Filtration & fluid control
Here, microfluidics allows single cells to be boxed within sub-nanolitre volumes. “When a cell secretes something or is lysed and releases something, it gets concentrated at a relatively high level and you can detect it more easily,” he adds.
Microfluidic single-cell analysis can achieve single- cell resolution in detecting metabolites, ions or small molecules. This generates detailed data into how these molecules regulate cellular processes, which are insights that could advance novel therapies.
Microfluidic droplets
Researchers employ different microfluidic approaches to trap single cells. These include cell-sized microwells and micropatterning, a technique to etch surface patterns with sticky spots to bind cells. However, these methods are limited by poor capture efficiency and non-uniform cell distribution, respectively. That’s why the focus of single-cell microfluidics research has shifted to droplet microfluidics in recent years. It involves capturing cells inside tiny droplets, with little risk of cross-contamination. These cells can then be isolated: varying the flow rates of the cell suspension and oil-based droplet fluid as they mix allows for the number of cells in each droplet to be manipulated, to the point where just one is present. Not only does this technique have a low sample requirement, but it has high sensitivity and multiplexability, and can be done at high speed. Droplets can be split or merged to facilitate downstream analyses. They can also be monitored over time, revealing the dynamics of their contents. For example, a pair of cells encapsulated together, such as a virus and its host cell, could provide insights into cell-cell interactions. The droplets can be lysed to extract their contents, while differences in metabolite concentrations between droplets can indicate the phenotypic variability between cells in a sample. Then there’s single-cell sequencing, which investigates genetic and transcriptional heterogeneity of cells in a population. In single-cell RNA sequencing (scRNA-seq), cells are isolated into droplets, tagged with unique barcodes and their pooled RNA is transcribed and sequenced. This quickly tells us which genes are being expressed in each cell. Early on, scRNA-seq was limited to cell populations with large numbers. “But then if you have to study rare populations, this is problematic because most droplets are empty,” says Miguel Xavier, a researcher at the International Iberian Nanotechnology Laboratory. Advances in scRNA-seq now make it possible to sort only occupied droplets, allowing researchers to study individual cells within rare cell populations. “Even if you’re looking at a population of 100 cells, like circulating tumour cells, you would be able to sequence all of them,” says Xavier.
This unlocks a new utility of droplet microfluidics in drug development. A major hurdle to developing therapies targeting circulating tumour cells is how rare yet diverse they are. If researchers can trap these cells in droplets, “then you can test different drugs or different concentrations of drugs for these cell populations”, says Xavier.
Other advances could further improve the performance of microfluidic single-cell analysis. In droplet microfluidics, detection relies on highly specific fluorescent labels, which limits what metabolites can be analysed. And in the future, AI-based imaging methods could enable label-free detection of droplets, automating ultrafast detection of all kinds of metabolites. Perturb-seq is a technique that combines CRISPR gene editing with single-cell RNA sequencing. “With Perturb- seq,” says Di Carlo, “you can link functional changes in libraries of cells with downstream transcriptomic changes.” It illustrates the cellular responses to complex gene modifications and the regulatory pathways involved.
The cellular context
What cells do depends on their environment, including the extracellular matrix and their location in the tissue. For instance, cues from the extracellular matrix determine how and when a stem cell differentiates. While microfluidic technologies can isolate single cells, they usually don’t account for signals from the extracellular matrix. As a result, existing microfluidic single-cell analysis leaves out many crucial details. “Except maybe some circulating immune cells, single cells are not in isolation in our body. Most cells are close to proteins and extracellular matrix and other cells,” says Di Carlo. New developments like producing single-cell microgels with droplet microfluidics are addressing this limitation.
For example, researchers are placing single cells inside little pockets in hydrogel, a biocompatible, cross-linked polymer. “Hydrogels mimic the native tissue environment,” says Castro Johnbosco, a PhD researcher at the University of Twente. In a 2024 study published in the journal Advanced Functional Materials, Johnbosco and his colleagues provided biomechanical cues to stem cells encapsulated in single-cell hydrogels. “You give each cell a unique microenvironment and by tuning it, you can regulate the stem cell phase,” he explains.
The location of different cells along a tissue also contributes to cellular heterogeneity. This is why there is a push towards spatial omics, an approach to combine genome or transcriptome analysis with mapping the position of individual cells. “Being able to look at the transcriptomics of single cells and
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