Standardizing Patient-Derived Organoid Generation Workflow to Avoid Microbial Contamination From Colorectal Cancer Tissues

Frontiers in Oncology

Oncology

Savas D. Soysal¹  Raoul A. Droeser¹  Otto Kollmar¹  Sebastian Staubli¹  Markus von Flüe¹  Lana Fourie¹  Martin Bolli¹  Ralph Peterli¹  Charlotte K. Y. Ng²  Jürg Vosbeck³  Stephan Rauthe⁴  Andreas Zettl⁴  John Gallon⁵  Federica Panebianco⁵  Gaia Bianco⁵  Mattia Marinucci⁵  Stephanie Taha-Mehlitz⁶  Caner Ercan⁷  Salvatore Piscuoglio⁷  Mairene Coto-Llerena⁷ 

[1] Clarunis, Department of Visceral Surgery, University Centre for Gastrointestinal and Liver Diseases, St. Clara Hospital and University Hospital Basel, Basel, Switzerland;Department for BioMedical Research, University of Bern, Bern, Switzerland;Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland;Institute of Pathology, Viollier AG, Allschwil, Switzerland;Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland;Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland;Clarunis, Department of Visceral Surgery, University Centre for Gastrointestinal and Liver Diseases, St. Clara Hospital and University Hospital Basel, Basel, Switzerland;Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland;Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland;

关键词: patient-derived organoids;    colorectal cancer;    Primocin;    antibiotics;    microbial contamination control;   

DOI  :  10.3389/fonc.2021.781833

 received in 2021-09-23, accepted in 2021-12-13,  发布年份 2022

来源: Frontiers

【 摘 要 】

The use of patient-derived organoids (PDO) as a valuable alternative to in vivo models significantly increased over the last years in cancer research. The ability of PDOs to genetically resemble tumor heterogeneity makes them a powerful tool for personalized drug screening. Despite the extensive optimization of protocols for the generation of PDOs from colorectal tissue, there is still a lack of standardization of tissue handling prior to processing, leading to microbial contamination of the organoid culture. Here, using a cohort of 16 patients diagnosed with colorectal carcinoma (CRC), we aimed to test the efficacy of phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), and Primocin, alone or in combination, in preventing organoid cultures contamination when used in washing steps prior to tissue processing. Each CRC tissue was divided into 5 tissue pieces, and treated with each different washing solution, or none. After the washing steps, all samples were processed for organoid generation following the same standard protocol. We detected contamination in 62.5% of the non-washed samples, while the use of PBS or P/S-containing PBS reduced the contamination rate to 50% and 25%, respectively. Notably, none of the organoid cultures washed with PBS/Primocin-containing solution were contaminated. Interestingly, addition of P/S to the washing solution reduced the percentage of living cells compared to Primocin. Taken together, our results demonstrate that, prior to tissue processing, adding Primocin to the tissue washing solution is able to eliminate the risk of microbial contamination in PDO cultures, and that the use of P/S negatively impacts organoids growth. We believe that our easy-to-apply protocol might help increase the success rate of organoid generation from CRC patients.

【 授权许可】

Unknown   
Copyright © 2022 Marinucci, Ercan, Taha-Mehlitz, Fourie, Panebianco, Bianco, Gallon, Staubli, Soysal, Zettl, Rauthe, Vosbeck, Droeser, Bolli, Peterli, von Flüe, Ng, Kollmar, Coto-Llerena and Piscuoglio