Cell Cultures

The cell culture collection of IZSLER Biobank includes over 600 cell types subdivided into the following five categories: established cell lines (CL), tumor cell lines (TCL), primary cell cultures (PRC), mesenchymal stromal cells (MSC) and hybridomas (HY). The established, tumor and primary cell cultures have been isolated from different tissues and organs of several different species. On the contrary, mesenchymal stromal cells have been isolated only from animals, mainly of equine, feline and canine species and obtained either from bone marrow or adipose tissue. All cell types are amplified, controlled in their proper characteristics and quality parameters and cryopreserved in nitrogen vapor tanks. Cells are handled by trained and qualified personnel in a cell culture laboratory with well standardized procedures.

Quality controls

All cell types are submitted to quality control tests in order to evaluate their characteristics, purity and identity. These controls are carried out by different techniques in the field of cell biology, by virology and microbiologic testing, throughout molecular biology reactions and by in vivo investigations.

Tests are carried out according to international guidelines such as those indicated by the European Pharmacopeia.

Cell characterization

• Viability: this is performed on all cell cultures before freezing and after thawing in order to evaluate the cryopreservation efficiency. Trypan bleu staining is usually performed along with cell culture proliferation;
• Mesenchymal stromal cell (MSC) differentiation: this is carried out in order to evaluate the preservation cability of MSC y to differentiate after in vitro amplification. This test is carried out by evaluating the adipogenic, osteogenic and chondrogenic evolution of MSC cultures.

Tumorigenicity

Detection of oncogenic features of cell cultures can be detected by in vivo tests that are carried out by inoculation of cells into laboratory animals as indicated in the European Pharmacopeia. Tumor formation is evaluated in the inoculated animals and confirmed by histology assay.

Moreover, the ability of cells to form colonies is detected by inoculation in an appropriate semi-solid medium.

Purity

The highest risk that can occur in in cell culture laboratories is contamination by microorganisms and viruses that can be present in the tissue / organ of the donors from which cells have derived, from animal reagents or that can result from improper laboratory practice.

• Contamination by bacteria, fungi and yeasts can be detected by sterility tests based on the inoculation of cell culture samples or supernatants in microbiological media.

Mycoplasma can be detected by the below indicated techniques:

• Isolation in culture media is the gold standard; however, some mycoplasmas strains are not cultivable and alternative and indirect methods can be performed such as DNA staining (Hoechst 33258);
• Real time PCR technology has been developed and allows to detect Mycoplasma DNA in cell cultures. It offers the advantage to be very competitive and can be routinely performed thanks to its sensitivity, specificity and rapidity.

A broad range of viruses can contaminate cell cultures and viral detection can be performed by the below indicated techniques:

• Inoculation in susceptible cell cultures and detection either following observation of cytopathic effect (CPE)
• PCR or Real time PCR reactions;

Identity

Another major risk in cell repositories is represented by the misidentified cell lines due to the circulation of false cells that are currently used in laboratories. Their identification can be confirmed by the following reported techniques:

• Species identification that can be carried out by PCR RFLP reaction;
• Authentication of human cell lines performed by STR-DNA typing.

Quality management system

The Cell Culture Laboratory perform in according to ISO EN IEC 17025 quality system and achieved ISO 9001 certification for its activity.

Updated to July 2022

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