Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
3D cell culture offers many advantages over traditional 2D techniques. 3D cultures typically have physiological and morphological characteristics that are closer to tissues in vivo. The greater physiological relevance of 3D cultures can result in different cell behaviors, and even experimental outcomes, than 2D cultures. Yet as researchers almost always learn their culture techniques on traditional 2D plates of cells, switching to 3D can be a challenge. Here are some tools to consider when making the change.
Special scaffolds and scaffold-free
Most 3D culture systems use a scaffold, which is a structural support on which the cells grow. When choosing a scaffolding material, Lara Cardy, product manager at B-Bridge International, recommends considering several factors, such as whether you want the scaffold to “mimic the cells’ native extracellular matrix,” whether you want the cells to “form functional multicellular structures” and whether you plan to image the cells. It can be challenging to image cells grown on some scaffolds; in these cases, says Cardy, confocal microscopy may be a good solution.
Some of the most common scaffolds are hydrogels. B-Bridge’s peptide hydrogel, PanaceaGel, for instance, is a chemically defined mixture of 13 self-assembling peptides intended for tissue engineering applications, while its dextran-based 3-D Life Biomimetic hydrogel is made of synthetic polymers and crosslinkers (originally created by Cellendes in Germany). Designed to enable researchers to control how the cells spread and migrate, the hydrogel also allows recovery of cells from the 3D culture, if desired.
The system is well suited to cell adhesion and migration studies. "Researchers choose crosslinkers that determine whether cells can locally degrade the hydrogel and create space to move,” says Cardy. “The inclusion of adhesion motifs like RGD peptides allows cells to spread and migrate.” The system is also suitable for studying co-cultures as well as functional cell aggregates such as epithelial cysts and spheroids.
TAP Biosystems’ collagen-based RAFT System represents another kind of hydrogel. Collagen is the main component of extracellular matrix, and the RAFT process uses it to construct scaffolding for 3D cultures with minimal time and effort. The researcher mixes cells at a specific “seeding density” with a collagen solution (plus additional matrix proteins, if desired). The solution gels within minutes, at which point an absorber is added, which removes water and “concentrates the collagen from a weak hydrogel to the same collagen concentration that you would find in a tissue,” says Grant Cameron, RAFT development director at TAP Biosystems (now part of the Sartorius Stedim Biotech Group). The cells can move, proliferate and form tissues within the gel.
Cameron says cell-seeding density is a critical consideration in 3D culture, just as in 2D culture. The density typically is higher for 3D, though, because the height of a 3D system increases its volume. “In some cases, the cell-seeding density has been increased dramatically to in excess of 100,000 cells in a 96-well plate culture, as the cells respond well to cell-cell interaction,” Cameron says.
Among other applications, the RAFT system can be used to model the multilayered blood-brain barrier, says Cameron. “The ability to create a single model in a permeable cell-culture insert, with not only an endothelial or epithelial cell layer but also with an attached stromal layer incorporating fibroblasts or smooth muscle cells, appears to be a unique ability of the RAFT system,” he says.
Some researchers prefer to make cell aggregates without a scaffold, so that the aggregates remain free in solution. MicroTissues makes micromolds (distributed by Sigma-Aldrich) that enable users to make their own 3D Petri Dish® from agarose. When cells are added to the 3D Petri Dish, they self-assemble to form multicellular spheroids or more complex cell assemblies, or microtissues, depending on the type of micromold used, no scaffolding required. Eight different mold designs are available, including spheroids, rods, toroids and a honeycomb pattern.
MicroTissues marketing manager Brian Morgan says the absence of scaffolding maximizes cell-to-cell interactions, which improves cell health. “Our culture system is very stable and enables long-term cell culture greater than 21 days,” he says. “We can grow single cancer stem cells into multicellular 3D spheroids.”
Special plates
Specialized cell-culture plates also can ease the transition to 3D culture—Stemcell Technologies’ AggreWell™ plates, for instance.
AggreWell plates contain microwells (either 400 or 800 microns in diameter) within each culture well. After a cell suspension—of embryonic or mesenchymal stem cells, for instance—is added to the wells, the cells settle and evenly distribute into the microwells, where they aggregate. Up to 4,700 uniform spheroids can be produced per well in this fashion. “By generating uniform spheroids using AggreWell, reproducibility is increased enormously, making it simpler for researchers to troubleshoot 3D cultures in the early stages of development,” says Simon Hilcove, product manager for pluripotent stem cell biology at Stemcell Technologies.
Another tool specialized for 3D culture is 3D Biomatrix’s Perfecta3D® Hanging Drop Plate, designed for spheroid growth in 96- or 384-well plate formats. The plate is designed such that when a drop of cell suspension is pipetted into a well, the drop hangs stably, and the cells within it can aggregate without contacting any surfaces or scaffolds. “The user can control the spheroid diameter with the type and number of cells in each well,” says MaryAnn Labant at 3D Biomatrix, whose products are distributed by Sigma-Aldrich. Access holes in each well enable media exchange and the addition of compounds, reagents or additional cells to establish co-cultures, she adds. Although scaffolding is not required to form aggregates, matrix material can be added to the drops, if desired.
Spheroids grown in Perfecta3D Hanging Drop Plates are used in cancer research, stem cell differentiation, toxicology and drug discovery, Labant says. One of the most common applications, she says, "is creating microtumor models for cancer research.”
Special magnets
n3D Biosciences offers a new take on 3D-culture formation with its magnetic nanoparticle-based NanoShuttle system. Cells are treated with a nanoparticle solution to magnetize the cells, and then magnetic drives move the cells to form 3D structures by magnetic 3D bioprinting and magnetic levitation. The structures can form in minutes, depending on cell type.
“Using a magnetic field to assemble 3D structures is like an invisible and imaginary scaffold,” explains Glauco Souza, president and chief science officer at n3D Biosciences. “By aggregating the cells and not directly controlling the environment, the cells interact with themselves to create their own extracellular matrix to facilitate the formation of the 3D structure.”
Conditions can vary from cell type to cell type, Souza notes. For instance, some cells can form a cohesive structure with as few as 600 cells per well of a 384-well plate, but another cell type may require 20,000 cells, he says.
The magnetic system is reproducible, Souza adds, because the magnetic field is of uniform strength. “One can easily print very reproducible structures in a 96- or 384-well plate,” he says.
The n3D’s BiO Assay™ kit uses magnetic 3D bioprinting to “print” cells onto plates for dose-response assays in automated, high-throughput viability testing. Souza says n3D’s customers especially value the BiO Assay for pre-clinical cardiovascular toxicity screening, where it can replace more expensive ex vivo aortic tissue assays. “Rings printed with vascular smooth muscle cells can contract and dilate with dose-dependent responses corresponding to their vasodilating or vasoconstricting nature,” he says.
Regardless of what type of 3D-culture system you use, remember to keep an open mind. Souza says many researchers are not prepared to accept results from 3D cultures that contradict their previous 2D findings. This may be due in part to differences between 2D and 3D in cell morphology or density. But Souza hopes researchers will press on, nonetheless. “Our hope is to bridge the gap from benchtop to bedside by reducing the dependence on poorly representative 2D and animal models before going to clinical trials,” he says.