Spheroid culture is a biological research method growing cells in three-dimensional (3D) clusters. This technique moves beyond traditional flat, two-dimensional cell cultures, allowing scientists to create more complex and physiologically relevant models of tissues outside the body. It represents an advancement in understanding cellular behavior and tissue function.
Formation and Characteristics of Spheroids
Spheroids are created by preventing cells from adhering to a flat surface, encouraging them to aggregate into spherical structures. One common method is the hanging-drop technique, where cells are suspended in small liquid droplets that hang from a surface, allowing gravity and surface tension to pull the cells together. Another approach uses low-adhesion plates, which have specially coated surfaces that inhibit cell attachment, prompting the cells to form aggregates in suspension. These methods facilitate cell-to-cell adhesion, which promotes the formation of cellular clusters.
Once formed, spheroids exhibit a distinct internal organization, mimicking aspects of natural tissues. They develop different zones based on nutrient and oxygen availability, as substances diffuse from the outer surface inward. The outermost layer contains rapidly proliferating cells, while a middle layer consists of quiescent cells with reduced metabolic activity. In larger spheroids, a necrotic core can form in the center due to limited access to oxygen and nutrients and the accumulation of waste products. Maintaining spheroid diameters between 100 and 150 micrometers is preferred to ensure uniform cell viability throughout the structure.
Advantages Over Traditional Cell Culture
Three-dimensional spheroid models offer advantages over conventional two-dimensional (2D) cell cultures, which involve cells growing as a flat monolayer. Spheroids accurately replicate the complex cell-to-cell interactions and the presence of an extracellular matrix found in living tissues. In a 2D environment, cells flatten and lose their natural morphology, altering their behavior and responses. Spheroids, however, maintain a natural morphology and function, which includes preserving normal signaling pathways.
The 3D structure allows for the development of physiological gradients, such as varying concentrations of oxygen, nutrients, and signaling molecules, which are characteristic of real tissues. These gradients influence cell behavior and gene expression in ways that cannot be replicated in uniform 2D cultures. Consequently, spheroids provide a representative microenvironment for studying cellular processes, making them predictive of how cells and tissues behave within a living organism. This mimicry enhances the relevance of in vitro studies for understanding biological phenomena.
Applications in Medical Research
Spheroid cultures have become versatile tools in medical research, offering insights that are difficult to obtain with simpler models. They are used in cancer research, where multicellular tumor spheroids (MCTS) replicate the characteristics of small, avascular tumors. These tumor spheroids mimic the cellular heterogeneity and the hypoxic core found in solid tumors, making them suitable for studying cancer cell behavior, resistance mechanisms to therapy, and the tumor microenvironment. Researchers can use patient-derived tumor spheroids to test different medications, potentially guiding individualized cancer treatments.
Spheroids are also employed in drug discovery and toxicology studies. Their ability to represent human tissue structures allows for accurate testing of new drug effectiveness and potential toxicity. By providing a physiologically relevant model, spheroids improve the correlation between in vitro test results and in vivo outcomes, potentially reducing the need for animal testing in early drug development phases. This application helps in screening compounds and identifying promising drug candidates more efficiently.
Beyond cancer and drug screening, spheroids contribute to regenerative medicine. Spheroids derived from stem cells are used to study tissue development and differentiation processes. They serve as building blocks for tissue engineering, offering a way to create more complex tissue constructs for research or therapeutic purposes. Their ability to maintain stem cell characteristics and guide differentiation makes them valuable for developing new regenerative therapies.
Spheroids and Organoids
While both spheroids and organoids are three-dimensional cell culture models, they differ in their complexity and organizational capabilities. Spheroids are simpler aggregates of one or more cell types that self-assemble through cell-to-cell adhesion. They offer a scaffold-free system where cells form a cluster. Spheroids are used to study fundamental cellular interactions or mimic basic tissue properties, such as those found in tumors.
Organoids, on the other hand, are more complex 3D structures that not only aggregate but also self-organize to resemble the architecture and function of a specific organ. They are derived from stem cells or progenitor cells and differentiate into multiple specialized cell types, recapitulating aspects of the native tissue’s complexity. Organoids require a supportive matrix, such as Matrigel, and specific growth factors to facilitate their self-organization and differentiation into miniature versions of organs like mini-intestines or mini-brains. This structural and functional mimicry distinguishes organoids from spheroids.