Abstract
Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT®3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: “Microbiological control of cellular products”), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT®3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT®3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT®3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT®3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.
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Acknowledgments
We thank Biomérieux for lending the BacT/ALERT®3D system for the duration of the study and Vincent Langeron for his assistance in writing the report.
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The material presented in this article is original research which has not been previously published and has not been submitted for publication elsewhere while under consideration. We certify that we have sufficiently participated in the work to take public responsibility for the appropriateness of the experimental design and method, and the collection, analysis, and interpretation of the data. All authors have reviewed the final version of the manuscript and approve it for publication. All the authors declare no personal or financial conflict of interest and have not entered into any agreement that could interfere with our access to the data on the research, or upon our ability to analyze the data independently, to prepare manuscripts, and to publish them.
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O. Damour and Céline Auxenfans: co-last authors.
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Plantamura, E., Huyghe, G., Panterne, B. et al. Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium. Cell Tissue Bank 13, 453–459 (2012). https://doi.org/10.1007/s10561-011-9281-1
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DOI: https://doi.org/10.1007/s10561-011-9281-1