Media Component Analysis during Human Primary T Cell Culture using a Triple Quadrupole Mass Spectrometer

Significance of the Topic

Comprehensive monitoring of T cell culture media is important to optimize cell expansion and ensure therapeutic efficacy in CAR T cell therapy

Objectives and Study Overview

This study aimed to develop a fast multicomponent LC MS method for profiling culture media during T cell expansion. Two parallel experiments compared continuous culture without media exchange and culture with fresh media exchanges at day 3 5 and 7 over a nine day period

Methodology

Primary human T cells were cultured with CD3 CD28 activator cytokines and IL 2 at 37 C with 5 percent CO2
Media samples from flask A without exchange and flask B with exchange were collected at defined intervals
Samples were protein precipitated with acetonitrile centrifuged and diluted before analysis
A reversed phase gradient on a Cell Culture Profiling column of 150 mm length and 2 1 mm ID with 3 micrometer particles was used
The 17 minute LC MS method employed water and acetonitrile with 0 1 percent formic acid
Multiple reaction monitoring in positive and negative electrospray modes quantified 144 compounds including amino acids nucleotides metabolites sugars and vitamins

Used Instrumentation

• Shimadzu LC MS 8060 triple quadrupole mass spectrometer
• Shimadzu Cell Culture Profiling column 150 mm x 2 1 mm 3 micrometer

Main Results and Discussion

• Flask B showed three fold higher live cell count by hour 210 relative to flask A
• Volcano plot identified compounds with altered levels. Glutamine decreased while O phosphoethanolamine increased suggesting PCYT2 enzyme downregulation
• Time course analysis revealed serine to glycine conversion is upregulated supporting T cell activation
• Metabolic shift toward glycolysis was evident from decreasing glucose and increasing lactate confirming enhanced energy demand

Benefits and Practical Applications

Real time media profiling enables timely interventions and enhances yield quality during CAR T manufacturing
This method can support process development QA QC and research

Future Trends and Opportunities

Integration with automated sampling and feedback control
Extension to additional metabolites lipids and cytokines
Coupling with transcriptomics proteomics for systems level understanding
Application of machine learning for predictive cell culture management

Conclusion

A rapid 17 minute LC MS method provided detailed insights into media consumption and secretion during T cell culture. Media exchange improved cell expansion and induced notable metabolic shifts. This approach offers a powerful tool for optimizing CAR T production workflows

Reference

1. Osawa Tsuyoshi et al Phosphoethanolamine accumulation protects cancer cells under glutamine starvation through downregulation of PCYT2 Cell Reports 29 1 2019 89 103
2. Ma Eric H et al Serine is an essential metabolite for effector T cell expansion Cell Metabolism 25 2 2017 345 357
3. Palmer Clovis S et al Glucose metabolism regulates T cell activation differentiation and functions Frontiers in Immunology 6 2015 1