Scaffold-free 3D culture systems for stem cell-based tissue regeneration

Abstract

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.

FIG. 1.

Cell sheet fabrication and harvesting systems. (a) Temperature-responsive system: cells cultured on the N-isopropylacrylamide (PIPAAm) -coated surface at 37 °C. By lowering the temperature to 20 °C, PIPAAm became hydrophilic and the cell sheets were detached. Similarly, cells were cultured on hydrophobic methylcellulose (MC)-coated surface at 37 °C. As the temperature declined below the lower critical solution temperature (LCST), MC turned hydrophilic and the cell sheets were detached. (b) Chemically defined culture medium: ascorbic acid was supplemented in the medium for facilitating extracellular matrix (ECM) production within cell sheets. (c) Photo-responsive system: TiO₂ nanodot (TN) films were applied to facilitate photo-responsive cell sheet detachment. Upon exposure to 365-nm ultraviolet (UV) light, the water contact angle significantly decreased, resulting in cell detachment. (d) Magnetic-responsive system: employing the magnetite cationic liposomes (MCLs) and magnetic force, assisted by low-attachment surfaces to minimize cellular adhesion. (e) pH-responsive system: electrochemically induced pH lowering at the interface altered the protein microstructure and disrupted cell–ECM interactions for detachment. (f) Ion-responsive system: incorporating functional copolymers coating, with their hydrophobicity determined by the divinylbenzene (DVB)/4-vinylpyridine (4VP) ratio, to facilitate cell sheet detachment through changing the concentration of cations. (g) Manipulation/stacking/transfer: techniques using pipettes, membranes and plungers for layering and transferring cell sheets precisely, facilitating the construction of multi-layered tissue structures.

FIG. 2.

Cell spheroid culture and fabrication techniques. (a) Force aggregation: utilizing pellet culture and centrifugation to promote cell aggregation. (b) Hanging drop: drops containing cells are suspended to facilitate spheroid formation. (c) Low attachment microenvironment: creating spheroids using ultra-low attachment surfaces or agarose-coated wells to prevent cell adhesion. (d) Dynamic culture: incorporating rotating wall vessels and rocking culture systems to enhance nutrient and gas exchange. (e) Magnetic levitation: employing magnets and magnetic nanoparticles to levitate cells, promoting spheroid formation without physical support. (f) Microfluidic: leveraging microfluidic channels to control the cell culture environment precisely. (g) Polymer scaffolds: using micropatterned thermally responsive cell culture platforms (TRCP) or chitosan surface to provide structural support for spheroids.