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.

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

“…However, despite advances in knowledge of genes implicated in the epileptogenic process, these studies have generated some unexpected findings related to unpredictable variability sources in experimental design. In differential gene expression studies, two types of variation can affect the results: i) biological variation among individuals, such as age, gender, ethnicity, body mass index (BMI), lifestyle, and other individual characteristics [ 10 – 14 ]; ii) technical variation due to sample processing, such as pipetting errors, reverse transcription efficiency, RNA quality and other [ 15 , 16 ]. Several strategies can be adopted to minimize these factors, including the use of numerous biological replicates in matched-pairs design for biological variances and the use of suitable normalizers for technical variations [ 17 – 20 ].…”

Combining culture and microbead‐based immunoassay for the early and generic detection of bacteria in platelet concentrates

“…Currently, culture methods are used to test their sterility according to the European and United States Pharmacopeias. For some advanced therapeutic medicinal products, a rapid method for bacterial detection is necessary. Our analytical approach could be adapted at several steps of the process to anticipate decisions during the manufacturing process and to reduce both time and cost.…”

Production and quality testing of multipotent mesenchymal stromal cell therapeutics for clinical use

“…According to the European Pharmacopeia and national/local regulations, the analytical methods must be validated reliably demonstrating the sensitivity, limit of detection, and robustness of the method; product specifications for microbial contamination and Mycoplasma are absolute values (i.e., detected/not detected), whereas other attributes will be reported as a range of values. Batches manufactured during development can be used to set the upper and lower limits of acceptance Identity.…”