Single cell spectrometry: Inkjet printing and ambient mass spectrometry for lipid profiling

Single cell printing

Single cell before LESA analysis

Bioprinting is one of the current buzzwords in science. It involves printing living cells layer-by-layer to create precise structures made up of living tissue. It is a fast-growing area of research and there have already been some astounding developments, like the printing of functional blood cells and cardiac tissue. Scientists are predicting that we will be able to print human tissue for toxicology tests in the near future, thereby reducing the need for animal testing. Bone scaffolds, in vivo skin printing for wound repair, and cosmetic repair are also anticipated. Once the problem of cell manipulation was solved, bioprinting started to expand rapidly and there are many organisations now working in this area. But there remains a keen interest in the analysis of individual living cells and bioprinting can help here by placing single cells alone or in microarrays on surfaces ready for analysis. The key to the printing process lies in the use of special bioink formulations which maintain cell viability and prevent the individual cells from aggregating or settling. In this way, they can be printed from several different types of commercial inkjet print heads.

This technology has been adapted by scientists from the University of Wollongong in Australia who have used their own printer design. Stephen Blanksby, Marc in het Panhuis and colleagues coupled their system with an automated ambient mass spectrometry technique for the specific analysis of phospholipids in single cells which had been printed in microarrays.

Linking single cell microarrays with mass spectrometry

     Single cell after LESA analysis  

Cells from three different mouse lines were each suspended in a bioink formulation consisting of a microgel suspension of gellan gum (a polysaccharide used as a gelling agent) in the DMEM cell culture medium containing two surfactants. The suspensions were loaded onto a commercial piezoelectric inkjet printing head that is normally fitted in a conventional printer for printing ink onto paper. The printing head was mounted 1-2 mm above a glass slide in a novel bioprinting setup, details of which are in press (the citation is listed below). The slide was marked with circles in an 11 x 4 grid and the cells were printed within these circular regions. For comparison, single droplets or squares of 3 x 3 or 10 x 10 droplets were deposited in each circle then allowed to dry under nitrogen gas.

The lipids on the cells were observed by liquid extraction surface analysis (LESA) linked directly to chip-based nano-electrospray ionisation mass spectrometry in a fully automated process. An extraction solvent was dispensed onto the single cell then withdrawn into the pipette after 7 seconds. A voltage was applied to the pipette tip to create a spray for electrospray ionisation and the ions were detected in a triple quadrupole mass spectrometer. A stable spray could be produced for up to 15 minutes from a single cell extract, which improves the sensitivity and reproducibility of the mass spectrometric analysis and allows ample time to carry out different types of scans, including various precursor ion scans and neutral loss scans.

Cell types differentiated by phospholipids

The bioprinting process placed an average of one cell per droplet, following the Poisson distribution. On average, 37% of the cells contained a single cell and they could be imaged using a light microscope. Cells that had been printed for several hours before analysis were found to be susceptible to oxidation via ambient ozonolysis, with the unsaturated lipids being affected. This was countered by turning off the cooling fan and conducting the mass spectrometric analyses with a covered ion source to eliminate the air flow. Under these conditions, different classes of lipids in the cell membrane could be detected. Using class-specific precursor ion scans, which are a common way to differentiate between phospholipid types, phosphatidylcholines (PC), sphingomyelin (SM), cholesterol esters and ceramides were all observed in microarrays containing up to 100 cells per spot. The high abundances of PC and SM in cell membranes persuaded the researchers to concentrate on these two classes while proving the technology.

When the lipids were analysed from single cells, the same PC and SM were detected with similar relative abundances to those from the multi-cell spots, but at a slightly lower signal-to-noise ratio. The spectra provided a unique fingerprint of cell type, allowing the three cell lines examined here to be distinguished. A principal components analysis on the spectra from spots containing up to 100 cells or just a single cell showed excellent grouping, proving that each cell had a unique lipid fingerprint. The printing process did not affect the lipid composition. When the data from the electrospray mass spectra of non-printed cell extracts were added to the PCA plots, they correlated well with the LESA data of printed cells. The research team point out that the combination of single-cell printing and LESA mass spectrometry can be applied to any cell types. It could be used, for example, to study how lipids are affected by the cell microenvironment or by the introduction of drugs. Single cells printed in large arrays would allow large comparative studies to be conducted in a short time at the cellular level.