title: Validation of shortened 2-day sterility testing of mesenchymal stem cell-based therapeutic preparation on an automated culture system.
abstract: Cell therapy products represent a new trend of treatment in the field of immunotherapy and regenerative medicine. Their biological nature and multistep preparation procedure require the application of complex release criteria and quality control. Microbial contamination of cell therapy products is a potential source of morbidity in recipients. The automated blood culture systems are widely used for the detection of microorganisms in cell therapy products. However the standard 2-week cultivation period is too long for some cell-based treatments and alternative methods have to be devised. We tried to verify whether a shortened cultivation of the supernatant from the mesenchymal stem cell (MSC) culture obtained 2 days before the cell harvest could sufficiently detect microbial growth and allow the release of MSC for clinical application. We compared the standard Ph. Eur. cultivation method and the automated blood culture system BACTEC (Becton Dickinson). The time to detection (TTD) and the detection limit were analyzed for three bacterial and two fungal strains. The Staphylococcus aureus and Pseudomonas aeruginosa were recognized within 24 h with both methods (detection limit ~10 CFU). The time required for the detection of Bacillus subtilis was shorter with the automated method (TTD 10.3 vs. 60 h for 10-100 CFU). The BACTEC system reached significantly shorter times to the detection of Candida albicans and Aspergillus brasiliensis growth compared to the classical method (15.5 vs. 48 and 31.5 vs. 48 h, respectively; 10-100 CFU). The positivity was demonstrated within 48 h in all bottles, regardless of the size of the inoculum. This study validated the automated cultivation system as a method able to detect all tested microorganisms within a 48-h period with a detection limit of ~10 CFU. Only in case of B. subtilis, the lowest inoculum (~10 CFU) was not recognized. The 2-day cultivation technique is then capable of confirming the microbiological safety of MSC and allows their timely release for clinical application.
authors.affiliation: Department of Hematology and Oncology, Medical School and Teaching Hospital in Plzen, Charles University in Prague, Alej Svobody 80, 304 60, Plzen, Czech Republic. lysak@fnplzen.cz.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lysák
authors.affiliation: Department of Hematology and Oncology, Medical School and Teaching Hospital in Plzen, Charles University in Prague, Alej Svobody 80, 304 60, Plzen, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Holubová
authors.affiliation: Institute of Microbiology, Medical School and Teaching Hospital in Plzen, Charles University in Prague, Plzen, Czech Republic.
authors.forename: Tamara
authors.initials: T
authors.lastname: Bergerová
authors.affiliation: ITEST-plus, s.r.o., Hradec Králové, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Vávrová
authors.affiliation: Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Giuseppina Cristina
authors.initials: GC
authors.lastname: Cangemi
authors.affiliation: Clinica Medica I, Fondazione IRCCS Policlinico San Matteo, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Rachele
authors.initials: R
authors.lastname: Ciccocioppo
authors.affiliation: International Clinical Research Center, St. Anne´s University Hospital, Pekarska 53, 656 91, Brno, Czech Republic. peter.kruzliak@savba.sk.|Masaryk University, Brno, Czech Republic. peter.kruzliak@savba.sk.
authors.forename: Peter
authors.initials: P
authors.lastname: Kruzliak
authors.affiliation: Department of Hematology and Oncology, Medical School and Teaching Hospital in Plzen, Charles University in Prague, Alej Svobody 80, 304 60, Plzen, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Jindra
mesh_terms: D001331:Automation; D001419:Bacteria / Q000254:growth & development; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D003114:Colony-Forming Units Assay; D005658:Fungi / Q000254:growth & development; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D015203:Reproducibility of Results; D013242:Sterilization / Q000379:methods*; D013997:Time Factors
keywords: Automated culture system; Cell therapy; Mesenchymal stem cells; Sterility; Validation