Simple Evaluation of Cultured Cells: Evaluation of Differentiation State of iPS Cells Using Benchtop MALDI-TOF MS

Significance of the Topic

An efficient, rapid method to assess the growth and differentiation state of induced pluripotent stem (iPS) cells is critical for regenerative medicine, basic research, and quality control in industrial applications. Traditional evaluation techniques are time-consuming and labor-intensive. The integration of benchtop MALDI-TOF mass spectrometry with automated analysis and statistical software offers a streamlined approach for characterizing cellular phenotypes and culture media profiles.

Objective and Overview

This study aimed to explore whether direct MALDI-TOF MS profiling, combined with automated data acquisition (SampleStation™/AuraSolution™) and multivariate analysis (eMSTAT Solution™), can reliably distinguish undifferentiated spheroids, differentiated spheroids, and adherent differentiated iPS cells, as well as corresponding culture media at different time points.

Methodology and Instrumentation

Sample Preparation:

• Cell samples: Undifferentiated spheroids, differentiated spheroids, adherent differentiated cells cultured for 7 days under varying medium conditions.
• Culture media samples: Supernatants collected at days 2 and 7 under undifferentiated and differentiated conditions, with or without gelatin coating.

Cell Processing:

• Twice washed in PBS, then treated with 10% trichloroacetic acid and ultrasonic cleaning.
• Pellets suspended in acetonitrile and mixed 1:1 with CHCA matrix (5 mg/mL in 50% acetonitrile + 0.1% TFA).
• Deposited 1 µL on MALDI target and air-dried.

Instrument and Data Acquisition:

• Shimadzu MALDI-8020 benchtop linear MALDI-TOF MS.
• Positive linear mode, mass range 2,000–30,000 Da, 200 Hz laser repetition, 20 shots/profile, 1,156 profiles (cells) or 676 profiles (media).
• Automated sample loading and spectrum collection via SampleStation™/AuraSolution™.
• Statistical grouping by eMSTAT Solution™.

Main Results and Discussion

Multivariate analysis of the cell spectra distinctly separated three cellular states: undifferentiated spheroids, differentiated spheroids, and adherent differentiated cells. Similarly, culture media spectra clustered according to differentiation status and culture time, with differentiated supernatants at day 7 grouping closely with media from adherent differentiated cells. These patterns demonstrate clear mass spectral fingerprints associated with each stage and condition, supporting the feasibility of rapid, label-free discrimination.

Benefits and Practical Applications

• Reduces time and complexity of cell state evaluation in research and manufacturing workflows.
• Enables non-destructive, direct profiling without extensive sample preparation or labeling.
• Supports high-throughput screening of multiple samples with automated instruments and software.

Future Trends and Applications

Emerging directions include integrating these MALDI-TOF workflows with online culture monitoring, extending the approach to other stem cell types, and coupling with machine learning for enhanced predictive analytics. Expanded libraries of mass spectral signatures may facilitate broader quality assurance in biomanufacturing and clinical research.

Conclusion

This preliminary study demonstrates that benchtop MALDI-TOF MS, combined with automated analysis and statistical software, offers a rapid, reliable platform for evaluating iPS cell differentiation states and culture media profiles. The method holds promise for simplifying workflows in regenerative medicine research and industrial applications.