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?

Advantages of 2D cell culture Advantages of 3D cell culture
Fast proliferation and colony formation (minutes to hours) 3D mimics tissue and organ structures
Simpler procedures In vivo-like cell-cell and cell-environment interactions
Lower reagent cost In vivo-like concentration gradients of essential compounds
More traditionally performed and accepted Preserved morphology and molecular mechanisms
Very good reproducibility Heterogenous cell polarities and phenotypes present, more representative of native tissue architecture
Suitable for high throughput Mimics tissue stiffness
  Good reproducibility
  Some models are suitable for high throughput applications

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

Disadvantages of 2D cell culture Disadvantages of 3D cell culture
Cells forced into planar shape, does not mimic native structures Slower culture formation due to physical restraints of the matrix (hours to days)
No cellular microenvironment More complex procedures
Lacking complex cell-cell and cell-environment interactions Higher reagent cost
Unrestricted access to essential compounds, unlike in vivo Fewer commercially available tests
Different cell morphology and molecular mechanisms compared to in vivo  
Stiffness of surrounding tissue not replicated  

What is 3D cell culture used for?


Drug discovery

3D cell culture can be used as a cost-effective screening platform for drug development and testing. The improved cell functionality and morphology of 3D cultures compared to 2D makes them a promising option for use in high throughput screening (HTS) and high content screening (HCS) applications.

Cancer research

Due their ability to mimic the body’s natural environment, 3D in vitro studies have revealed insights into tumorigenesis that have not been detectable with traditional 2D models. This means that 3D cell culture could play a central role in unravelling remaining unknowns around cancer mechanisms.

Differentiation studies

3D cultures have been widely used in stem cell research, as cell differentiation and the cell microenvironment can be modelled in much more detail in 3D compared to 2D. In 3D, the environment stiffness, ECM (extracellular matrix) proteins and cell-cell interactions can be tuned. In addition to improving our understanding of stem cells, stem cells grown in 3D models can be used for tissue engineering applications.


Gene and protein expression studies

Due to the vast variety of proteins involved in maintaining the 3D structure of cells and in mediating intercellular binding, the transcriptional and translational profiles of 2D cultures is significantly altered which can impact the behaviour of your mechanism of interest. Therefore, 3D cell culture is a much safer option for mimicking the in vivo expression of genes and proteins.

Cell physiology studies

3D cell culture provides an improved platform for studying physiological aspects such as the cell cycle, cell proliferation, apoptosis, cell adhesion and cell motility.



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. 


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?
  • Do you want to use animal-derived or animal-free matrices?