Doxorubicin hcl

The ex vivo experiments performed in this study involved the cytotoxic drugs CIS (Cisplatin Teva^®, Teva, Ulm, Germany) and DOX (Doxorubicin HCl Teva^®, Teva, Ulm, Germany), which are very toxic and present a potential hazard for the personnel involved. A risk analysis was performed and standard operating procedures were developed. All personnel involved were trained in these procedures. All experiments were performed in a class-3 safety hood certified for manipulating cytostatic drugs. Air measurements were performed under real conditions in the lab by an external, independent institution (DEKRA, Stuttgart, Germany), and platin contamination was prevented. The safety of the NCPP laboratory was audited successfully by Unfallkasse Baden-Württemberg, Stuttgart, Germany in Fall 2016.

Chemotherapeutic drugs were obtained from the in-house clinical pharmacy department, at 2 mg/mL doxorubicin-HCl (06581630, Teva GmbH, Ulm, Germany) and 1 mg/mL MMC (11213532, medac, Wedel, Germany) stock solutions. For HIPEC treatment, the peritoneal tissue was treated with 10 µM doxorubicin or 10 µM MMC diluted in culture medium for 90 min at either 37 °C or 41 °C in a 5% CO₂ incubator. Afterwards, the tissue was washed gently with PBS and medium containing doxorubicin or MMC was replaced by normal culturing medium. For immunofluorescent evaluation of doxorubicin entry into peritoneal cells, the tissue was embedded immediately after treatment and fresh-frozen tissue sections of 3 µm were stained with Vectashield with DAPI (BIOZOL Diagnostica GmbH, Eching, Germany) and analyzed by a Leica TCS SP8 fluorescence microscope. For analysis of cell survival three days after hyperthermal treatment, the peritoneal tissue was digested with 2 mg/ml collagenase (Sigma-Aldrich Chemie GmbH, St. Louis, Missouri, US) for 24 h and the luminescent signal of HT29/GFP-luciferase cells was measured using a Bright-Glo™ Luciferase Assay System (Promega, Madison, Wisconsin, US) according to the manual.