Cell and Tissue Banking

, Volume 13, Issue 3, pp 453–459

Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium

  • E. Plantamura
  • G. Huyghe
  • B. Panterne
  • N. Delesalle
  • A. Thépot
  • M. E. Reverdy
  • O. Damour
  • Céline Auxenfans
Article

DOI: 10.1007/s10561-011-9281-1

Cite this article as:
Plantamura, E., Huyghe, G., Panterne, B. et al. Cell Tissue Bank (2012) 13: 453. doi:10.1007/s10561-011-9281-1

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.

Keywords

BacT/ALERT®3DEpithelial cell sheetMicroorganismEuropean pharmacopoeiaFA and SN bottles

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • E. Plantamura
    • 1
  • G. Huyghe
    • 2
  • B. Panterne
    • 2
  • N. Delesalle
    • 2
  • A. Thépot
    • 3
  • M. E. Reverdy
    • 4
  • O. Damour
    • 1
  • Céline Auxenfans
    • 1
  1. 1.Banque de Tissus et cellules, Hospices civils de Lyon, Hôpital Edouard HerriotLyon Cedex 03France
  2. 2.Unité Microbiologie et Unité Produits Sanguins et Thérapie Cellulaire Direction des Laboratoires et des ContrôlesAFSSAPSSt Denis CedexFrance
  3. 3.CellSeed France S.A.R.LLyonFrance
  4. 4.Centre de Biologie et de Pathologie Est, Institut de microbiologieBron CedexFrance