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.
Studying the complexities of cells and tissues and the regulatory and signaling processes that govern them is no easy task. Just as difficult may be the attempts to mimic cellular or tissue environments to create the most genuine experimental system possible. This is the challenge of three-dimensional (3D) cell culture, in which cells are grown in environs constructed to better resemble their original in vivo homes. Increasing evidence suggests that 3D cell culture systems might be more physiologically relevant than traditional 2D cell culture systems. Perhaps the most valuable reason to approximate in vivo conditions is to facilitate the development of therapeutics. “The major advantage of 3D cell culture is better duplication of in vivo behavior in vitro early on in the research process,” explained Laura Schrader, CEO of 3D Biomatrix. “Conventional 2D cell culture often fails to capture the cellular functions and responses that are present in tissue. As a result, drug assays and biological research findings based on conventional 2D cell cultures can be skewed and offer limited predictive capability. 3D cultures can lower the costs of getting a new drug to market by providing more realistic early-stage information about biologic or drug candidates.”
The recent explosion in 3D cell culture options gives more researchers access to this tool. The type of 3D cultures they choose, however, depends on their individual requirements. Some determining factors might include cell type, experimental design, scale, whether co-cultures will be used, types of analysis used, whether cells need to be removed or harvested for analysis, required throughput, and possible clinical use. Learning about the types of 3D culture systems available can help the researcher choose the one best suited to his or her research.
Systems without scaffolds
Scaffolds are structures onto which cells can attach, grow, and/or spread. However, some types of experiments demand that cells remain unattached—for example, if one wants to encourage cells in solution to aggregate into tissue-like clusters called spheroids, or spheroid bodies. Spheroids are thought to mimic solid tissues, avascular tumors, and embryoid bodies, according to Schrader. “With inherent metabolic (oxygen, carbon dioxide, nutrients, wastes) and proliferative gradients, spheroids serve as excellent physiologic models and are used in cancer and stem cell research,” she said.
Cell culture plates
One challenge has been to study spheroids in long-term culture, in co-culture with other cell types or in conjunction with liquid handling systems. 3D Biomatrix solves these issues with the Perfecta3D ® Hanging Drop Plates, which generate spheroids consistent in size and shape. The user can also influence the spheroid size by adjusting the seeding density (from 50 to 15,000 cells). The plates (96- and 384-wells) work with manual and robotic liquid handling systems. “Cells grown in the Perfecta3D Hanging Drop Plates generate and organize their own 3D extracellular matrix, so spheroids closely resemble in vivo tissues,” said Schrader. “It is also possible to generate co-cultures with other cell types, such as endothelial, stromal and epithelial cells.”
Another 3D culture system for researchers using spheroids is the SCIVAX 3D NanoCulture Plate from InfiniteBio, which contains nano imprinting technology on the bottom of the well. This forms a surface for attachment, upon which spheroids form. “The uniqueness [of our system] is that the spheroids are attached to the plates while they maintain the three dimensional structure, giving unique advantages such as easy handling and high cell viability,” stated Akiko Futamura, President and CEO at InfiniteBio.
Other 3D culture systems lacking in a scaffold structure are offered by Microtissues and Nano3D Biosciences. The system from Microtissues, the 3D Petri Dish® includes micro molds with which the researcher creates, from agarose, 3D “petri dishes” that fit into microplates. The Bio-Assembler™1, offered by Nano3D Biosciences, uses the magnetic levitation method, in which magnetic nanoparticle assemblies are used to magnetize cells, which are then levitated with magnetic fields.
Systems with a scaffold
If an experiment calls for a 3D culture system with a scaffold for the cells to grow on, several options are available. 3D Biotek, which offers many 3D polymer scaffolds for cell culture and tissue engineering, recently released the 3D Nano-mesh scaffold. “This scaffold offers dual geometry advantages because it merges micro-scale and nano-scale geometries for complex stroma and epithelial cancer models,” said Carlos Caicedo-Carvajal, R&D Manager at 3D Biotek. The homogeneity of the interconnected open pores within the scaffold gives optimal exchange of nutrients and waste. “The technology also can generate controlled length scales for cells, such as changing fiber size, number of scaffold layers and changing fiber-to-fiber spacing, to meet the demands of the cellular system,” he continued. “For example, some cells like to be in close proximity. However, bone cells require larger pore sizes, about 400 [to] 500 µm, for potential blood vessel formation.” Another notable feature of this system is its adaptability, so it can be adjusted to better approximate in vivo conditions using in vitro models.
Two other vendors offering scaffolding tools include Life Technologies and Reinnervate. The Life Technologies AlgiMatrix® 3D culture system uses a bioscaffold; the structure is made of raw materials derived from brown seaweed that form a 3D macroporous alginate structure. Reinnervate’s polystyrene Alvetex® Scaffold also helps to keep cells organized in three dimensions.
Gels and extracellular matrices
Gels provide a type of cell-growth matrix that is structurally akin to the extracellular matrix surrounding cells in vivo. “A representation of the rapid change [in 3D culture techniques] is in the movement from tissue-derived 3D culture materials to synthetic and chemically defined hydrogels,” according to Lara Cardy, Product Manager at B-Bridge International. B-Bridge distributes 3D Life Biomimetic Hydrogels from Cellendes, which are made to be flexible so that the user can better design the cells’ environment. By varying the composition of synthetic polymers and crosslinkers, different 3D environments can be created. “The choice of crosslinker determines whether cells can locally degrade the hydrogel and create space to move,” continued Cardy. “The addition of RGD Peptide provides a cell adhesive extracellular environment, a prerequisite for most cells to spread and migrate. This makes 3D Life Biomimetic Hydrogels ideal for studying functional cell aggregates like epithelial cysts and spheroids, as well as cell spreading and migration. Live or fixed cells can be easily recovered from the degradable polymer; this process is protease-free and does not harm cells. Furthermore, 3D Life Hydrogels are injectable and amenable to automation.”
When designing a 3D culture environment, it is important for researchers to decide what they are most interested in exploring, said Cardy. This might be cell–cell interaction, cell–environment interaction, or cell mobility, for example. “I think those are some of the most important initial questions to ask when selecting a 3D culture platform, and in particular, which of the 3D Life Hydrogels polymers and crosslinkers are ideal for your first experiments.”
Instead of designing the environment and then introducing cells, TAP Biosystems’ RAFT (Real Architecture for 3D Tissue) System allows the user to encapsulate cells with an in vivo-strength collagen matrix by removing liquid after combining collagen with cells, thereby concentrating the collagen around the cells. “A collagen environment is critical in understanding cellular behavior, as it is the major extracellular matrix protein,” said Grant Cameron, RAFT Development Director at TAP Biosystems. “Rather than it being a static bystander in cellular processes, the cells have receptors for collagen and interact with it, including in remodeling during cell invasion and migration.” A RAFT-based corneal therapy is on its way to human testing soon in the United Kingdom. Other vendors supplying gels and extracellular matrices include Amsbio, BD Biosciences, and Sigma-Aldrich.
Bioreactors
Bioreactors are vessels that contain cultured cells, culture media, and sometimes scaffolds. For example, the 3D Perfusion Bioreactor from 3D Biotek was designed for use with the company’s porous 3D polymer scaffolds. “It has a bioreactor chamber where the 3D polymer scaffolds are placed, and bottom-to-top media recirculation to recreate in vivo-like, flow-induced shear stress profiles,” explained Caicedo-Carvajal.
Synthecon uses rotating wall vessel technology in its Rotary Cell Culture System (RCCS) bioreactors, which can be used with or without scaffolds. The cylindrical RCCS is filled completely with media and rotated along its horizontal axis. “Gas exchange occurs via a silicone membrane incorporated into the vessel,” said Stephen Navran, Chief Scientific Officer at Synthecon. “Cells introduced into this environment rapidly aggregate into 3D spheroids. Alternatively, a variety of scaffold materials can be added to which cells can attach. Unlike spinner flasks or sparged bioreactors, the mechanical forces needed to maintain the 3D constructs in suspension in the RCCS are very low, minimizing cell damage. Additionally, the rotational dynamics provide much better access to nutrients and oxygen than static culture systems, which depend on diffusion.” According to Navran, a limitation of any type of 3D culture system is a lack of blood supply. “This usually means that the center of a 3D cell construct is anoxic and nutrient-starved and will eventually become necrotic. A dynamic culture system such as the RCCS can partially address this problem but is not a total solution.”
The future of 3D cell culture
A considerable body of experimental evidence suggests that 3D cell culture, while not a replica of in vivo conditions, is certainly a closer approximation than traditional 2D cell culture. The future holds an exciting period of exploring 2D results in 3D. “While there is a huge body of scientific literature based on 2D cell culture that has resulted in valuable findings, it’s time to evolve the process,” said Schrader. “Researchers taking in vitro research to the next level using 3D cell culture are going to reap the earliest rewards by getting therapies to market faster.”
The image at the top of the page is from 3D Biomatrix.