ECM and rapid self-assembly into a cell sheet using a simple pH trigger and without the need to use responsive surfaces or applying external stimuli such as electrical and magnetic fields, only with routine tissue culture plates. This technique can be used with cells that are capable of syncytialization and fusion such as skeletal muscle cells and placenta cells. Using C2C12 myoblast cells we show that the pH trigger induces a rapid delamination of the cells as a continuous layer that self-assembles into a thick dense sheet. The delamination process has little effect on cell viability and maturation and preserves the ECM components that allow sheets to adhere to each other within a short incubation time enabling formation of thicker constructs when multiple sheets are stacked (double- and quadruple-layer constructs are formed here). These thick grafts can be used for regeneration purposes or as in vitro models.