Active empowerment of cell sheets: culture-stage intervention strategies driving functional enhancement and...

Cell sheet technology (CST) represents a pivotal tool in regenerative medicine, enabling the fabrication of cell layers that retain an intact extracellular matrix (ECM) and intercellular connections. These cell layers can substitute damaged or missing tissues to restore their structural and functional integrity. However, two major challenges restrict the broader applicability of CST: the intrinsic limitations of cell sheet performance and insufficient adaptability within complex regenerative environments. Currently, intervention strategies predominantly concentrate on the harvesting and application phases, such as developing environmentally responsive hydrogels to optimize harvesting efficiency or simply integrating scaffolds to improve adaptability in complex regenerative contexts. Nevertheless, these strategies frequently exhibit inherent shortcomings of being retrospective and non-integrative. Recent studies indicate that proactive intervention during the cell sheet cultivation phase, a stage characterized by high plasticity, may be pivotal for overcoming these limitations. This review systematically evaluates two classes of cultivation-phase intervention strategies: firstly, endogenous reinforcement achieved by incorporating bioactive molecules or micro/nanomaterials that can be internalized by the cell sheets; secondly, co-cultivation of cell sheets with exogenous minerals or polymer scaffolds to form functionally integrated composite systems that address limitations posed by complex regenerative scenarios. We critically assess the design principles, implementation approaches, and both in vitro and in vivo outcomes of each strategy, discussing persistent challenges and possible improvements. By clarifying the physiological characteristics of cell sheets during cultivation and exploring effective intervention methodologies, this review seeks to resolve the two principal challenges facing CST and pave the way for fully realizing its regenerative potential. The insights presented here will support the development of more effective strategies to facilitate the widespread adoption of CST in regenerative medicine.