Microbial occurrence in liquid nitrogen storage tanks: a challenge for cryobanking?

Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N2 vapour phase or in liquid nitrogen (LN) at temperatures below - 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and - 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. KEY POINTS: • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.