If We Are 3-D, Should We Grow Cells In The Same Dimensions? Microscopic Analysis In 3-D.

“Do we use a monolayer or 3-D cell culture for our experiments?” A simple, yet puzzling question asked by my group leader while I was working on a drug development project at the University of Abertay Dundee. How do you answer such a question? Just read on to find out!

Monolayer vs spheroid

One of the aims of the project I was working on was to measure the size of cardiomyocytes over a long period of time (approximately three days).

Have a look below. Which is easier to measure: A or B?

A is a monolayer and B is a spheroid in a 3-D culture.

Given a choice A or B? I opted for B. Here’s why:

• 3-D cell cultures are easy to handle under the microscope. Just find the blob of cells! If you have to search for a certain group of cells on a monolayer, it takes a lot of time and patience.
• When you have to measure the size of a 3-D cell culture (usually in the form of a spheroid) you make one measurement per image. This saves valuable time and efforts.
• Furthermore, a beating heart-like cell culture is ideal for Ca⁽²⁺⁾ imaging of large numbers ofcardiomyocytes (we also had to do that).

Advantages of 3-D

In addition, here are some of the advantages of 3-D cell cultures over monolayer cell cultures as listed in many research papers on Google Scholar:

• Cells grow in a tissue-like structure in vitro, which closely resembles the anatomy and physiology of a tissue in vivo.

In 3-D cell cultures in vivo–like cell-cell interactions are a precursor of increased cellular survival and more realistic gene expression and cellular behavior. This is vital when you do basic research with, for example, neurons, muscle cells or hepatocytes. All of these cells rely on a vast and dense cell network in order to survive and function properly.

• Great potential as disease models for drug testing (e.g. in vitro 3-D cancer tumors).

“Cancer is not 2-D, it is 3-D.”, said Dr. Christoph Wiesner, CSO at SeaLife Pharma, at a talk about drug discovery at our University. “At SeaLife Pharma we use 3-D cancer cell cultures to test the anti-cancer properties of various marine substances. We have found out that such cultures mimic closely the in vivo environment of cancer tumors. We have also noticed that a higher drug concentration is needed to kill the cancer cells in 3-D than 2-D culture, which is often the case for drug response of in vivo cancer cells when compared to a traditional 2-D cancer cells grown in vitro.”

• Great potential in tissue engineering (e.g. replacement of damaged tissue).

A few months ago a friend of mine, who has recently worked on a tissue engineering project, shared his experience with a few colleagues and me. “The difficult part is actually growing the cartilage cells in such a way so that they form a tissue (on a biodegradable scaffold with the right amount of growth factors and nutrients)”, he said, “once the tissue is fully formed it can used to replace damaged or worn out cartilage in the human body.”

What dimension will you choose?

Conversely, some researchers still consider 2-D cell cultures a paradigm of biomedical research. Sure, 3-D cultures are difficult to make, but there is a great variety of commercially available 3-D cell cultures (with user manuals included!) and they are no more expensive than their 2-D counterparts. So what will you choose: 2-D or 3-D?

Remember you are in 3-D, so should your cells be!