What is the difference between 2D versus 3D cell culture?

For mammalian cells to be studied, they need to be grown in an environment that mimics the body as closely as possible. This has traditionally been done by growing the cells on a flat surface, in other words, in two dimensions. This has many limitations, namely that the cells inside the body do not grow in 2D, but in 3D, which has surprisingly big implications on how the cells behave.

You might be familiar with the rapidly growing popularity of 3D cell culture, but you still have some questions left unanswered. On this page you will find what the main differences are between 2D and 3D, their advantages and disadvantages, why you might want to choose 3D, which 3D culture format might best suit your needs, and finally, how to get started on 3D cell culture.

What is the difference between 2D and 3D cell culture?

• In 2D cell cultures, cells are grown in a flat plane on top of a flat surface, whereas in 3D cell culture they are grown in a three-dimensional space, usually embedded within a gel-like matrix, or grown in a solid scaffold.
• While the formation of the cell monolayer is faster in 2D and has lower reagent costs, 3D cultures mimic in vivo environments more closely.
• On a cellular level, a key difference is the nature of interactions between cells and other cells, cells and the culture medium, and cells and the petri dish surface or the 3D scaffold surface.

What are the advantages of 2D vs 3D culture?

What are the disadvantages of 2D vs 3D cell culture?

Modelling physiological events in 2D vs 3D cell culture

When the aim is to accurately model physiological events in vitro, a 3D cell culture model is often considered to be superior to 2D models. The main advantages of 3D cell culture include more in vivo-like cell interactions, cell division, and morphology, as the 3D shape more accurately mimics the natural environment of cells. This also means that gene expression and morphology are more representative of the human body. Another key advantage is that cells have variable access to oxygen, nutrients, metabolites and signalling molecules, creating environmental niches and microenvironments whereas in 2D, cells have unrestricted access to these and in equal amounts.

How is 3D cell culture done?

3D cell culture is usually done in one of two ways, either using scaffold-based techniques such as using a hydrogel for support, or scaffold free techniques (e.g., magnetic 3D cell culture). Scaffold-based 3D culture techniques are usually recommended since they allow for better transfer of oxygen, nutrients, and waste. Hydrogels especially, are often favoured since they provide more in vivo-like tissue stiffness that can easily be adjusted depending on cell type.

Furthermore, 3D cell culture can be used for growing both spheroids and organoids. While these terms are often used interchangeably, the main difference is that spheroids are simpler, spherical, and usually contain just one cell type, while organoids are more complex, containing a variety of cellular phenotypes and thus mimic organs in miniature form. Learn more about the differences of spheroids and organoids.

Is it time to start transitioning from 2D to 3D culture?

Interest in 3D cell culture is rising exponentially. The number of articles released about the subject each year has more than tripled in the last decade. However, despite its potential, there are always challenges to overcome, as with any new technology. Some of these include cost, handling, reproducibility, and automation. Hydrogels such as GrowDex® provide an easy to use, animal-free and affordable solution that overcomes many of these remaining challenges. Given the numerous advantages of 3D cell culture and the constant improvements in technologies and protocols, researchers currently working with 2D cell culture should consider transitioning from 2D to 3D culture.

How to transfer from 2D to 3D cell culture

There are several aspects that you need to consider when transitioning from 2D to 3D cell culture and when choosing the right type of matrix. These include:

• What tissue type are you modelling?
  • This is important for choosing the right stiffness for your matrix. Visit our application notes page for further information on stiffness optimization.
• What assays will you run?
  • This is important since some assays require the cells to be easily recovered by first breaking down the cell culture matrix.
• What type of well plate are you using?
  • Unlike 2D cell culture, 3D cell culture does not require protein coated wells since cells are embedded in the matrix and do not need to adhere to the walls. With adherent cells, typically low adhesion coated plates are used with hydrogels.
• Does the culturing method need to be automation compatible?
  • Most animal-derived 3D cell culture matrices need to be handled on ice which makes automation difficult. Read more about how wood-based GrowDex® hydrogels overcome this problem.
• Do you want to use animal-derived or animal-free matrices?
  • Animal-derived matrices cannot be used for clinical purposes and they suffer from batch-to-batch variability due to foreign molecule bias. Read more about animal-free matrices.