Most cells are cultured in two dimensions: height and length (think about a Petri dish for instance). However, for several decades (since the 1970s), researchers have been able to grow cells in three dimensions.
This is what is called a 3D cell culture, an artificially created environment in which cells can grow or interact with their surroundings in all directions (and thus the “three dimensions”: height, length and depth), similar to how they would in normal organisms.
These cultures are usually grown in what is called a culture flask displaying a flat bottom, in which the cells can grow freely and horizontally on a plan surface.
Why are 3D cell cultures so important?
While 2D cell cultures (or monolayer) are not accurate representations of cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells, 3D cultures allow researchers to reproduce a better in-vitro representation of cells and their behaviour; it often makes them more reliable to predict toxicity and efficacy (this is more true for 3D organs, and less so for endothelial tissue and lymphocytes, for instance).
This culture format has proven instrumental in further understanding tissue homeostasis and cancer, it has thus been a tremendous help with research in cancer biology and tissue engineering.
How it works is that, quite simply, being closer to how actual tissue grows in organisms in terms of cellular communication and the development of extracellular matrices (that help cells move within the culture, just how cells move in actual organisms), they give us a better representation of reality for cell migration, differentiation, survival and growth.
3D cell cultures are different from 2D cell cultures in that they can have a different, more accurate gene expression – how genes influence the growth of cells, from genotype (the genetic material) to phenotype (the end, observable, result). In pre-clinical trials, researchers have to estimate drug toxicology. In that setting, testing gene expression of cells grown in 3D can be more efficient.
Finally, 3D cell cultures also have greater stability and longer lifespans than 2D ones. This means that they are more suitable for long-term studies and for demonstrating long-term effects of drugs for instance. 3D environments also allow the cells to grow undisturbed.
In 2D, the cells must undergo regular “maintenance” (through trypsinization, using an enzyme that breaks down protein to dissociate adherent cells from the vessel in which they are being cultured) to provide them with sufficient nutrients for normal cell growth, which is not needed in 3D cultures, allowing the cells to grow undisturbed.
How are cells grown in 3 dimensions?
There are a wide range of solutions claiming to grow cells in 3D. But they can all be classified into two broad types: scaffold and scaffold-free techniques.
As the name implies, scaffold techniques let cells grow in three dimensions using “scaffolds” to help them occupy space better – the objective being that the cells do not adhere directly to the plastic support but instead form a 3D structure that can stand on its own. These solutions either use solid scaffolds, hydrogels or other materials.
Natural cells grow throughout all dimensions thanks to what is called an “extracellular matrix” (ECM), which is a network of macromolecules and minerals (like collagen or enzymes) that provide structural and biochemical support to cells around it (think of it as “the natural scaffold” of cell growth).
This is why the “scaffold techniques” try to mimic this natural structure. And to do so, they can use hydrogel: interconnected pores with high water retention that can thus help move nutrients and gases throughout the cell culture.
There are multiple types of hydrogels using a wide range of source materials such as animal ECM extracts, proteins, peptides, etc. – those can be classified into two main categories: synthetic (which are standardised and simple) and animal-derived (which are highly complex and hardly standardised).
Scaffold-free techniques however try to forego this requirement to stimulate cell growth in different dimensions without the structures. Spheric 3D cell cultures are generally called spheroids.
They are most often produced using either low cell adhesion plates (cells do not spread over two dimensions and instead “clump” together in the rounded bottom of the plates), the hanging drop method (the cell aggregates are hanging from the surface of a plate, letting gravity form the spheroids) or rotating the bioreactors’ wall (as it spins, the cells are in a constant “free fall” and thus gather in layers of ultra-low attachment coating).
What is the future of 3D cell culture?
As technology progresses, so do applications for 3D cell cultures. On top of the advances in cancer research:
Creating cultures with cancerous cells to observe how it progresses and how to counteract it or creating “personalised” cultures with cells from specific patients to understand how their specific cells act and craft specific therapies for them.
And even further on that, scientists can now create organoids from patients’ stem cells (unlike spheroids, these cultures present more properties of the organs modelled; the stem cells are gathered from biopsies) for personalised medicine, to gain a better understanding of how their organs would react to therapies and diseases. To that end, researchers use 3D cell cultures derived from single-donor induced pluripotent stem-cells (iPSCs).
The most notable advance has been the creation of 3D cellular micro platforms with enhanced physiological settings, or “complex 3D cell cultures”, to model properties of organs in order to better understand how they would react to certain drugs and certain drug-drug interactions.
Not only do 3D cell cultures provide us with a better understanding of how diseases and drugs work, but they also have the potential to make drug development simpler by reducing or entirely replacing animal studies (“animal model testing”). In the far future, 3D cell cultures could enable pre-clinical trials to answer most of our research questions, and even derive entire organs from iPSCs to be transplanted.
However, to do so, scientists first needed to find ways to better control the size of the spheroids (thanks to developments on the front of ECMs or other scaffold-free techniques); now, they have been obtaining more consistent and reproducible results thanks to starting cell cultures being more uniform in size and shape.
However, they still need to perfect the organoids being created; but at some point, it will be possible to use them for organ transplants and diagnostic tools to detect and treat cancers, not only helping cancer research and medicine but also regenerative medicine (by growing tissues or even entire organs). In the meantime, these models are extremely useful to Reduce, Reuse and Recycle (the 3 R’s of waste management) and reduce overall research costs by adhering to the “fail early, fail cheaply” philosophy.