Development of pyrogen-free utensils for cell culture by combining ozone/hydrogen peroxide mixed gas sterilization with steam-washing technology

Objective

The aim of this study was to develop disposable cell culture utensils with improved cleanliness.

Methods

Pyrogen contamination levels were evaluated using a direct human cell-based pyrogen test (HCPT). Pyrogen inactivation tests were performed using low-temperature ozone/hydrogen peroxide mixed gas treatment. Residual hydrogen peroxide was quantified using a commercially available measurement kit, and the effectiveness of vacuum drying and steam washing in removing the residual hydrogen peroxide was evaluated. The physicochemical properties of the materials were analyzed by measuring FI-IR spectra, static contact angles, and tensile strength.

Results

The HCPT of commercially available pyrogen-free products showed that two out of eight pipette chips were contaminated with pyrogen exceeding the limit of quantitation. For the well plates and conical tubes, two out of eleven and four out of ten of the products, respectively, were identified as pyrogen-positive. Some products were contaminated with trace amounts of pyrogen. Pyrogens present in these products were inactivated to below the limit of detection by ozone/hydrogen peroxide mixed gas treatment at 50 °C. Residual hydrogen peroxide on the surface of the products after the treatment, that exhibits strong oxidative effects to cultured cells, was removed to almost the limit of detection by sequential vacuum drying (80 °C/24 h) and steam-washing at 50 °C. Polypropylene and polystyrene, as raw materials for the products, were physicochemically stable for mixed gas treatment.

Conclusion

These results suggest that the production of cell culture utensils containing no pyrogen and with no influence on the cell culture is now possible by installing these technologies in the manufacturing process, instead of the radiation sterilization currently used.