Deep freeze

Here’s a challenge: grab a handful of brightly coloured jelly beans, put them all in your mouth and chew. Then try and figure out all the individual flavours. Although you might be able to spot distinctive cherry or tangy lemon, you would probably struggle to identify every single taste. But pop them into your mouth one at a time and each flavour is easy to distinguish.

This scenario is very similar to the problem experienced by researchers investigating the changes in gene activity that happen in individual cells during the development of diseases such as Alzheimer’s and cancer. Previously, scientists had to mash up tissue samples containing many thousands or millions of cells and look at the average overall result – just like eating a whole handful of jelly beans at once.

Thanks to improvements in technology, researchers can now look at gene activity patterns in single cells taken from healthy or abnormal tissue and get a true readout of its individual ‘flavour’. But getting the right kind of samples for single cell analysis isn’t easy.

“We need to start with living cells extracted from fresh material,” explains Holger Heyn, leader of the Single Cell Genomics team at the CNAG-CRG. “Here in Barcelona we are right next to the hospital and have all the machines we need to separate the cells. But this simply isn’t possible for many medical or research facilities.”

Instead, tissue samples taken from a patient are often preserved with formaldehyde so they can be sent off for analysis elsewhere. But this preservative effectively glues all the cells together so they can’t be separated. Alternatively, samples can be snap-frozen with dry ice or liquid nitrogen, although this damages the cells so much that they disintegrate upon thawing. And without single cells, researchers can’t do single cell analysis.

So Heyn decided to develop an alternative method that could be used to preserve samples gathered from anywhere in the world while still allowing single cells to be separated out at a later date.

To develop their new method, Heyn and his team took his inspiration from cryopreservation. This technique is usually used to preserve living cells and tissues such as human eggs or embryos in IVF clinics, although some adventurous people choose to cryopreserve their whole body or brain after death (but although there is a good success rate from thawing IVF embryos there are no examples of cryopreserved humans being brought back to life!)

During cryopreservation, the tissue sample is mixed with a special solution containing a gentle preservative chemical called DMSO, along with a protein-rich serum derived from blood, and is then slowly cooled down to -80˚C in a laboratory freezer or -200 ˚C in liquid nitrogen. The first cooling steps can even be done in a coolbox or portable chiller, making it possible to gather samples from far-flung locations outside a hospital setting.

Once safely frozen, the cryopreserved samples can be stored for at least six months, waiting to be thawed, minced and broken down with enzymes to release single cells for analysis. After testing the technique with cells grown in the lab, blood, bowel and cancer samples, the researchers found that although some of the cells are damaged and lost, a significant proportion survive intact.

After perfecting their technique, Heyn and his team can now recover around 90 per cent of cryopreserved blood cells, although this figure is lower for solid tissues such as tumours. Importantly, they have proved that the freezing process doesn’t affect the patterns of gene activity in cryopreserved cells compared with fresh tissue, opening up the possibilities of single cell analysis to research teams who don’t have direct access to such complex facilities.

“Our method is very cheap and easy, and you don’t need anything fancy,” explains Heyn. “We didn’t expect that it would work this well – there are no signs of ‘shock’ from the freezing and we are pretty confident we are getting a representative sample of the tissue back as it was in life.”

Because the cryopreservation technique is so simple, many hospitals are now switching to collect samples in this way in the hope that they can set up a collaboration with Heyn or another single cell genomics lab in the future. For example, doctors can gather cells from a tumour at the point of diagnosis, then take more samples as the disease responds to treatment or develops resistance and relapses, using single cell analysis to map the detailed changes across all the genetically distinct pockets of cells that make up a tumour.

Furthermore, researchers working in developing countries now have a way of gathering samples for research projects involving single cell analysis, which would have been impossible in the past.

“All samples are complex and composed of different cell types,” Heyn says. “But this fine-grained analysis on a single cell level enables us to generate new knowledge about what’s really going on inside tissues, how they are composed and how they are functioning. Our cryopreservation method is going to open up a whole world of single cell samples – it’s a real gamechanger.”