Abstract
Three-dimensional (3D) cell culture systems have promising applications compared with conventional two-dimensional cell culture systems. Herein, we report a facile method for the formation of 3D spheroids using novel thermo-reversible polysaccharide-based hydrogels. A series of thermo-reversible hydrogels consisting of N-acyl glycol chitosans (NAGCs) are synthesized through a simple N-acylation reaction, and the degree of acylation is finely tuned to obtain adequate thermo-reversible properties and gel stability. Among the NAGCs, N-hexanoyl glycol chitosan is the most thermo-sensitive and is highly effective for forming multi-cellular spheroids when used to coat the surfaces of cell culture dishes. Cell spheroids are effectively formed at various cell concentrations, and their spheroid shape and cellular functions are well maintained for longer times. The hydrogel culture system is also useful for co-cultures that mimic a biological microenvironment. Our thermo-reversible hydrogels may offer a convenient method for the development of in vitro 3D cell culture systems to provide enhanced performance in tissue regeneration, organ-on-chips, drug screening research and other biomedical applications. Modifying the crustacean-derived molecule chitosan produces thermosensitive hydrogels for realistic, spherical cell growth studies. Three-dimensional (3D) cell environments are better platforms for exploring cell structure and function than traditional Petri dishes, but typically require complex ‘hanging drop’ substrates or micromoulds. Researchers from the Korea Institute of Toxicology and Chungnam National University have developed a simpler 3D cell culture using a water-filled polysaccharide that can transform into a gel on demand thanks to the thermo-responsive property. In experiments, cells such as cardiomyocytes spontaneously aggregated to form spheroids after only 24 hours immersion in the chitosan-based hydrogel. Harvesting was performed using temperature-reduction techniques suitable for large-scale processes. Cytotoxicity tests revealed that these cultures were safe and could be used to co-culture different cells to realize better biomimicking. We developed glycol chitosan-based thermo-reversible hydrogels that can provide a facile, convenient and reproducible method for the formation of 3D cell spheroids. The spontaneous formation of spheroids on HGC-coated plates was completed within 1 day. Furthermore, this system can be useful for co-culturing heterotypic cells to form spheroids that mimic biological systems.
Highlights
Three-dimensional (3D) spheroid culture is an important process for understanding biological mechanisms and obtaining transplantable cells for therapy
N-acyl glycol chitosans (NAGCs) were synthesized with various carboxylic anhydrides in a simple one-step reaction procedure under mild conditions at room temperature (Figure 1a)
The degree of acylation (DA) values for the NAGCs were in the range of 73.1–91.6% for N-acetyl glycol chitosans, 48.3–74.5% for N-propionyl glycol chitosans, 36.3–61.4% for N-butyryl glycol chitosans, 26.7–48.0% for N-valeryl glycol chitosans and 19–36.5% for hexanoyl glycol chitosan (HGC)
Summary
Three-dimensional (3D) spheroid culture is an important process for understanding biological mechanisms and obtaining transplantable cells for therapy. Non-cell adherent polymers have been utilized for 3D spheroid formation and culture.[7,8,9] In this case, cells may not adhere to the substrate but could form spheroids on the non-adherent substrate because cell–cell interactions are favored over cell–substrate interactions In this regard, chitosan, a deacetylated chitin, has been widely studied as a non-adherent polymer to promote the formation of self-assembled 3D cellular spheroids.[10,11,12] chitosan is soluble only in an acidic environment, which may be potentially cytotoxic to cells. Chitosan, a deacetylated chitin, has been widely studied as a non-adherent polymer to promote the formation of self-assembled 3D cellular spheroids.[10,11,12] chitosan is soluble only in an acidic environment, which may be potentially cytotoxic to cells This method requires an extensive washing process to remove cytotoxic conditions before the addition of cells for spheroid formation. The non-cell adherent property of chitosan itself seems insufficient for successful 3D spheroid culture
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