Influence of Structure-Property Relationships of Polymeric Biomaterials for Engineering Multicellular Spheroids

Abstract

Two-dimensional cell culture systems lack the ability to replicate the complex, three-dimensional (3D) architecture and cellular microenvironments found in vivo. Multicellular spheroids (MCSs) present a promising alternative, with the ability to mimic native cell-cell and cell-matrix interactions and provide 3D architectures similar to in vivo conditions. These factors are critical for various biomedical applications, including cancer research, tissue engineering, and drug discovery and development. Polymeric materials such as hydrogels, solid scaffolds, and ultra-low attachment surfaces serve as versatile platforms for 3D cell culture, offering tailored biochemical and mechanical cues to support cellular organization. This review article focuses on the structure-property relationships of polymeric biomaterials that influence MCS formation, growth, and functionality. Specifically, we highlight their physicochemical properties and their influence on spheroid formation using key natural polymers, including collagen, hyaluronic acid, chitosan, and synthetic polymers like poly(lactic-co-glycolic acid) and poly(N-isopropylacrylamide) as examples. Despite recent advances, several challenges persist, including spheroid loss during media changes, limited viability or function in long-term cultures, and difficulties in scaling for high-throughput applications. Importantly, the development of MCS platforms also supports the 3R principle (Replacement, Reduction, and Refinement) by offering ethical and physiologically relevant alternatives to animal testing. This review emphasizes the need for innovative biomaterials and methodologies to overcome these limitations, ultimately advancing the utility of MCSs in biomedical research.

Keywords: hydrogel; multicellular spheroids; polymer; scaffold; ultra-low attachment surfaces.

Figure 2

Effect of hydrogel/scaffold composition on mechanical properties, morphology, and adipogenic differentiation. (A) Schematic illustrating how increasing matrix density/crosslinking compacts the network (porosity ↓), elevates stiffness (↑), and limits spheroid size (↓). (B) Scaffold composition and impact on adipogenesis of human adipose-derived stem cells (hASCs). (a) Scaffold compositions detailing variations in collagen concentration, elastin-like polypeptide (ELP) addition, and EDC/NHS crosslinking. (b) Mechanical characterization of hydrated scaffolds showing an increased elastic modulus and compressive strength with a higher collagen content, ELP incorporation, and chemical crosslinking. Error bars represent 95% confidence intervals (* p ≤ 0.05 vs. 2C; # p ≤ 0.05 vs. non-crosslinked scaffold of same formulation). (c) Adipogenic differentiation of hASCs encapsulated in scaffolds visualized by Oil Red O staining for lipid accumulation on days 5 and 11. Scale bar: 200 μm. (C) Mechanically tunable polyethylene glycol-diacrylate (PEGDA) hydrogels support time-dependent Huh7.5 spheroid formation. (a) All the Huh7.5 cell-laden hydrogels exhibited viscoelastic properties. (b) Stiffness mimicking normal (soft) and cirrhotic (stiff) liver environments modulated Huh7.5 mechanotransduction and spheroid growth. Increased PEGDA concentration significantly elevated the complex modulus (one-way ANOVA, p < 0.001), with no significant difference between PF10% and P12.5% (p > 0.05). (c) Spheroid size increased over 20 days in a stiffness- and time-dependent manner. One-way ANOVA showed significant differences in spheroid diameters across conditions from day 6 onward (p < 0.05 to p < 0.001), with PF10% and P12.5% differing significantly at days 6, 13, and 20 (p < 0.01). Reprinted from open-access publications [7,29].

Figure 3

Comparison of spheroid culture methods. (A) Schematic representation of spheroid culture techniques, including ultra-low attachment (ULA) plate and microwell-based aggregation formats. (B) Brightfield images showing cell morphology and spheroid formation at day 0 (5 h), day 3, and day 7 for a (a) 2D monolayer culture, (b) microwell-based spheroid culture, and (c) spontaneous aggregation in ULA plates. Scale bar = 1 mm. (C) Effect of initial seeding density (2500–20,000 cells/well) and culture duration (24–72 h) on spheroid formation in HCT-116 cells using ULA plates. Spheroid diameter increases with both cell density and culture time. Scale bar = 100 μm. Reprinted from open-access publications [76,77].

Figure 1

Models to recapitulate the underlying mechanisms of cellular behavior. Using adipocytes as an example, the figure illustrates three experimental models used to study cellular processes: in vivo animal models, providing a more complex, systemic context for examining cellular interactions in the living organism; 2D in vitro cell culture, representing a simplified cellular environment; and 3D in vitro cell culture, serving as an intermediate approach that bridges the gap between 2D culture and in vivo systems by mimicking the spatial and mechanical properties of tissues. These models collectively offer insights into the mechanisms of cellular behavior, providing a more comprehensive understanding of biological processes.

Figure 4

Schematic representation of biomaterial properties, processing parameters, and cellular responses in tissue engineering. Material characteristics (stiffness, porosity, hydrophobicity, surface chemistry) and processing factors (polymer structure, polymerization, temperature) influence spheroid morphology, differentiation, and longevity, ultimately shaping cell–material interactions and tissue regeneration outcomes.