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

Abstract

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.

Toward reproducible tumor organoid culture: focusing on primary liver cancer

“…Marinucci et al. ( 151 ) attempted to standardize washing protocols and antibiotic use to prevent contamination. They found that non-washed samples had a contamination rate of 62.5%, which decreased to 50% and 25% with PBS or p/s-containing PBS, respectively.…”

“…They found that non-washed samples had a contamination rate of 62.5%, which decreased to 50% and 25% with PBS or p/s-containing PBS, respectively. Interestingly, none of the organoid cultures washed with PBS/primocin were contaminated ( 151 ). Moreover, adding p/s to the washing solution reduced cell viability compared to primocin ( 151 ).…”

“…Interestingly, none of the organoid cultures washed with PBS/primocin were contaminated ( 151 ). Moreover, adding p/s to the washing solution reduced cell viability compared to primocin ( 151 ). Establishing easy-to-follow protocols for preventing microbial contamination could improve organoid generation success rates.…”

“…Researchers use washing with PBS or antibiotics such as primocin, penicillin/streptomycin (p/s), normocin, gentamicin/amphotericin B, or plasmocin, either individually or in combination, to prevent contamination (30, 142-150). Marinucci et al (151) attempted to standardize washing protocols and antibiotic use to prevent contamination. They found that non-washed samples had a contamination rate of 62.5%, which decreased to 50% and 25% with PBS or p/s-containing PBS, respectively.…”

Tumor organoid model of colorectal cancer (Review)

“…The success rate of culture may be further improved by obtaining multiple biopsies and direct evaluation by pathologists ( 119 ). CRC organoids with different histological subtypes have been described to be successfully constructed and clinical parameters, such as tumor size or presence of ulcers, were not associated with the occurrence of contamination ( 55 , 80 , 120 ). However, the establishment of organoids from tumors with rare CRC histologic subtypes (e.g., poorly differentiated adenocarcinoma, mucinous adenocarcinoma and neuroendocrine carcinoma) is more difficult ( 33 ).…”