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

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.

Multi tissue processing for single cell sequencing of human immune cells v1

This protocol has been developed for the simultaneous processing of multiple human tissues to extract immune cells for single cell RNA sequencing using the 10X platform, and ideal for atlasing projects. Included in this protocol are the steps needed to go from tissue to loading the 10X Chromium for single cell RNA sequencing and includes the hashtag and CiteSeq labelling of cells as well as the details needed to stimulate cells with PMA+I.

Comparative Study of Microwave Oven-Assisted Tissue Processing with Isopropanol and Mineral Oil Mixture and Propylene Glycol Methyl Ether on Morphological Quality of Tissue Sections

Background: Over several years, xylene has been traditionally utilised as the clearing agent of choice in tissue-processing due to effectiveness in rapidly clearing tissue, facilitating the paraffin infiltration process. However, xylene use adversely impacts the health of personnel with long term exposure due to toxicity. In order to overcome these effects and replace it with a safer alternative agent, the present study aims to compare quality of tissue sections processed using an isopropanol and mineral oil mixture and propylene glycol methyl ether (PGME) as xylene substitutes. Methods: Rat skeletal muscle tissue samples (n=20) were prepared for each processing protocol with xylene substitutes. Tissue specimens were processed according to the proposed microwave protocol. The clearing steps were performed using isopropanol and mineral oil mixture, and PGME, replacing xylene. From each paraffin-embedded block, one section of 4-5µm thickness tissue was obtained and conventionally-stained with Haematoxylin and Eosin (H&E). The histological sections were microscopically assessed and scored by a pathologist. A qualitative analysis was performed with the results obtained. Results: The overall score obtained for xylene processed tissue was 100% with a score of 2 for all the 3 parameters assessed. However, the outcome for tissue processed with isopropanol and mineral oil mixture was 28.6% unsatisfactory, 28.6% satisfactory and 42.8% good. In PGME-treated tissues, 14.3% were unsatisfactory sections, 71.4% were satisfactory and 14.3% produced good quality sections. Overall, tissues processed using both substitutes exhibited sufficient staining quality in terms of the aforementioned parameters as compared to xylene-processed tissues, though significant difference in scores were observed. Conclusion: Despite several challenges faced in the study, isopropanol and mineral oil mixture and PGME can be suggested as alternative clearing agents to xylene, provided having access to a more sophisticated microwave oven with precise temperature control for complete tissue-processing.