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Establishment of porcine and monkey colonic organoids for drug toxicity study.

ABSTRACT: Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity, but high cost limits their uses. Organoids have been shown to be promising models for drug test as they reasonably preserve tissue structure and functions. However, colonic organoids of pig and monkey are not yet established. Here, we report a culture medium to support the growth of porcine and monkey colonic organoids. Wnt signaling and PGE2 are important for long-term expansion of the organoids, and their withdrawal results in lineage differentiation to mature cells. Furthermore, we observe that porcine colonic organoids are closer to human colonic organoids in terms of drug toxicity response. Successful establishment of porcine and monkey colonic organoids would facilitate the mechanistic investigation of the homeostatic regulation of the intestine of these animals and is useful for drug development and toxicity studies.

SUBMITTER: Li H

PROVIDER: S-EPMC8486901 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Establishment of porcine and monkey colonic organoids for drug toxicity study.

Li Haonan H Wang Yalong Y Zhang Mengxian M Wang Hong H Cui Along A Zhao Jianguo J Ji Weizhi W Chen Ye-Guang YG

Cell regeneration (London, England) 20211002 1

Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity, but high cost limits their uses. Organoids have been shown to be promising models for drug test as they reasonably preserve tissue structure and functions. However, colonic organoids of pig and monkey are not yet established. Here, we report a culture medium to support the growth of porcine and monkey colonic organoids. Wnt signaling and PGE2 are important for long-term expansi ...[more]

PMID: 34599392

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Project description:BackgroundThe study of ovine lactation and mastitis is limited by the lack of physiologically relevant in vitro models. Compared to mammary epithelial cells, mammary gland organoids (MaOs) offer a better model for studying lactation, as they more accurately replicate the tissue structure and interactions of cells in vivo. Here, we optimize a protocol for the isolation, culture, characterization, and transfection of ovine MaOs derived from ewe mammary tissue.ResultsWe compared different enzymatic digestion methods and found that type IV collagenase digestion for 60 min yielded the highest number of MaOs compared to a mixed enzyme solution of type II collagenase and hyaluronidase. Culture medium optimization revealed that medium B supported superior growth, with increased budding structures, a higher number of MaOs over time, and a greater proportion of EdU-positive proliferating cells compared to media A and C. Cell-type characterization confirmed the presence of both luminal and myoepithelial cells, as shown by RT-PCR analysis of CK14, CK18, and CK7 expression, and further validated by immunofluorescence staining for CK14 and CK18. MaOs in medium B exhibited significantly higher mRNA expression levels of milk lipid metabolism-related genes (XDH, FABP3, SREBP1), lactose metabolism-related genes (GLUT1, GLUT4), and milk protein synthesis genes (EIF4E, CSN2) at multiple time points. In all media, XDH, FABP3, SREBP1, GLUT1, GLUT4, EIF4E, and CSN2 mRNA expression peaked at Day 7 before declining. Additionally, at Day 7, supernatant analysis confirmed that triglyceride, lactose, and CSN2 concentrations were significantly elevated in medium B compared to media A and C. Finally, we demonstrated that the ovine MaOs could be transfected using electroporation with 27.3% becoming GFP-positive.ConclusionsThis study establishes a protocol for the isolation, culture, characterization, and genetic manipulation of ovine MaOs. These organoids serve as a physiologically relevant model for studying the regulatory mechanisms of lactation and mastitis in sheep, providing a tool for future research in veterinary and agricultural sciences.

Project description:BackgroundPatient-derived organoids (PDO) have been proposed as a novel in vitro method of drug screening for different types of cancer. However, to date, extrahepatic biliary tract carcinoma (eBTC) PDOs have not yet been fully established.MethodsWe collected six samples of gallbladder carcinoma (GBC) and one sample of extrahepatic cholangiocarcinoma (eCCA) from seven patients to attempt to establish eBTC PDOs for drug screening. We successfully established five GBC and one eCCA PDOs. Histological staining was used to compare structural features between the original tissues and cancer PDOs. Whole exome sequencing (WES) was performed to analyze the genetic profiles of original tissues and cancer PDOs. Drug screening, including gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, infigratinib, and ivosidenib, was measured and verified by clinical effects in certain cases.ResultsDifferent PDOs exhibited diverse growth rates during in vitro culture. Hematoxylin and eosin staining demonstrated that the structures of most cancer PDOs retained the original structures of adenocarcinoma. Immunohistological and periodic acid-schiff staining revealed that marker expression in cancer PDOs was similar to that of the original specimens. Genetic profiles from the four original specimens, as well as paired cancer PDOs, were analyzed using whole exome sequencing. Three of the four PDOs exhibited a high degree of similarity when compared to the original specimens, except for GBC2 PDO, which only had a concordance of 74% in the proportion of single nucleotide polymorphisms in the coding sequence. In general, gemcitabine was found to be the most efficient drug for eBTC treatment, as it showed moderate or significant inhibitory impact on cancer growth. Results from drug screening were confirmed to a certain extent by three clinical cases.ConclusionsOur study successfully established a series of eBTC PDOs, which contributed to the field of eBTC PDOs. Additional enhancements should be explored to improve the growth rate of PDOs and to preserve their immune microenvironment.

Dataset Information

Establishment of porcine and monkey colonic organoids for drug toxicity study.

Publications

Establishment of porcine and monkey colonic organoids for drug toxicity study.

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