Using hydrophobic surfaces to isolate rare cells

Wilko Duprez
Meysam Rezaei
Single-cell Research Using hydrophobic surfaces to isolate rare cells

Manually identifying and picking up rare cells from large samples can be a serious challenge, with the risk of missing and damaging precious material. A team at the Warkiani lab in the School of Biomedical Engineering, at the University of Technology of Sydney, has come up with an elegant solution using hydrophobic surfaces and chips to isolate and recover genetic material from such rare cells.

Fighting a time-consuming, frustrating cell selection process

“We wanted to develop a non-invasive prenatal diagnosis tool, which meant purifying, isolating, and picking up fetal cells from blood samples to perform genetic analysis to detect genetic disorders in the early stages of pregnancy,” explains Meysam Rezaei, first author on the paper.

“Initially we used a microfluidic device to exclude white blood cells and enrich the sample with fetal cells based on size. Despite our size exclusion and enrichment steps, we would still end up with around 5,000 white blood cells per mL of blood, for a 20mL sample. I would then use a microscope and a cocktail of antibodies and spend hours looking for fetal cells using fluorescence, then trying to pick them up with micromanipulators. When cells are floating in layers of media or PBS, it is very hard to separate specific rare cells.”

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“It is a nightmare to manually pick individual cells in PBS. If you miss, if you move even slightly, you lose the cell and you have to spend time manually scanning for it again. This step could take me up to 6 or 7 hours!” – Meysam Rezaei

The team had then to come up with a solution, an extra purification step to remove the unwanted remaining white blood cells and make it easier to select fetal cells. “We initially added a size-exclusion filter trap to our purification method, and we were successful in easily isolating fetal cells. The issue was, we could not pick them up off the filter. We attempted to lyse the cells directly on the filter, but we realized we were losing a lot of genomic material in the process.”

The researchers then turned towards the possibilities offered by hydrophobic surfaces.

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Wondering how many cells you should use? Check out our toolbox about sample size and sequencing depth!

Etching glass with acid to isolate single-cell droplets

Inspired by a previous publication in the lab, the team tried their hand at creating hydrophobic surfaces on a slide that would allow them to easily separate individual cells and identify the rare cells they needed.

Using a static droplet generator, they randomly distributed the pre-enriched sample into hundreds of droplets on a slide. The trick was to find a surface hydrophobic enough to prevent droplets from merging with each other, but not too hydrophobic so that droplets would skate off the surface at the slightest vibration, such as by opening of the lab doors.

They find a compromise by roughening a silicon-coated glass slide with sulfuric acid, reducing the contact area of the droplet with the surface of the slide while maintaining it in place.

Once hundreds of droplets are randomly spread on the surface, the slide is scanned using fluorescence to identify fetal cells, a step easily automated. Existing scanning software can also easily identify multiplets, meaning two or more cells ending up in the same droplet.

With the exact position of the droplets containing the rare cells of interest being known, a lysis buffer can be directly injected into the droplet using micromanipulators. The resulting freed genetic material from the lysed cell can then be pipetted out from the droplet into a tube for reverse transcription, amplification, and sequencing.

On top of a simple method to successfully isolate rare cells, the reduction in the required amount of lysis buffer was another success.

Rezaei adds: “Using less lysis buffer meant that we ended up with less diluted genetic material, which helped us a lot for downstream processing such as next-generation sequencing.”

A second, welcome improvement is the high hydrophobicity of the surface reducing the non-specific adsorption of freed RNA transcripts onto the slide, thus limiting the loss of important genetic material.

“We had satisfactory results from the start with this approach, but there still was a lot of room for improvement in terms of the spacing and how many cells you can actually analyze,” comments Payar Radfar, co-author on the paper.

Designing chips with hydrophobic surfaces for manual handling

Pushing the idea further, the researchers then designed static droplet array (SDA) chips based on the same acid-roughened hydrophobic surface, but with dedicated 2mm pockets to host the droplets with individual cells (compared to the previous random distribution on a slide). The pockets were also designed so that once a rare cell has been identified, the lysis and DNA/RNA recovery process can be performed with a classic hand-held pipette, bypassing the need for micromanipulators.

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“The idea was to eliminate the need for the cytospin and the micromanipulators step which in many settings, especially clinically, is a bit troublesome.” Payar Radfar (co-author).

Automating the process could reduce unwanted gene expression linked to stress.

It is established that lengthy sample processing time induces spurious gene expression from stress-related markers, altering the resulting dataset from the original physiological conditions. The use of the hydrophobic surfaces and the SDA chip enables partial automation and speeding up the process.

“Using microfluidics, it takes up to 45 minutes to enrich fetal cells from 20mL of blood for the first step,” lists Rezaei. “Add another 90 minutes for staining, and between 30 minutes to an hour to scan your slide and find your rare cells. Then you can lyse them and recover genetic material. The whole process now takes only up to 4 hours, maybe half a day”.

A definite step-up from sitting in front of the microscope for hours trying to pick eluding cells floating in medium.

A polyvalent approach open for multiple improvements.

For now, this methodology is restrained to ultra-low throughput objectives, picking a few rare cells and recovering the genetic material manually. However, it has a lot of potential for development, including the potential use of genetic barcodes to label the mRNA from individual cells.

“While you will never run thousands of cells with this technique, the barcoding can be easily integrated into the process. If you are already running a high-throughput single-cell sequencing experiment in parallel, you could add barcode beads to your isolated rare cells on the SDA chip, recover the mRNA, and process it along with the rest of your sample.”

The team is currently working on a new device and methodology, using a two-step process to add easily add barcode beads to droplets featuring rare cells.

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