Three-dimensional (3D) cell culture involves growing biological cells in an artificially created environment, allowing them to interact in all three dimensions. Unlike traditional two-dimensional (2D) monolayer cultures, where cells grow on a flat surface, 3D cultures enable cells to develop a more rounded, histotypic morphology and establish complex cell-cell and cell-extracellular matrix interactions, closely resembling their natural state within living tissues. This approach aims to faithfully recreate the structural architecture and specialized functions observed in vivo, offering a more physiologically relevant model than conventional 2D methods, which often lack the complexity needed to fully understand biological processes or predict drug responses.
Selecting the Appropriate 3D Culture System
Choosing the right 3D cell culture system influences experimental outcomes. These systems are categorized into scaffold-based and scaffold-free approaches, each offering distinct advantages depending on research objectives and cell types. Scaffold-based systems utilize a physical structure or matrix to provide structural support for cell growth, allowing cells to migrate and attach within a predefined three-dimensional space. These scaffolds can be derived from natural materials like collagen, gelatin, or basement membrane extracts such as Matrigel, which provide bioactive cues that promote cell adhesion and proliferation. Synthetic polymers like polylactic acid (PLA) or polycaprolactone (PCL) also serve as scaffolds, offering tunable mechanical properties and degradability.
Scaffold-free methods rely on cells’ inherent ability to self-assemble into aggregates or spheroids without external structural support, promoting cell-cell adhesion and forming compact, spherical multicellular structures. Common scaffold-free methods include the hanging drop technique, ultra-low attachment (ULA) plates, and spinner flasks. The choice between scaffold-based and scaffold-free systems often depends on the specific cell type being cultured; some cells readily form spheroids, while others require a supportive matrix.
Research questions also guide this selection; for instance, studying cell migration might benefit from a hydrogel scaffold that allows cell movement, whereas investigating tumor formation or drug penetration into a dense tissue might favor spheroid models. Cost and available equipment are practical considerations, as some advanced scaffold materials or specialized bioreactors can be more expensive than simpler spheroid formation techniques.
Essential Preparations for 3D Culture
Successful 3D cell culture requires careful preparation of cells and materials. Cells must be healthy and actively dividing, typically in their logarithmic growth phase. Prior to harvesting, cells should achieve an appropriate confluency, often between 70-90%, to ensure a sufficient number of viable cells for seeding. Cells are harvested using standard enzymatic dissociation methods, such as trypsinization, then counted to determine the precise cell density needed.
All reagents and materials must be prepared to maintain sterility. This involves sterilizing all equipment that will come into direct contact with the cells or culture media, typically through autoclaving or sterile filtration. Culture media and any supplemental solutions, such as growth factors or antibiotics, should be pre-warmed to 37°C before use to prevent temperature shock. When using hydrogels or other matrix components, it is often necessary to prepare stock solutions on ice to maintain their liquid state, as many hydrogels polymerize rapidly at warmer temperatures.
A Scaffold-Based Protocol Using Hydrogels
A common scaffold-based approach involves embedding cells within hydrogels, such as basement membrane extracts like Matrigel, which mimic the extracellular matrix found in tissues. This protocol begins by thawing the hydrogel, supplied as a frozen liquid, on ice at 4°C overnight or for several hours. Once thawed, the hydrogel should be kept on ice throughout the mixing process to prevent premature gelling.
Cells are harvested from monolayer culture, counted, and resuspended in cold culture medium or a suitable buffer at a concentrated density (1 x 10^6 to 1 x 10^7 cells/mL), depending on the desired final cell density within the gel. This suspension is then gently mixed with the cold liquid hydrogel at a predetermined ratio, typically yielding a final cell density of 1 x 10^5 to 1 x 10^6 cells/mL of gel. This mixing must be performed carefully to avoid air bubbles, which can disrupt the uniformity of the final gel. The cell-hydrogel mixture is then plated into appropriate cell culture vessels, such as multi-well plates, forming a dome-like structure or a uniform layer.
Following plating, the mixture is incubated at 37°C in a humidified CO2 incubator for approximately 30-60 minutes to allow the hydrogel to polymerize, encapsulating the cells within the solidifying matrix. Once the gel has fully formed, fresh, pre-warmed culture medium is carefully added to the wells, ensuring the gel remains submerged and undisturbed. The medium provides nutrients and maintains the necessary pH for cell viability and growth within the newly established 3D construct.
A Scaffold-Free Protocol for Spheroid Formation
Generating spheroids without a scaffold relies on encouraging cells to self-aggregate, a process often achieved through methods like the hanging drop technique or the use of ultra-low attachment (ULA) plates. For the hanging drop method, a concentrated single-cell suspension (1 x 10^5 to 1 x 10^6 cells/mL) is prepared in a small volume of culture medium. Small drops (20-50 microliters) are dispensed onto the inner surface of a sterile petri dish lid, where surface tension holds them.
The petri dish lid is then inverted over the bottom part of the dish, which contains a small volume of sterile phosphate-buffered saline (PBS) or water. This creates a humidity chamber, preventing evaporation and maintaining cell viability. Due to gravity, cells within each hanging drop settle to the lowest point and begin to aggregate, forming a single spheroid at the bottom of the droplet over 24-72 hours. This method allows for the production of tightly packed spheroids with reproducible sizes.
Alternatively, ULA plates utilize a specialized polymer coating that prevents cells from adhering to the well surface, thereby promoting cell-cell aggregation. To use ULA plates, cells are harvested, counted, and resuspended in culture medium at a desired concentration, often 5,000 to 10,000 cells per well, depending on the target spheroid size. This cell suspension is then directly seeded into the wells of the ULA plate. The round or U-shaped bottom of these wells helps to guide the cells to the center, facilitating their aggregation into a single spheroid per well. The cells begin to form distinct spheroids within 24-48 hours, with spheroid size influenced by the initial cell seeding density.
Post-Seeding Maintenance and Validation
After initial setup, regular maintenance ensures the long-term health and stability of 3D cell cultures. Media changes are performed periodically, often every two to three days, to replenish nutrients and remove metabolic waste products. When changing media in hydrogel-embedded cultures, care must be taken to gently aspirate the old medium from the sides of the well without disturbing the delicate gel structure, followed by slow addition of fresh, pre-warmed medium. For spheroid cultures in ULA plates or hanging drops, the medium can be carefully removed from the periphery of the well or drop, minimizing disturbance to the spheroid.
Validating 3D cultures involves several observations. Brightfield microscopy is a primary method for visual confirmation, allowing researchers to observe spheroid formation, assess morphology, and monitor growth over time. For scaffold-based cultures, microscopy verifies uniform cell distribution within the hydrogel and maintenance of three-dimensional morphology.
Beyond visual inspection, cell viability assays are commonly employed to confirm the health of cells within the 3D construct. Simple live/dead stain assays provide a quick assessment of cell viability and cytotoxicity. These assays use fluorescent dyes to differentiate between live and dead cells, allowing microscopic visualization of healthy cells versus those with compromised membranes. These validation steps confirm the establishment of a viable 3D cell culture model for further experimentation.