Today, the vast majority of single-cell high-throughput technologies rely on microfluidics instruments for cell analysis, from scRNA-seq to spatial transcriptomics. However, researchers are exploring other innovative avenues in which the advantages of microfluidics could be instrumental at single-cell resolution. Tissue dissociation, transfection, organellar transfer, high-throughput compartmentalization, we summarize a few of these new(-ish) applications from their recent progress published in 2021.
Microfluidics for tissue dissociation into single cells
Many proved microfluidic methods already exist for cell sorting, sample enrichment and cell analysis, including scRNA-seq. However, they still require an initial cell suspension with clearly dissociated cells. Tissue dissociation remains the clear bottleneck in single-cell analysis to this day, being labor-intensive, often tissue-specific, and adding mechanical, chemical, or enzymatic stress impacting cell viability and triggering spurious gene expression.
Lombardo et al. have designed a microfluidic tissue-processing two-device platform that can be fully automated. Combining shearing forces, enzymatic digestion and nylon-based filtering membranes, it aims to automate splitting tissue samples into single cells while limiting stress.
The team tested their device with adult murine kidney cells for their natural heterogeneity, and checked the resulting cell viability and diversity with flow cytometry and scRNA-seq. A strong advantage of their device relies in the flexibility provided by the microfluidic aspect: the dissociation can be modulated through many parameters, such as digestion times – or even modes, such as continuous or intermittent – or flow rates for shearing and filtering to adapt to the fragility (or robustness) of the cell types of interest. After optimisation, this enabled the researchers to recover far more kidney cells compared to traditional dissociation, while limiting chemical and enzymatic stress on the cell by shortening digestion times.
What is the effect of digestion time and other sample preparation parameters on cell viability? Check out our article on technical artifacts in single-cell preparation!
Single-cell microfluidics for transfection
Traditional transfection methods are typically applied in bulk, or by hand on a few selected cells using labour-intensive and time-consuming steps. While not a new idea, microfluidics have come a long way forward towards the transfer of exogenous material into single cells at a high-throughput rate. Here are a few possibilities offered by microfluidics to inject material into cells individually:
- By needle micro-injection: instead of a mobile needle moving from cell to cell, cells are injected into microfluidic devices with a stationary needle on one end of a channel. Once the cell is physically injected with transfection material, a stream takes the cell further down a secondary channel to be collected.
- By mechanoporation: cells are injected into microfluidic channels with constriction section, where the diameter of the channel is substantially smaller than the cell itself. Forcing the cell through the constriction create shearing and compression forces opening pores in the cell membrane, allowing exogenous material to enter the cytoplasm at the exit of the section. The technique has also been combined with electroporation to further increase the transfer efficiency.
- By electroporation: electrodes can be integrated into microfluidic device to deliver a combination of millisecond- and nanosecond-long pulses to create pores in the cell membrane. The technique has been demonstrated to efficiently transfer DNA plasmids, and the single-cell configuration of microfluidics also limits the damage inflicted to cells from repeated pulses – leading to poor cell viability – in traditional electroporation settings.
Single-cell microfluidics for organellar transfer
External injection is not the only way to transfer exogenous material into single cells: Wada et al. demonstrated efficient organellar transfer – the demonstration is mainly based on peroxisomes – through an intercellular tunnel.
For their device, the researchers designed a microfluidic chip with 105 pairing structures. In each structure, a cell is captured on each end of a microfabricated aperture, before triggering cellular fusion to the edges of this tunnel. The paired cells are then effectively connected, and cytoplasmic transfer occurs, including the passage of organelles through the microtunnel. However, the researchers never observed transfer of nuclear material, probably due to the aperture width. Finally, the paired cells are disconnected from the tunnels and recovered at the exit of the flow channel.
Interestingly, the team measured that the quantity of organellar transfer was directly linked to the length of the microtunnel. Testing with shorter (4 um) or longer ( 10 um) microtunnels, they were able to modulate the intercellular transfer of organelles.
Using larger channels can also open new possibilities for single-studies, such as individual cultures and communities. Head to our millifluidics article to know more!
Single-cell compartimentalization for enzymatic assays
Compared to scRNA-seq or most proteomics assays which are extracting information already present in a single-cell, enzymatic assays require the accurate exposure of a cell to multiple reagents (e.g. an increasing concentration of substrate), media exchange for washing steps and real-life monitoring of enzymatic activity. To perform the assay evenly across single cells, this require efficient compartmentalization, with a throughput limited to the number of compartments you can afford in your device.
While microfluidics seem the perfect solution to the issue, increasing the number of compartments signifies increasing the number of microvalves, meaning maintaining higher pressure for the correct distribution of cells across all compartments. This extra pressure can put microvalves at risk of rupturing. Briones at al. just reported their in-depth study on valve design for microfluidic compartmentalization, looking at size, geometry and pressure distribution. They demonstrated that specific enhancements, such as the use of circular valves, enabled them to manufacture a microfluidic device with 5,000 valves, efficiently trapping either 3,922 Jurkat cells or 2730 PBMC cells and measuring the enzymatic activity of the granzyme B protease.
- Lombardo JA, Aliaghaei M, Nguyen QH et al. Microfluidic platform accelerates tissue processing into single cells for molecular analysis and primary culture models. Nat Commun 12, 2858 (2021). https://doi.org/10.1038/s41467-021-23238-1
- Kaladharan K, Kumar A., Gupta P, Illath K; Santra TS, Tseng FG. Microfluidic Based Physical Approaches towards Single-Cell Intracellular Delivery and Analysis. Micromachines 12, 631 (2021). https://doi.org/10.3390/mi12060631
- Chang AY, Liu X, Tian H et al. Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy. Sci Rep 10, 6061 (2020). https://doi.org/10.1038/s41598-020-63172-8
- Wada KI, Hosokawa K, Ito Y and Maeda M, A Microfluidic Device for Modulation of Organellar Heterogeneity in Live Single Cells Anal Sci 37, 3 (2021). https://doi.org/10.2116/analsci.20SCP11
- Briones J, Espulgar W, Koyama S et al. A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis. Sci Rep 11, 12995 (2021). https://doi.org/10.1038/s41598-021-92472-w