Spheroids are three-dimensional (3D) cellular aggregates utilized as in vitro models to study biological processes with greater accuracy than traditional laboratory methods. These spherical formations occur when cells self-assemble and adhere, creating a miniature structure that closely mimics the architecture and function of living tissues. By providing a more natural environment for cells to interact, spheroids have become powerful tools for advancing the understanding of human health and disease. Their significance stems from their ability to bridge the gap between simplistic in vitro studies and complex in vivo systems, leading to more predictive research outcomes.
The Shift to Three Dimensions
For decades, biological research relied heavily on two-dimensional (2D) cell culture, where cells grow as a flat monolayer adhered to a plastic surface. This conventional method, while simple and cost-effective, fails to replicate the complex environment of the human body. Cells cultured in this manner exhibit an unnatural, flattened morphology and lack the intricate cell-to-cell and cell-to-extracellular matrix interactions that occur in native tissues.
This artificial environment often leads to altered cellular behavior, including changes in gene and protein expression profiles, compromising the physiological relevance of experimental results. The spherical architecture of a spheroid resolves these issues by allowing cells to interact in all three spatial dimensions. This restores natural cell polarity and communication pathways, encouraging cells to behave and function more like they would within a living organ. The shift to 3D spheroids represents a move toward creating laboratory models that more faithfully reflect the complexity of human tissue organization.
Creating and Characterizing Spheroids
Spheroids are typically generated using scaffold-free techniques that encourage cell self-assembly. The hanging drop method is a common approach, where cell suspensions are placed on the inverted lid of a plate, allowing gravity to pull the cells together into a single sphere. Another widely used technique involves ultra-low attachment (ULA) microplates, which are coated with a material that prevents cells from adhering to the surface, forcing them to aggregate with each other at the well’s rounded bottom.
Once formed, the internal structure of a spheroid develops characteristics that are crucial for its utility as a tissue model. As the sphere grows, it develops concentration gradients for oxygen, nutrients, and metabolic waste, similar to an unvascularized tissue mass. In larger spheroids (exceeding 500 micrometers in diameter), this gradient results in a distinct, three-layered organization. This organization includes an outer layer of highly proliferative cells, a middle layer of quiescent cells, and a core where nutrient and oxygen deprivation can lead to cell death.
Spheroids in Drug Discovery and Disease Modeling
The capacity of spheroids to form an internal structure with gradients and distinct cell zones makes them highly valuable for disease modeling, particularly for solid tumors. Multicellular tumor spheroids (MCTS) are extensively used in cancer research because they accurately mimic the tumor microenvironment, including the density and 3D arrangement of cancer cells. This structural complexity contributes to chemoresistance, where the drug must penetrate multiple layers of cells to reach the core.
Testing drug candidates on MCTS provides more predictive data than 2D cultures, which often overestimate drug effectiveness due to easy access to all cells. The hypoxic core and cell-cell interactions within the spheroid are factors that reduce a drug’s efficacy, mirroring the challenges seen in human clinical trials. Incorporating spheroids into high-throughput screening (HTS) campaigns allows researchers to assess a compound’s toxicity and therapeutic window, helping to filter out drug candidates likely to fail in later stages of development.
Role in Regenerative Medicine and Personalized Treatment
Beyond drug screening, spheroids play an expanding role in the fields of regenerative medicine and personalized treatment. Stem cell spheroids are particularly important in tissue engineering, where they are used as building blocks to create larger, functional tissue constructs. When aggregated, stem cells within the spheroid maintain their intrinsic properties, including the ability to differentiate into specialized cell types like bone, cartilage, or nerve cells.
Spheroids also exhibit enhanced paracrine signaling, secreting higher levels of beneficial growth factors and anti-inflammatory molecules compared to single cells in 2D culture. This enhanced secretion capability is leveraged for therapeutic applications, such as improving tissue repair and promoting the formation of new blood vessels. In personalized medicine, patient-derived spheroids are created from a patient’s own cells, allowing clinicians to test various treatment options on a model of their specific disease, tailoring therapeutic strategies for improved clinical outcomes.