Vanessa Lobo Vanessa is an Applications Scientist with Dolomite Bio. Prior to joining Dolomite Bio, Vanessa pursued her Master’s in Bioinformatics and System Biology from the University of Manchester where she researched various “locked” forms of SARS-CoV-2 Spike protein structures using Clustering algorithms. Vanessa is now part of the Dolomite Bio Applications team where she is responsible for handling customer queries and guiding users with our technology.

5 things you need to know about 3D Cell Culture

3 min read

A (Very) Brief History of 3D cell culture

The earliest studies related to 3D cell culture in the 80s highlighted the importance of 3D techniques for creating accurate in vitro culture models. The importance of the tissue microenvironment and 3D culturing techniques for cancer research was first proposed in the early 1980s by Mina Bissell and the idea was to focus on the importance of mimicking the natural extracellular matrix (ECM) structure and shifting toward more in vivo –like cell culturing.

Consequently, 3D cell culture techniques have an important role to play in the future of cell culture research and so, we created a list of five things you need to know about it:

 

1. Hydrogel scaffolds can mimic a tissue micro-environment

3D cell culture microdroplet imagery- nadia3D on the Nadia Instrument
Two hydrogels of specific interest in the field of single cell microdroplet encapsulation are agarose and collagen, due to their versatility, compatibility and biological origin (2).

The natural ECM in living tissue is a complex network of protein and carbohydrate polymers which allows cell-cell and cell-matrix interactions and helps cells to survive, differentiate and migrate (1). Hydrogels can be thought of as 3D networks of crosslinked polymers. They have interconnected pores which enable hydration and the transport of nutrients and gases, which are essential aspects of a cell’s microenvironment. When hydrated, hydrogels are soft and rubbery, resembling the properties of living tissue.

 

2. A paradigm shift from 2D to 3D is happening

When cells are put onto coated flat surfaces to adhere and spread, it is not representative of an in vivo cell micro-environment. Cells growing in a standard 2D culture consume large amounts of growth media and produce a lot of waste leaving other cells prone to a damaged environment. Consequently, results from 2D cell culture experiments can have poor predictability of in vivo behavior. This has led to wasting large amounts of money in pharmaceutical and biotechnology industries, as a result of failed drug development.

So, why is 2D culture still such a popular choice?

They have been used since the 1900s and are well established. It is easier to compare results from existing literature. It is an inexpensive method and cultures are easy to analyse in comparison to some 3D culture systems. In general, it is also an easier technique to perform in comparison to some 3D cell culture methods. Despite the evidence demonstrating that 3D culture is better than 2D for several reasons (3), many labs have been slow or hesitant to adopt 3D cell culture technologies.

The 3D cell culture industry is slowly but steadily starting to catch up. Several fields and research groups are undertaking 3D cell culture to better mimic the in vivo tissue environment. 3D cell cultures are more accurate models of disease especially of cancerous tumors (4), causing animal models, which are not necessarily a reliable way to predict treatment response and difficult to ethically justify, to become outdated. Screening drugs against organoids is a much more reliable method.

 

3. Why keep it single?

Both clinical and basic research is increasingly focused on the growth and interactions of single cells to understand the complex heterogeneity within a tissue or tumor. This requires the separation of cells to maintain monoclonal identity.

Most 3D cell culture models for tumors, start with a large bulk of cells that are used to “seed” the culture vessel. Cells within a seed may have originated from the same population but can be phenotypically different from each other at the single-cell level. Designing effective therapeutics requires a better understanding of cellular heterogeneity. Therefore, the ideal method would also facilitate the compartmentalization of cells to maintain single-cell identity (5).

The technical challenges of 3D cell culture
Studying the heterogeneity of single cells answers many biological questions but can be technically difficult.

 

4. The benefit of generating free-floating spheroids

The nadia3D kit provides a complete solution for free-floating spheroid culture using the Nadia platform. The nadia3D kit is supplied with an all-rounded collagen matrix possessing wide-ranging properties to promote cell growth.

 

3D cell culture with the nadia3D kit on the Nadia Instrument

 

On the Nadia platform, cells can be continuously captured in droplets containing a collagen-based matrix. Using droplet microfluidics, we are able to capture thousands of cells in individual microenvironments of collagen-based matrix within a biocompatible oil shell, at an accelerated rate. The collagen-containing spheroids, once hardened, and removed from their oil shells, are spherical scaffolds which remain stable in various culture media.

These free-floating spheroids benefit both from the free perfusion of nutrients and the support provided by a three-dimensional matrix.

 

 

 

 

 

 

 

 

 

5. Taking advantage of instrumentation

Generating these spheroids on a microfluidic platform means that throughput scalability can be achieved. The Nadia’s temperature-controlled environment not only helps to keep cells alive and happy, but seamlessly facilitates the solid-liquid state transition during the scaffold generation process. Droplets production on the Nadia instrument is controlled by delivering pressure in a precise manner which produces monodisperse droplets ensuring each cell is grown in the same volume of hydrogel.

Free floating spheroids on the Nadia – an innovative technique

With the advent of 3D cell culture, in vitro studies are now able to better replicate diseases than 2D or animal models. A major challenge has been achieving throughput as well as automating individual cell picking (6). The ability to generate discrete biological culture vessels at an accelerated rate is yet to be taken advantage of by researchers.

 

Find out more about the nadia3D kit for 3D cell culture

Download the app note

 

 

 

Watch the webinar

 

 

 

Read the datasheet

 

 

 

 

Discover other Dolomite Bio reagent kits

Breaking down cost barriers for single cell research: Introducing RNAdia

Using hydrogels in single cell research- getting started with agarose encapsulation

References

1. El-Sherbiny I, Yacoub M. Hydrogel scaffolds for tissue engineering: Progress and challenges. Global Cardiology Science and Practice. 2013;2013(3):38.
2. Lee J, Cuddihy M, Kotov N. Three-Dimensional Cell Culture Matrices: State of the Art. Tissue Engineering Part B: Reviews. 2008;14(1):61-86.
3. Mishra D, Sakamoto J, Thrall M, Baird B, Blackmon S, Ferrari M et al. Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture. PLoS ONE. 2012;7(9):e45308.
4. Thoma C, Zimmermann M, Agarkova I, Kelm J, Krek W. 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Advanced Drug Delivery Reviews. 2014;69-70:29-41.
5. Dagogo-Jack I, Shaw A. Tumour heterogeneity and resistance to cancer therapies. Nature Reviews Clinical Oncology. 2017;15(2):81-94.
6. Jain R, Chittiboyina S, Chang C, Lelièvre S, Savran C. Deterministic culturing of single cells in 3D. Scientific Reports. 2020;10(1).

Vanessa Lobo Vanessa is an Applications Scientist with Dolomite Bio. Prior to joining Dolomite Bio, Vanessa pursued her Master’s in Bioinformatics and System Biology from the University of Manchester where she researched various “locked” forms of SARS-CoV-2 Spike protein structures using Clustering algorithms. Vanessa is now part of the Dolomite Bio Applications team where she is responsible for handling customer queries and guiding users with our technology.