Controlling endotoxin contamination in PDMS-based microfluidic systems for organ-on-chip technologies

In the past decade, organ-on-chip (OoC) systems have gained significant attention as advanced platforms for replicating the physiological microenvironments of various organs. These microfluidic devices allow in vitro cell cultures at the microscale, integrating biophysical and biochemical cues that traditional cell culture models cannot reproduce. A critical aspect of their functionality is sterilization, which is necessary to eliminate bacterial contamination. However, conventional sterilization methods often fail to remove bacterial endotoxins, such as lipopolysaccharides (LPS) from Gram-negative bacteria. These endotoxins are potent pyrogens that can induce fever and profoundly alter cellular behavior, thereby compromising the reliability and accuracy of OoC models. This issue is particularly challenging for OoC systems fabricated with hydrophobic materials like polydimethylsiloxane (PDMS), which readily adsorb endotoxins. In this study, we developed an analytical method to quantify endotoxin levels in PDMS-based microfluidic devices using a Limulus Amoebocyte Lysate (LAL) assay. We tested two groups of devices: those made with PDMS from a batch opened for over a year (“1-year-old PDMS”), and others made with PDMS from a batch opened for just one month (“1-month-old PDMS”). We also compared contamination levels after 1 h and 1 week post-sealing by oxygen plasma treatment. Storage time (period from sealing to testing for endotoxin) displayed critical for contamination level, revealing that oxygen plasma treatment is effective in reducing endotoxin from PDMS surfaces. This result was also confirmed by FTIR analysis. Our findings emphasize the critical need for rigorous contamination control in the manufacturing of OoC systems to ensure they are not only sterile but also endotoxin-free. Achieving this dual standard is essential for maintaining the reliability and performance of these innovative platforms in biomedical research and therapeutic development. • PDMS adsorbs endotoxins, risking contamination in OoC device fabrication. • Oxygen plasma sealing reduces endotoxins but effects diminish over time. • PDMS hydrophobic recovery reintroduces endotoxins to device surfaces. • Endotoxin detection optimized via LAL assay and FTIR confirms plasma efficacy.