7 Tips for Successful Development of 3D Cell Culture Assays

Cell studies traditionally involved placing cells on 2D surfaces. While these studies have been tremendously helpful at advancing biomedical research, it is now accepted that cells in 2D fail to faithfully represent many key features of the physiology of cells in living organisms. Not surprisingly, it is mainly because cells in living tissues evolve in three dimensions!

In 3D, cells are forced to interact more heavily through their entire surface area with their micro-environment, either with other cells or an extracellular matrix (ECM). In contrast, cells cultured in 2D typically have half of their surface area exposed to glass or plastic, the other half is exposed to media.

Recreating more complex micro-environmental interactions in 3D is of utmost importance as we now know that cells in vivo are heavily affected by their surroundings, especially by the ECM.¹ Notably we now know that the micro-environment can affect the response of cells to drugs.^2,3 This highlights how important it is to mimic the micro-environment for in vitro drug testing.

The shift in developing 3D cellular models is therefore helping create more physiologically relevant in vitro models. This serves two main purposes: testing drugs in vitro more efficiently⁴ or pre-conditioning cells better prior to their implantation in patients.⁵

While the shift to 3D cell culture has been under way for the last decade, it is still evolving rapidly to meet the ever growing and dynamic pharmaceutical demand. As of today, 3D cell culture relies on a myriad of formats (often combined), relying on the use of hydrogels, bioreactors, engineered scaffolds or microfluidic platforms.⁶ Here we provide tips which we deem essential for anyone carrying out 3D cell culture.

Figure out early on how to make your cells fluorescent

While in 2D, bright field imaging is often good enough to visualize your cells, in 3D things become harder to resolve as cells evolve in more than one plane. Making your cells fluorescent can immensely facilitate their 3D visualization. It is not trivial to obtain fluorescent cells. You can either buy fluorescent cell lines or transfect them yourself if your lab has the expertise. In either case, these cells would have the advantage of stably expressing a fluorescent reporter and thus permanently emit fluorescence.

If you are dealing with primary cells, that are either sensitive or that need to be isolated fresh, repeatedly transfecting them every time might not be ideal. In that case, consider using dyes that can be up-taken by cells making them temporarily fluorescent (e.g. CellTracker by Invitrogen). This method however only enables fluorescence for a short time and can be toxic to cells. If you do transfect your cells, it is a good idea to fluorescently sort them so that you isolate a homogeneous population of cells that are highly fluorescent. Otherwise, the heterogeneity in fluorescence amongst your cells will make it hard to adjust your imaging settings to detect them all.

In parallel, think carefully of what you want to analyze: there are ways of staining for particular regions of the cells, for example, just the nucleus or the membrane or the whole cytoplasm. Staining for the whole cytoplasm might not be ideal if you are trying to analyze fine protrusions or membrane dynamics. In any case, remember that rendering cell fluorescent can affect their physiology, so it is always wise to compare them to identical cells that are non-fluorescent.

Choose the appropriate platform for imaging needs

There are many platforms amenable to 3D cell culture, ranging from conventional well-plates, to bioreactors or microfluidic platforms. An important criterion for choosing the right platform for your experiments will be your imaging needs: if you need to image, and especially at high resolution, you want to make sure that the platform you are using offers the appropriate imaging depth. In other words, the cells need to be close enough to the objective to achieve the resolution you need. This is relevant for 2D cell culture experiments too, but 3D cell culture adds an extra depth to your assay. Therefore, you need to ensure that the cells in your 3D construct are now not too far from the objective.

Choose the appropriate ligands for your scaffold

In vivo, cells interact with adhesion sites on the proteins that make up the ECM (called integrins) which greatly affects the cell’s behavior.¹ Different organs are made up of distinct combinations of ECM proteins, and each ECM protein is composed of unique sets of integrins. To decide which ECM to use in your assay (such as collagen, fibrin, laminin or Matrigel) a good starting point consists in deciding which organ you are trying to mimic: for example, not all organs are particularly rich in collagen I, such as the brain. Note that you can also functionalize your polymer yourself by adding ligand sites,⁷ or one can create a hybrid hydrogel by mixing natural and synthetic polymers. The natural polymer provides biological functions while the synthetic polymer provides tunable mechanical properties.⁸

Control the stiffness and pore size of your scaffold

You need to consider the concentration and cross-linking of the matrices your cells are embedded in, as this will affect their stiffness and pore size. Stiffness and pore sizes are known to affect cell migration and physiology and response to drugs. It also varies between organs; one should therefore tune these variables according to the tissue they are trying to recreate. An important problem lies with the fact that stiffness and pore size variables are not independent from each other. Often, to increase stiffness one increases the concentration of the polymer; this however will most likely decrease the pore size of your hydrogel. Therefore, it can be hard to isolate the effect of these variables independently, although a few groups have successfully done so.^9,10

Think of coating the surfaces of scaffolds

Cells do not like all surfaces equally. While this is especially important for 2D cell culture, where most cells are in direct contact with surfaces, it is also relevant for 3D cellular assays. 3D cellular assays can contain cell monolayers that are part of a larger and more complex tissue-like model. For example, cells have been seeded on top of polydimethylsiloxane (PDMS) membranes to mimic the lung-air interface.¹¹ or cells have been seeded on PDMS microfluidic channels to re-create a cylindrical endothelial monolayer covering a 3D hydrogel.¹² In these cases, one needs to pre-coat these surfaces with ECM such as fibronectin to facilitate cell attachment. Alternatively, surface coating can also ensure hydrogel attachment¹³ to your platform.

Do not forget diffusion now matters!

In 2D monolayers, all your cells are exposed to media: they are all equally and directly nourished and oxygenated. In 3D things change. Cells that are embedded into a 3D scaffold must rely on the diffusion of the media through the scaffold. Diffusion is inversely proportional to distance. This means that cells at different depths in the 3D construct can be exposed to different (and insufficient) levels of nutrients or oxygens, establishing stable or transient gradients. This for example is a well-known phenomenon in large tumor spheroids, where a necrotic core forms due to a reduced amount of oxygen, nutrients and ability for CO2 and waste removal.¹⁴ This type of gradient is found in the body in selected processes and can be therefore interesting to reproduce in vitro. However, one must be conscious of the establishment of these gradients in 3D cell culture as they might not always be desired if they cause some cells in your scaffold to be insufficiently nourished. In the body, vascularization of tissues allows cells that are in proximity to the vessels to remain nourished and viable. Thus, unless you decide to vascularize your 3D cellular assay or design other strategies to over-come hypoxia,¹⁵ make sure you control the depth of your construct and acknowledge the possibility of these gradients, as they can affect the physiology of the cells.

KEEP CALM and KEEP IT SIMPLE

Most important of all, try to keep your assay as simple as possible. In the current race towards implementing the most physiologically relevant models, one can easily be tempted to add many cell types, or other variables, even though it does not directly address your biological question. Make sure you try to answer as many preliminary questions as you can with the simplest assay possible, and only then move towards more sophisticated experiments.

References 

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12. Zervantonakis, I. K. et al. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc. Natl. Acad. Sci. 109, 13515–13520 (2012). 
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