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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Barreto, Sara (Keele University)


Introduction: Human pluripotent stem cells (hPSCs) proliferate indefinitely and produce cells from the three germ layers. Such abilities have tremendous applications in the biomedical field. Large amounts of hPSCs are needed for such purposes, which require prolonged in vitro culture. Unfortunately, long-term culture is associated with genomic and phenotypic changes in hPSCs, and thus, routine characterisation is required to ensure the maintenance of a healthy state1. Furthermore, the presence of hPSCs in cell therapies has severe implications as these cells are associated with a risk of tumorigenicity. Any residual hPSCs must be identified and eliminated before clinical translation. Characterisation and, consequently, identification of hPSCs, can be done by measuring the cells’ metabolism (i.e., the concentration of metabolites in a cell). Volatile organic compounds (VOCs) are metabolites that offer information regarding the metabolic activities in cells. A strategy to detect VOCs is selected ion flow tube-mass spectrometry (SIFT-MS). SIFT-MS analyses humid gaseous samples for several compounds simultaneously and in real-time2. Here, we used SIFT-MS to investigate the VOC profile of various hPSC lines to find differences and/or commonalities between the lines.

Methodology: The hPSC lines SHEF-1, SHEF-2 and ZK2012L were cultured in xeno-free conditions. Each line was seeded at a specific split ratio, so confluency at 70-80% would be reached after 3 days in culture. hPSC conditioned media (CM) was collected on day 1 and day 3 post-passaging and transferred into 150mL glass bottles for SIFT-MS measurements. Media-only controls were also created, corresponding to media incubated on vitronectin-coated culture vessels without cells for the equivalent time as the cellular counterparts. The bottles’ headspace was purged with dry and sterile air (20% oxygen and 80% nitrogen mixture), and bottles were incubated for 16 hours at 37°C. The gaseous headspace above the cells was then measured and analysed using multiple ion monitoring (MIM) mode with H3O+ and NO+ precursor ions. Samples were measured for approximately 50 seconds. Samples were normalised to 4% water level.

Results: Overall, MIM data showed that the concentration of VOCs generally decreased in hPSC CM collected at day 3 compared to day 1. Additionally, each cell line displayed distinct amounts of individual VOCs. hPSCs were also positive for pluripotent markers (NANOG, OCT4, SSEA-4 and alkaline phosphatase), analysed by immunohistochemistry and flow cytometry, which validated the results observed from SIFT-MS.

Conclusions: The distinct VOC profiles observed for individual hPSC lines may reflect the inherent high variability associated with hPSCs1. Furthermore, these differences were detected without apparent changes in protein expression. Therefore, SIFT-MS can identify smaller changes in hPSCs behaviour that pass unnoticed when using standard characterisation techniques. SIFT-MS represents a non-invasive method that provides a detailed characterisation of hPSCs. This is a significant advantage over current non-invasive methods, like live staining, which are limited in the information they offer regarding hPSC status. SIFT-MS can be a valuable resource in the monitorisation of hPSCs for cell manufacturing and clinics.

1Kato, R. et al., Sci. Rep. 6, 1-12 (2016).
2Rutter, A. V. et al., Analyst. 138, 91-95 (2013).

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