Not all 3D Cultures are Created Equally – Which is the best one for you?


2D cell culture has been used since the early 1900s and over the decades has been vital not only for advancing our understanding of the biology of cells, but it has become an important component of drug screening and toxicity testing. Traditionally this monolayer system involves cell growth on a plastic or glass surface. However, compelling evidence suggests that cells cultured in such conditions are not representative of cells residing in the complex microenvironment of a tissue.  Cells are forced to adapt and generate artificial responses resulting in flattened morphology and poor replication of the complicated cellular signals between cells and their matrix.  As result, data generated by 2D cell culture methods could be misleading, and this discrepancy is thought to make these systems relatively poor models of in vivo response.This can be particularly problematic for predicting drug responses and is a contributing factor to the high failure rate in drug discovery. Despite these drawbacks, 2D cultures remain very attractive because of their simplicity and low cost.

In terms of improving physiological relevance, animal models provide a useful tool to study biology. However, animal models are expensive, time-consuming, have obvious ethical considerations, and are not always able to accurately recapitulate human tissues/diseases, and in the case of some diseases, they are impossible to model.  In vitro 3D cell culture models bridge the gap between unrealistic in vitro 2D culture and animal models, allowing the study of human cells in a physiologically-relevant environment with the convenience and speed of an in vitro model.

With 3D cell culture the aim is to eliminate the stress and artificial responses cells experience as a result of culture on flat, 2D growth surfaces and to create more amenable surroundings which more closely mimic the natural environment for optimal cell growth, differentiation and function. 3D cell culture allows individual cells to maintain their normal morphology, allows complex cell-cell and cell-matrix interactions, and provides oxygen and nutrient gradients, thereby providing an environment which more closely mimics the natural ECM, promoting the creation of native architecture found in tissues.

3D culture systems can be divided into 2 broad categories, scaffold-free and scaffold-based methods.

Scaffold-Free Methods

Scaffold-free techniques are cell aggregate-based, such as hanging drops, ultra-low attachment plates, and magnetic biolevitation, and rely on physical forces to bring the cells together, and cell-cell adhesions to form the aggregate.

  • Hanging Drop Method

Hanging drop plates exploit the fact that cells, in the absence of a surface to attach to, will self-assemble into a 3D aggregate (spheroid). Each plate instead of containing normal wells with a traditional bottom have an opening designed to form a discrete droplet of media sufficient for cellular aggregation. Cells suspended in the media accumulate at the tip of the drop at the air-liquid interface, spontaneously aggregate over the course of hours to days, and finally form spheroids.

3D Cell Culture_Scaffold-Free Methods_Hanging Drop Method

Advantages of the technique include maintaining the natural cell-cell interactions that would occur in vivo, the fact that spheroids can be created from a relatively low number of cells (advantageous when working with rare patient-derived cells) and importantly that spheroids created can be done so in uniform size (useful in terms of experimental reproducibility). The use of hanging drop plates is simple and can be integrated with liquid handling robots, however it can nevertheless be relatively expensive.

  • Ultra-low Attachment Plates

It is also possible to use microplates where the bottom surface is pre-coated with hydrophilic material to form such cell aggregates. This coating (e.g. poly-HEMA) prevents adherence of the cells to the surface, instead forcing them to be in suspension resulting in 3D spheroid formation. The coating is stable, non-cytotoxic, and non-degradable. Spheroids can be cultured for extended periods and can be recovered after culture. However, unlike the hanging-drop method, formation of spheroids from small number of cells is challenging, and the resulting spheroids can be too heterogeneous in terms of size and composition.

  • Suspension Culture

This is an agitation-based approach to 3D cell culture in which a cell suspension is placed into a vessel where the cells are agitated continuously to prevent them adhering to the vessel walls promoting cell-cell interactions, and spheroid formation. There are 2 main methods used in suspension culture; using of a spinner flask in which a stirring element keeps the cell suspension in motion, or a bioreactor in which agitation is achieved by the vessel rotating.

3D Cell Culture_Scaffold-Free Methods_Suspension Culture

These simple approaches generate large yields of spheroids, and since the culture fluid is in constant motion, nutrients and oxygen are easily transported to spheroids. On the other hand, the spheroids formed are often non-uniform, which can be problematic for their use in drug screening where reproducibility is key. In addition to this, with regards to spinner flasks, the shear force experienced by the cells as a result of the motion of the stirring bar can affect normal cellular functions. There are options to circumvent such issues, such as forming spheroids in ultra-low attachment plates, selecting spheroids of appropriate size, and then transferring them to a spinner flask to continue suspension culture, however in some instances such an additional step maybe undesirable.

  • Magnetic Biolevitation

In this method, cells are treated with magnetic nanoparticles (a combination of iron oxide and gold) and incubated overnight to allow uptake. A magnetic is placed on top of the plate containing the treated cells causing the cells to rise to the air–medium interface and promoting cell-cell interactions.

3D Cell Culture_Scaffold-Free Methods_Magnetic Biolevitation

Spheroids start forming within a few hours and can be incubated for a few days until they reach a suitable size. Benefits of this method include the high rate of spheroid growth compared to other methods, and the sizes of spheroids achievable have been found to better replicate in vivo characteristics of tumours. On the other hand, the magnetic beads used are expensive, and they have been found to be toxic to cells at higher concentrations.

The aggregate-based, scaffold-free methods described above are relatively simple, the resulting spheroids are easy to visualise and recover, and the lack of an artificial supports means any matrix effects are minimised. Having said this, the requirement of specialised equipment means that some of these techniques can become expensive. In some instances, the formation of heterogenous spheroids can be an issue, and often the aggregates formed in such ways are sensitive to movement of the culture dish, making automation a challenge.

Scaffold-based Methods

Turning to scaffold-based techniques, in this case cells are grown in presence of a support. Scaffold-based 3D culture models can be developed using a range of natural and synthetic materials. There are two major types of support:

  • Polymeric Hard Scaffolds

Seeding cells into a solid scaffold provides a 3D space to support cells, allowing them to create natural 3D tissue-like structures (tissue being organized, well-defined, and consisting of discrete layers of alternative cell types). With regards to their use for 3D cell culture, such scaffolds can be broadly divided into fibrous or porous matrices manufactured using a range of different techniques (e.g. electrospinning or particulate leaching).

3D Cell Culture_Highly porous Polystyrene Scaffold

These methods allow for the preparation of scaffolds with defined void/pore dimensions which are critical parameters that can influence how cells will behave in the scaffold. Synthetic materials used in 3D scaffolds include biocompatible polymers such as polystyrene or PLA, porous titanium, and ceramic-based materials such as bioglass. Such scaffolds can be manufactured with control over their chemical and physical properties, thereby minimising batch variation, and offering reproducibility and consistency, a vital requirement of 3D culture models for in vitro drug screening applications.

  • Hydrogel Scaffolds

As highlighted previously, cell-matrix interactions play an important role in determining cell function and response. As such, a 3D culture model that can replicate the role of the ECM would be advantageous. In this context, both natural and synthetic hydrogels have been investigated for the encapsulation of cells due to their potential to better mimic the mechanics, composition, and structural cues of native tissues over polymeric scaffolds.  Naturally-derived hydrogels such as those based on ECM protein collagen or commercially available Matrigel (a mixture of basement membrane proteins) possess an inherent bioactivity and they can promote many cellular functions, leading to increased viability, and proliferation. However, the ability to control the properties of such hydrogels is limited, meaning the ability to tailor a 3D model for specific cell/tissue types is limited. Additionally, these animal-derived materials often suffer from poor batch-to-batch reproducibility and complex handling which are major limitations for their use in drug screening assays. On the other hand, synthetic hydrogels (e.g. PEG, PLA, peptide-based) can be consistently manufactured, and can be designed to provide both the optimal physical environment and in some cases chemical cues for specific cell types. However, in some instances, such synthetic materials can often pose significant challenges with respect to biological compatibility and cell viability.This limitation can be overcome by combining natural and synthetic materials to achieve semi-synthetic composite materials, or alternatively peptide-based hydrogels can be designed to incorporate biomimetic sequences from ECM proteins to create totally synthetic hydrogels which are able to mimic in vivo functionality.

For 3D cell culture experiments, scaffold-based systems provide a high degree of reproducibility, and provide a physical stability not achieved with scaffold-free methods, and which is required for routine assays. However, it should also be noted that for both polymeric scaffolds and hydrogel matrices visualization of cells/cell aggregates as well as or complete recovery of viable cells, whilst achievable, can be challenging.

Advancements in technology, materials science and our understanding of cell biology have led to new opportunities for culturing cells in a more physiologically-relevant 3D environment. Whilst a growing range of 3D cell culture techniques are available and can “bridge the gap” between traditional 2D culture and in vivo, for the most part, standard 2D models are still used in applications such as drug screening. When it comes to developing a 3D cell culture model, it is important to consider your ultimate requirements and thus which of the above 3D culture technologies is best suited. For generating large numbers of spheroids, scaffold-free methods might be more suitable, but for applications requiring high levels of reproducibility and control such as high-throughput screening assays, then scaffold-based culture might be more desirable.



“Halfway between 2D and Animal Models: Are 3D Cultures the Ideal Tool to Study Cancer-Microenvironment Interactions?” J. Pasquier et. al., Int. J. Mol. Sci., 2018, 19, 181.

“3D Cell Culture: A Review of Current Approaches and Techniques”, J. W. Haycock, Methods Mol. Biol., 2011, 695, 1-15.

“3D Cell Culture Systems – Advantages and Applications,” M. Ravi et. al., J. Cell Physiol.,2015,230, 16-26.

“Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro,” E. Knight and S. Przyborski, J. Anat.,2015,227, 746-756.


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