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.
Most cultured cells grow on the surface of a plastic dish—that is, in two dimensions. But in the body, cells are generally surrounded on all sides by other cells, tissues and extracellular matrix.
Researchers who grow cells in 3D cell culture, in an attempt to more closely approximate the cells’ normal physiological environment, observe differences compared to 2D cultures, including changes in gene expression, signal transduction, cell proliferation, physical shape, cell-cell interactions and reactions to drugs.
Fortunately, changing from 2D to 3D cell-culture systems isn’t as tricky as it sounds. And the payoff is likely to be worth the fuss. “The gain is that you are studying cells that are closer to being in their real environment, and that has to be good for your biology,” says Grant Cameron, RAFT development director at TAP Biosystems.
Here are some tips to keep in mind as you contemplate making the switch.
Types of 3D Systems
There are several main types of 3D cell-culture substrates, or scaffolds, available today, all of which add a third dimension to the cells’ environment. These include hydrogel scaffolds derived from natural materials, such as the extracellular matrix protein collagen. The collagen-based RAFT technology from TAP Biosystems forms a “hydrogel with tissue-like properties” says Cameron, enabling researchers to pursue cell biology, drug discovery and tissue-engineering applications. (RAFT is short for Real Architecture for 3D Tissue.)
Life Technologies’ Geltrex® and BD Biosciences’ BD Matrigel™ are made from murine tumor basement membrane preparations, while Sigma-Aldrich’s MaxGel™ is human extracellular matrix, and Life Technologies’ AlgiMatrix, derives from brown seaweed. Microtissues Inc. uses agarose-based hydrogel in its 3D Petri Dishes®. “The user seeds the agarose 3D Petri Dish with cells,” says Brian Morgan, Microtissues’ director of marketing. “The cells aggregate and self-assemble hundreds of uniform-sized multicellular spheroids.”
A second type of scaffolding used in 3D cell culture is a synthetic hydrogel, sometimes including biomimetic peptides that are added to enhance the binding of integrins (which encourage the cells to form adhesions). BD Biosciences’ BD™ PuraMatrix™ Peptide Hydrogel and Sigma-Aldrich’s HydroMatrix™ fall into this category, as does B-Bridge International’s dextran-based Cellendes 3D Life Biomimetic hydrogel, which is made from synthetic polymers and crosslinkers.
“Researchers choose crosslinkers that determine whether cells can locally degrade the hydrogel and create space to move,” says Lara Cardy, product manager at B-Bridge International. “The inclusion of adhesion motifs like RGD peptides allows cells to spread and migrate.”
B-Bridge also offers PanaceaGel, a synthetic hydrogel made from 13 types of self-assembling peptides intended for tissue-engineering applications.
Other types of artificial scaffolds made from tissue-culture plastics, or even biodegradable plastic, are available as well. There is even a system that suspends cells using magnetic levitation, where cells generate their own extracellular environments, no plastic scaffolding required.
The Bio-Assembler™ system from n3D Biosciences enables researchers to culture cells in 3D using magnetic levitation. “We use magnetic nanoparticles to enable the formation of 3D cell culture without the need for scaffolding,” says n3D Biosciences’ chief scientific officer Glauco Souza [1]. “You can move the cells [using a magnetic pen] to create layers of tissue, and then when you remove the magnet, the cells are free.” Souza led a research group that used these methods to grow a four-layered cell structure that approximates lung tissue [2].
Finally, there are systems to induce spheroid body formation, such as B-Bridge’s Lipidure-Coat plates, 3D Biomatrix’s Perfecta3D® hanging drop plates, and STEMCELL Technologies’ AggreWell™ for human induced pluripotent and embryonic stem cell growth.
When to Change from 2D to 3D
Given the strong evidence showing that cells grown in 3D culture systems show more physiological phenotypes, interactions and responses, you should probably consider switching to 3D culture whenever the physiological relevance of your results are important—or if you cannot accurately recapitulate the behavior you want any other way.
“That cells are growing on a flat, plastic surface is a situation that is far from their natural physiological environment and needs a certain degree of adaptation,” says Peter Rettenberger, business unit manager at AMSBIO. “Associated with this is a certain selection pressure towards cells that can adapt.”
3D culture is probably irrelevant in, say, transient transfection assays looking at reporter gene expression. But the calculus would be different if you were studying complex drug interactions. “Drug sensitivity [to anti-cancer drugs] of cells in 3D is more like in vivo tumors than cells in 2D,” says Morgan. “Drug transport and diffusion into 3D microtissues is more like 3D organs and tissues that have multiple cell layers.”
When you’re ready, these tips can smooth the transition. First, pay attention to cell-seeding density, which may need to be higher than in 2D culture. “Many cells want to be in close proximity to one another or even require co-culture with additional cell types,” says Cameron. “Cell number per well will likely be increased, and a factor of 10 to 20 is a reasonable factor to consider.” He also recommends allowing the cells some transition time before jumping in with experiments.
Choosing the best 3D scaffolding materials for your work is also important for a successful transition. Cardy recommends considering several key questions: “Do I need to mimic the cells’ native extracellular matrix? Will I need cells to form functional multicellular structures? Do I need to analyze my 3D cultures microscopically and/or recover the cells for further analysis? Do I want to have control over gel stiffness and bioactive components?” Answers to such questions can help you narrow your choices.
Potential Pitfalls
Scaffold choice bears directly on one of the potential pitfalls of transitioning to 3D cultures—it can complicate some methods of analysis. Microscopy can be challenging, because the multiple cell layers of 3D cultures scatters the light reaching the objective (compared with the relatively more straightforward imaging of cell monolayers formed in most 2D cultures). Confocal microscopy is a good solution to this problem, says Cardy, but other types of analysis may prove challenging. “Many scaffolds are not amenable to immunochemistry techniques or cell recovery,” she says.
Sometimes the complications encountered in 3D culture stem from the very reason for making the switch in the first place. “The cells may behave in a manner different from how they behave in 2D—for instance, forming higher-order structures, such as a cancer tumoroid. However, this is the very point of the change!” says Cameron. “3D cell culture opens up a host of new cellular responses and possibilities, and these may surprise the researcher.”
References
[1] Souza, GR, Molina, JR, Raphael, RM, Ozawa, MG, Stark, DJ, Levin, CS, Bronk, LF, Ananta, JS, Mandelin, J, Georgescu, MM, Bankson, JA, Gelovani, JG, Killian, TC, Arap, W, Pasqualini, R, “Three-dimensional tissue culture based on magnetic cell levitation,” Nat Nanotechnol, 5(4):291-296, 2010. [PubMed]
[2] Tseng, H, Gage, JA, Raphael, RM, Moore, RH, Killian, TC, Grande-Allen, KJ, Souza, GR, “Assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation,” Tissue Eng Part C Methods, 19(9):665-675, 2013. [PubMed]
Image: the 12-well Spheroids Mix Pack from Microtissues Inc.