When examining cellular regulatory events, testing drug compounds and performing tissue research, three-dimensional (3D) cell-culture systems have provided scientists with a viable tool that has the potential to more closely mimic in vivo conditions in an in vitro setting. In contrast to traditional 2D culturing systems, in which cells typically grow as monolayer sheets adhered to a matrix, 3D cultures create an environment that enables cells to contact and interact in all dimensions with other cells in culture. Although establishing 3D cultures can be tricky, navigating the transition from 2D cultures will be smoother with the tips and tools presented here.
Transitioning from 2D to 3D
Paramount among the steps to a successful transition to 3D cultures is doing your homework. Most importantly, search the literature to learn which type of 3D cultures best suit your needs, and which types of assays work well with them. Cindy Neeley, senior staff scientist in field applications at Thermo Fisher Scientific, recommends a thorough literature search on experimental design, cell types and experiments following the 3D cell-culture process. Advance planning could save you big headaches. “Not all cell types are suitable for scaffold-free 3D culture systems,” she says. “Cells such as SW480, which don’t produce enough adhesive molecules on their own, may not effectively form 3D aggregates spontaneously without the physical confinement of the scaffold material. On the other hand, scaffold-based 3D culture systems may limit how cells can be harvested and analyzed downstream.”
Another factor to consider is the time required for analyses, because some types of assays are read on conventional 2D plates. “Such assays need to be relatively short,” says Laura Schrader, president and CEO of 3D Biomatrix. “Once a sample is transferred to a 2D plate, eventually the 3D cellular construct will start to adhere, and the biology will begin to change.”
With interest growing in 3D cultures as more physiologically relevant tissue models, vendors are making strides in ease of use. 3D cultures were historically much more labor-intensive than traditional 2D cultures, but some newer systems are reducing this discrepancy. For example, Neeley says that Thermo Fisher Scientific’s Nunclon Sphera 3D culture platform facilitates the transition from 2D to 3D culture by allowing most researchers to use the same media, culture conditions and microscope that they used for 2D cultures. “It doesn’t require much change in protocol for seeding, growing, observing and harvesting,” she says. Lubna Hussain, senior product manager for primary cells and 3D culture at Lonza, also says that “many standard 2D analysis methods, such as immunocytochemistry, can be applied directly or with only minor modifications to [Lonza’s] RAFT™ 3D cultures.”
Rising above the plate
Scaffold-based platforms grow cells within extracellular matrix or synthetic scaffolding materials. Lonza’s RAFT™ 3D Cell Culture System uses a collagen I scaffold, along with the company’s patented RAFT Absorbers, which increase the density of the collagen matrix. “If used in combination with cell-culture inserts with permeable membranes, the RAFT System is an ideal tool for the generation of barrier models, including air-lift models,” says Hussain. “The RAFT System has been used successfully to develop blood-brain barrier models, tumor models, corneal models and hepatocyte models.” Vivo Biosciences’ialso offers 3D HuBiogel, a patented biomatrix material for growing tumor models.
Lena Biosciences’ SeedEZ 3D cell-culture system is particularly useful for drug-screening applications. A clear, paper-like microfiber scaffold, SeedEZ wicks up cell-suspension drops that are added to it and then uniformly distributes cells throughout the scaffold. Because cells become embedded in the scaffold, multiple media exchanges and the addition and removal of drugs and assay reagents don’t disturb the cultures. This is advantageous for drug screening, repeat drug dose efficacy and toxicity testing, for example.
By layering the paper-like scaffolds, researchers can use SeedEZ to create multilayered tissue models and co-cultures. Jelena Vukasinovic, president of Lena Biosciences, says that that company’s “spot-a-culture technology” enables researchers to screen a 3D cell culture as easily as dried blood spot samples in point-of-care testing. “You can spot multiple cell populations side by side, add them to different scaffolds and stack to form multilayered tissues; deliver viscous extracellular matrix suspensions; and still handle the scaffold using sterile forceps, without worrying that it will come apart,” says Vukasinovic.
Spheroids—cell aggregations that form in the absence of scaffolding materials—are commonly used in 3D culture systems. Scaffold-free spheroid systems have the advantage of being free of scaffolding materials like extracellular matrix, which may possibly complicate downstream analysis, depending on your experiments.
Thermo Scientific™ Nunclon™ Sphera microplates are designed to encourage cells to spontaneously aggregate to form cell clusters rather than attach to the microplate surface. The resulting spheroids “have been used for modeling tumor development in cancer research and for studying embryonic stem cell differentiation,” says Neeley.
Another spheroid-based tool, the Perfecta3D®eHanging Drop Plates (HDPs) from 3D Biomatrix, uses hanging drops stabilized by surface tension to assemble spheroids. Because only one spheroid forms per well in the multiwell HDPs, researchers can control the spheroid size by using particular numbers or types of cells per well. “This also allows the user to create or avoid a necrotic core in the spheroid, as experimentally required,” says Schrader.
InSphero also offers the GravityPLUSTM Hanging Drop System and the GravityTRAPTM ULA Plates for scaffold-free cultures. Both culture platforms are currently available from Revvity. InSphero also offers assay-ready 3D microtissues derived from live, islet, tumor, heart, skin and brain tissue, as well as 3D-focused screening services on these organotypic model systems.
Additional tools and assays
Thermo Fisher Scientific’s Cell Culture Insert is a porous-membrane platform for growing 3D cultures of epidermal skin cells in multiwell dishes. “It allows for an air-liquid interface culture that promotes proper differentiation and maturation of skin cells,” says Neeley. Another 3D system from Thermo Fisher Scientific is UpCell™, which enables researchers to generate tissues by growing and stacking cell sheets. “UpCell has been used to generate corneal tissues for treatment of eye diseases and to create myocardial tissues to restore damaged heart[s],” says Neeley.
Off-the-shelf assays adapted for 3D cultures are also important tools; these include Promega’s CellTiter-Glo® 3D Cell Viability Assay, which can be used with many 3D culture formats. Terry Riss, Promega’s global strategic marketing manager in cell health, says the company has also modified protocols for assays such as the Caspase-Glo® 3/7 Assay for use with 3D cultures. “For some reagent formulations, modifying the detergent content is limiting because of the stability of the marker being measured,” says Riss, so the company added a more physically rigorous lysis procedure.
Avoiding potential pitfalls
Here are a couple tips and considerations to help guide you through a smoother transition from 2D to 3D cell culturing.
Different tissue thickness
3D cultures are usually several cell layers thick, so assays designed for 2D cell monolayers must be adapted and validated. Potential problems include the greater diffusion barriers; it may be harder for reagents or detection probes to penetrate 3D cultures to reach sites of action, so adjustments in concentrations or times may be required. “This especially relates to 2D assays that [were] not previously validated for 3D cultures,” says Schrader.
Different cell numbers and growth habits
Cells often grow and behave differently in 3D culture. This may entail changes in cell titration or timing, which could affect your data, depending on your experiments. “For certain 3D methods, higher cell numbers may be required [for 3D cultures] compared to 2D cultures,” says Hussain. “Some cells might also need some time to re-adapt from standard 2D culture to 3D culture, and they might grow initially a little bit slower in 3D.”
Cell density when first seeding cultures is important with any system, but 2D conditions may need revising to account for the larger volumes required for 3D culturing. “Low cell-seeding densities may reduce cell-cell interactions, deprive cells from paracrine signaling molecules and influence cell phenotype, doubling time and proliferation rate,” says Vukasinovic.
Opportunities in 3D
Especially useful for tumor and tissue research, 3D cell cultures add valuable physiological relevance. Riss cautions, however, that 3D culture models likely differ from in vivo environments. “The dynamics of cell proliferation, quiescence, potentially apoptosis and necrosis, which may exist as a gradient from the outer layers to the innermost area of large 3D structures, can complicate interpretation,” says Riss.
Researchers and tool providers continue to test and develop better options for culturing cells. Although traditional 2D culturing may serve your needs, 3D cell culturing offers many advantages—including the opportunity to do better research and gain more information.