Non-cell adhesive hexanoyl glycol chitosan hydrogels for stable and efficient formation of 3D cell spheroids with tunable size and density
Graphical abstract
Schematic illustration of 3D cell spheroid formation using non-cell-adhesive hydrogel-coated dishes.
Introduction
Over the past decade, three-dimensional (3D) culture systems that allow cells to grow and interact with their surroundings have attracted much attention for various biomedical applications. This has been because they may provide a more physiologically similar in vivo environment than two-dimensional (2D) culture systems and yield better predictive information for in vitro animal studies [1], [2]. Conventional 2D cell culture systems are still considered to be a standard method for analyzing physiological phenomena because of their simplicity, reproducibility, and cost-effectiveness, rather than large-scale experiments with animals [3], [4], [5]. Nevertheless, the demand for an efficient in vitro 3D culture system that is more reliable and controllable has been on the rise to mimic the growth, organization, and differentiation of cells [6], [7], [8]. Spheroid formation is an effective method for multicellular 3D culture systems owing to its ease of handling, reproducibility, and similarity to physiological conditions [6], [9], [10]. Several culture systems allow cells to self-assemble to form spheroids in circumstances where cell-cell interactions dominate cell-substrate interactions [10]. The 3D spheroids resemble real tissues with regard to structural and functional properties, show enhanced viability, and are less apoptotic during maintenance for long culture periods [11], [12].
Ultra-low attachment (ULA) surfaces are one of the most popular methods for 3D cell spheroid formation. In general, ULA systems often use cell culture dishes coated with non-cell adhesive biomaterials to inhibit the attachment of cells or spheroids and facilitate the cell-cell interaction for spheroid formation [13], [14]. Several non-cell adhesive biomaterials, such as chitosan, have been observed to be effective and widely used in the formation and culture of 3D spheroids [15]. Recently, a new class of non-cell adhesive biomaterials, N-acyl glycol chitosans, which were synthesized from a series of N-acylation reactions of glycol chitosan (GC) [16]. They demonstrated excellent spheroid-forming performance as well as thermo-reversible sol-gel transition properties. In particular, N-hexanoyl glycol chitosan (HGC) is the most effective for the formation of 3D cell spheroids. The HGC could be conveniently introduced onto the surface of cell culture dishes by a simple wet coating process without the use of any organic solvents or additional processes, owing to its thermosensitive sol-gel transition behavior, providing a facile method for the efficient formation of 3D spheroids. Despite its excellent ULA properties, the HGC hydrogel as a non-cell adhesive coating material has several limitations with physical stability and mechanical properties, especially for long-term spheroid culture. The thin HGC hydrogel coating layers on dish surfaces are vulnerable to dissolution or mass loss during typical cell culture processes that often accompany temperature changes for microscopic observation or medium exchange.
In this study, chemically crosslinked hydrogels were prepared by photocrosslinking of methacrylated HGCs (M-HGCs), and their spheroid-forming abilities were evaluated for long-term 3D cell culture. The chemically crosslinked HGC hydrogel coating was introduced on culture dishes using photocrosslinkable M-HGCs to overcome the inherent weakness of the HGC-based system for stable and efficient 3D cell spheroid formation. The non-adhesive properties and spheroid-forming abilities of the M-HGC-coated dishes were investigated and compared to those of the HGC-coated dishes together with a commercial ULA product that is known to be convenient, simple and easy to maintain for 3D culture. Furthermore, we report for the first time that the cell size and density of spheroids can be customized by manipulating the chemical composition and structure of the HGC-based hydrogels. There has be no report so far that typical ULA dishes can control the size and density of spheroids. Our M-HGC-coated dish system that can produce diverse cell spheroids with customized sizes and densities could be useful for various biomedical applications, such as tissue engineering, organ-on-a-chip, and drug screening areas.
Section snippets
Materials
GC, with a degree of polymerization ≥200, was purchased from Wako Pure Chemical Industries, Ltd. (Japan); hexanoic anhydride (97%) and glycidyl methacrylate were purchased from Sigma-Aldrich (India) and Sigma-Aldrich (St. Louis, MO, USA); and 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, Irgacure 2959 (I2959), was supplied by BASF (Switzerland). Distilled water and methanol (Samchun Chemical, Korea) were used as the solvent, and acetone (Samchun Chemical, Korea) was used during
Synthesis and characterization of HGC and M-HGC
In this study, HGC was synthesized by the N-hexanoylation reaction of GC according to a previously reported one-step reaction procedure [17]. The synthesized HGC was further reacted with glycidyl methacrylate to produce photocrosslinkable M-HGCs as shown in Fig. 1.
In the first step of the reaction, the hexanoyl groups were introduced to GC by the reaction of hexanoic anhydride with the amine groups of the GC via amide bond formation. In the second step, the methacrylation reaction of HGC was
Conclusion
In this study, photocrosslinkable thermogelling M-HGCs were prepared by the sequential reactions of N-hexanoylation and N-methacrylation of GC and evaluated as new coating materials for 3D cell culture systems. The M-HGCs maintained thermo-sensitive sol-gel transition behaviors similar to those of HGC. The photocrosslinked M-HGC hydrogels exhibited enhanced physical stability and mechanical properties compared to the HGC hydrogel itself. The photocrosslinked M-HGCs were coated on a petri dish
CRediT authorship contribution statement
Bo Seul Jang: Methodology, Data curation, Visualization, Writing – original draft. Kyoung Hwan Park: Methodology, Software, Formal analysis. Eun Yeong Suh: Investigation, Data curation, Writing – original draft. Byoung-Seok Lee: Conceptualization, Methodology, Investigation, Writing – review & editing. Sun-Woong Kang: Conceptualization, Methodology, Writing – review & editing, Funding acquisition. Kang Moo Huh: Conceptualization, Project administration, Supervision, Writing – review & editing,
Declaration of competing interest
The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by Basic Science Research Program (NRF-2020R1A2C2100794) and Bio&Medical Technology Development Program (NRF-2019M3A9H1103331) of the National Research Foundation of Korea (NRF) funded by the Korean government.
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These authors contributed equally to this work.