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.

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures

An organ-on-chip platform equipped with microsensors for long-term microfluidic cultivation and metabolic monitoring (O2, Glu, Lac) of 3D tumour organoid cultures grown from patient-derived single cancer stem cells.

TAMI-16. THREE-DIMENSIONAL ORGANOID CULTURE UNVEILS RESISTANCE TO CLINICAL THERAPIES IN ADULT AND PEDIATRIC GLIOBLASTOMA

BACKGROUND Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent a significant challenge to effective treatment. Traditional culture methods may mask the complexity of such interactions while three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. Our objective was to determine whether organoid cultures show increased patterns of resistance to potential clinical therapies compared to traditional sphere cultures. METHODS Adult and pediatric surgical specimens were collected and established as 3D organoids. We created organoid microarrays and visualized bulk and spatially defined differences in cell proliferation using immunohistochemistry (IHC) staining, as well as cell cycle analysis by flow cytometry with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy using proliferative and viability assays. RESULTS Compared to sphere cultures from the same patient, organoids showed diverse proliferative cell populations and broad resistance to all therapies tested, albeit with both intraspecimen and interspecimen variability in the extent of resistance. Organoid specimens demonstrated a blunt response to current GBM standard of care therapy (combination temozolomide and radiotherapy) and maintained both cellular proliferation in their outer rim and overall structure and viability compared to the matched sphere specimens. CONCLUSIONS Our results suggest that growth of tumor specimens as organoid cultures may better reflect the cellular diversity and clinical reality of GBM therapeutic response. Patient-derived GBM organoids offer a valuable complement to traditional culture methods and may have powerful predictive capability of personalized drug sensitivities and therapeutic resistance.

Patient-derived endometrial organoids from MRKH patients: Insight in disease causing pathways

The uterus is responsible for the nourishment and mechanical protection of the developing embryo and fetus and is an essential part in mammalian reproduction. The Mayer-Rokitansky-Kuester-Hauser (MRKH) syndrome is characterized by agenesis of the uterus and upper part of the vagina in females with normal ovarian function. Although heavily studied, the cause of the disease is still enigmatic. Current research in the field of MRKH mainly focusses on DNA-sequencing efforts and, so far, failed to decipher the nature and heterogeneity of the disease, thereby holding back scientific and clinical progress. Here, we developed long-term expandable organoid cultures from endometrium found in uterine rudiment horns of MRKH patients. Phenotypically, they share great similarity with healthy control organoids and are surprisingly fully hormone responsive. Transcriptome analyses, however, identified an array of dysregulated genes that point at potentially disease-causing pathways altered during the development of the female reproductive tract. We consider the endometrial organoid cultures to be a powerful research tool that promise to enable an array of studies into the pathogenic origins of MRKH syndrome and possible treatment opportunities to improve patient quality of life.

Human pluripotent stem cell-derived organoids and their potential to reduce the use of animal models in research

Efficient and reproducible generation of tissue-specific organoids from Human Pluripotent Stem Cells (hPSCs) represents one of the key tools for reducing the use of animals in research. STEMCELL Technologies is committed to optimizing workflows that efficiently support the generation and maintenance of multiple types of organoid cultures derived from hPSCs.

Organoid and Primary Epithelial Cultures of Human Prostate Show the Key Role of the Epithelial-to-Mesenchymal Transition in Generation of Tissue-Specific Stromal Cells

The prostate gland (PG) is a small organ in the male reproductive system that is currently the focus of biomedical research due to its leading position in morbidity and mortality from the tissue-specific prostate cancer (PC). The PG epithelium, which undergoes a cancerous transformation, is formed and functions under the control of androgens. At the beginning of the disease, epithelial cells produce an androgen receptor (AR) and are sensitive to androgen-deprivation therapy. However, such therapy inevitably leads to the transition of the disease to the castration-resistant prostate cancer (CRPC), which manifests itself in metastasis and rapid mortality. In CRPC, the cells of the prostate epithelium change their phenotype, that may be associated with AR mutation and loss the sensitivity to specific therapy. The mechanism of PG phenotypic transformation may be hidden in the interaction and formation of the stromal and epithelial cells, which are evident during the establishment of the primary cultures. The aim of this study was to investigate the generation of human PG stromal cells in primary stromal and organoid cultures. We found that, in contrast to the rapid appearance and formation of a homogeneous population of mesenchymal cells in primary stromal cultures of most tissues, human PG cell cultures are formed initially from epithelial cells. They appear in the second week of cultivation and produce cytokeratins (CKs). A homogeneous population of mesenchymal cells producing vimentin is formed only at the end of the fourth week of cultivation. It is accompanied by the disappearance of epithelial cells. At the same time, some epithelial cells simultaneously produce CKs and vimentin. In PG organoid cultures, there is often a concomitant growth of epithelial, but not mesenchymal, cells on culture plastic. During the cultivation of epithelial cells arising from the organoid cultures, they, like the cells of the primary epithelium, exhibit the ability to spontaneous transformation into mesenchymal cells and simultaneously produce CKs and vimentin. Our data suggest that in primary and organoid PG cultures, stromal cells can be formed from epithelium due to the epithelial-to-mesenchymal transition (EMT). The tendency of PG epithelium toward spontaneous EMT may contribute to the mechanism of high sensitivity of prostate tissue to malignant transformation and metastasis. Understanding this mechanism may contribute to the development of effective antitumor therapy of prostate cancer.

Correlation Between Drug Sensitivity Profiles of Ex Vivo Expanded Circulating Tumor Cells and Clinical Treatment Response in Pancreatic Ductal Adenocarcinoma Patients

BackgroundPancreatic ductal adenocarcinoma (PDAC) is highly aggressive and has poor prognosis. There are few biomarkers to inform treatment decisions, and collecting tumor samples for genomic or drug sensitivity testing is challenging.MethodsCirculating tumor cells (CTCs) were prepared from the liquid biopsies of PDAC patients. These cells were subsequently expanded ex vivo to form CTC-derived organoid cultures, using a laboratory-developed biomimetic cell culture system. The CTC-derived organoids were tested for sensitivity to a PDAC panel of nine drugs, with tests conducted in triplicate, and a weighted cytotoxicity score (CTS) was calculated from the results. Clinical response to treatment in patients was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria at the time of blood sampling and 3 months later. CTS was then correlated with clinical response, and analyzed using 2 × 2 contingency tables.ResultsA total of 41 liquid biopsies were collected from 31 patients, with 87.8% of liquid biopsies from patients with Stage 4 disease. CTC-derived organoid expansion was achieved in 3 weeks, with 87.8% culture efficiency. CTC-derived organoid cultures were positive for EpCAM staining and negative for CD45 staining in surface marker analysis. All patients had received a median of two lines of treatment prior to enrollment, and prospective utility analysis indicated significant correlation of CTS with clinical treatment response. Two representative case studies are also presented to illustrate the relevant clinical contexts.ConclusionsIn this study, CTCs were expanded from the liquid biopsies of PDAC patients with a high success rate. Drug sensitivity profiles from CTC-derived organoid cultures correlated meaningfully with treatment response. Further studies are warranted to validate the predictive potential for this approach.Trial RegistrationTaipei Medical University Hospital Protocol Record N201803020, registered on July 10, 2018; ClinicalTrials.gov Identifier: NCT04972461, retrospectively registered on July 22, 2021.

Assessing donor-to-donor variability in human intestinal organoid cultures

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high confidence data using human primary cultures through thorough characterization of variability.