Cell culture is a foundational technique in biological research, where cells are grown outside of a living organism in a controlled laboratory setting. Traditionally, this involved growing cells as a single, flat layer on plastic dishes, known as two-dimensional (2D) culture. The spheroid assay represents a modern advancement, moving researchers from this simplistic flat environment to a complex, three-dimensional (3D) structure.
These 3D models aim to accurately simulate tissues found in the body to study disease progression and test potential treatments. This shift toward 3D modeling is driven by the necessity for results that more closely mirror the biological reality of the body, allowing researchers to better understand how cells behave and respond to drugs before human trials.
Why Cells Need Three Dimensions
Living tissue involves cells interacting with each other and with the surrounding structural components, which is poorly represented in a flat, 2D culture. In the body, cells are embedded within the extracellular matrix, a 3D scaffold that provides biochemical and structural support. When cells are grown in a 2D monolayer, they lose these natural cell-to-cell and cell-to-matrix connections, altering their shape, function, and genetic activity.
Spheroids allow cells to self-assemble into a compact, spherical mass, restoring a more natural architecture and cell signaling. This re-established cell-cell communication governs processes like cell differentiation, metabolism, and sensitivity to external stimuli. Another element is the formation of internal gradients, which are impossible to achieve in a flat culture.
As the spheroid grows, the outer layer consumes nutrients and oxygen, creating a progressive gradient toward the center. This results in distinct zones: an outer layer of actively dividing cells, a middle layer of quiescent cells, and an inner core where nutrient scarcity leads to necrosis (cell death). This layered structure accurately mimics the environment found in solid tumors and other dense tissues, providing a more physiologically relevant system for research.
Creating Spheroids: The Methods
The fundamental principle behind spheroid formation is preventing cells from attaching to a flat surface, forcing them to aggregate. This self-assembly process is driven by the cells’ natural tendency to maximize cell-to-cell contact when adhesion is blocked. Researchers employ a variety of scaffold-free techniques designed to suspend the cells and encourage spherical organization.
Hanging Drop Method
One common approach is the Hanging Drop method, where small droplets of cell suspension are placed on the inverted lid of a culture dish. Gravity pulls the cells to the bottom of the drop, and surface tension holds the droplet shape. This concentrates the cells into a tight, self-assembling sphere, effective for forming spheroids of a highly consistent size and shape.
Ultra-Low Attachment (ULA) Plates
Another widely used technique involves specialized Ultra-Low Attachment (ULA) plates. These plates have a coating, often a hydrogel or polymer, that prevents cell adhesion to the well surface. When cells are seeded into the round-bottom wells, they spontaneously aggregate in the center, forming a single, uniform spheroid.
Magnetic Levitation
A third method is Magnetic Levitation, which involves coating the cells with magnetic nanoparticles. A magnet placed beneath the culture plate lifts the cells, suspending them in the culture medium and forcing them together into a 3D aggregate. This technique allows for the rapid formation of a stable, cohesive spheroid structure.
Applications in Drug Discovery and Disease Modeling
The realistic architecture of spheroid assays has made them invaluable tools in drug discovery and disease modeling. For drug screening, spheroids offer a better prediction of a compound’s effectiveness compared to traditional 2D cultures. This predictive power is due to the physical barrier and cellular complexity inherent in the 3D structure.
The compact nature of the spheroid accurately models the challenges a therapeutic compound faces in penetrating solid tissue, such as a tumor. Drugs must diffuse through the outer layers to reach the inner, less accessible cells, a process missed in flat culture. By exhibiting differential drug penetration and zones of varying cell proliferation, spheroids help researchers select effective drug candidates.
In disease modeling, spheroids are extensively used in cancer research as Multicellular Tumor Spheroids (MCTS) to mimic the tumor microenvironment. These models recreate the heterogeneous cell populations and gradients that contribute to drug resistance and tumor growth. Spheroids are also gaining traction in toxicology testing, such as using liver-derived spheroids (hepatospheres) to test the toxicity of new chemical compounds.
The use of these advanced 3D models addresses the need for better in vitro (laboratory) models that can reduce the reliance on animal testing. Because the spheroid structure more closely resembles human tissue, they provide a more accurate assessment of how a drug will behave in a patient. This improved accuracy in preclinical testing helps streamline the development of safer and more effective medical treatments.