Budget-Friendly Generation, Biochemical Analyses, and Lentiviral Transduction of Patient-Derived Colon Organoids.

For the past decade, three-dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two-dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ-restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so-called patient-derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient-derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget-friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D-culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost-effective techniques to achieve immunoblot and high-resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of PDCOs from biopsies Basic Protocol 2: Long-term maintenance and expansion of PDCOs in BME domes Basic Protocol 3: Cryopreservation and thawing of PDCOs Basic Protocol 4: Lentiviral transduction of PDCOs Basic Protocol 5: Immunoblot and evaluation of variability between donors Basic Protocol 6: Immunofluorescence labeling and high-resolution microscopy of PDCOs Basic Protocol 7: Transcriptomic analyses of PDCOs by RT-qPCR.

Servier in oncology: bringing innovation to patients.

Interview conducted with Claude P Bertrand, PharmD, PhD, Executive Vice President of Servier Research and Development, Chief Scientific Officer, Servier, France. Claude Bertrand speaks to Roshaine Wijayatunga, Senior Editor: Oncology. Dr Claude Bertrand graduated in pharmacy (PharmD) from Strasbourg University, France, and obtained his PhD in Strasbourg with research in the fields of immunopharmacology and neurogenic inflammation. After a 2-year postdoctoral appointment at the University of California, San Francisco, USA, Claude joined the allergy and asthma unit at Ciba-Geigy (later Novartis) in Basel, Switzerland. In 1996, he moved to the Inflammatory Disease Unit at Roche Bioscience, CA, USA, where he became the head of the in vivo pharmacology group and was responsible for supporting projects in rheumatology and respiratory diseases. In 1999, he was recruited as Director of Biology for Inflammation, GI and Pain at Parke-Davies, which later became part of Pfizer where he headed drug discovery. In 2004, Dr Bertrand joined AstraZeneca as Vice President of Discovery for Respiratory and Inflammation Research at Alderley Park, UK, and, in 2005, he was appointed Global Senior Vice President for Respiratory and Inflammation Research Area overseeing research and development activities at three sites in the UK and Sweden. In 2009, Dr Bertrand joined Ipsen, France, as Executive Vice President, Chief Scientific Officer, and from June 2011 was the Executive Vice President for Research and Development, Chief Scientific Officer with a focus on oncology, neurology and endocrinology. In March 2017, he was appointed General Director R&D, Chief Scientific Officer at Servier and has recently been promoted to Executive Vice President R&D. As such, he joined the Executive Committee on 1 November 2018. Claude sits on the Board of Directors of Eclosion2 and ABIVAX, and is also part of the Scientific Advisory Board of MEDALIS. He was the President of ARIIS from 2011 to 2016. Since 2014, he has been on the Board of Hcéres. Claude has published more than 70 papers in peer-reviewed journals, authored 20 chapters and presented more than 100 communications at scientific meetings. Since 1996, he has been a visiting lecturer for PhD student programs at universities in London, Strasbourg, Nancy, Rennes, Orléans and Paris.

Laparoscopic ultrasonography as an alternative to intraoperative cholangiography during laparoscopic cholecystectomy.

AIM: To assess the role of laparoscopic ultrasound (LUS) as a substitute for intraoperative cholangiography (IOC) during cholecystectomy. METHODS: We present a MEDLINE and PubMed literature search, having used the key-words "laparoscopic intraoperative ultrasound" and "laparoscopic cholecystectomy". All relevant English language publications from 2000 to 2016 were identified, with data extracted for the role of LUS in the anatomical delineation of the biliary tract, detection of common bile duct stones (CBDS), prevention or early detection of biliary duct injury (BDI), and incidental findings during laparoscopic cholecystectomy. Data for the role of LUS vs IOC in complex situations (i.e., inflammatory disease/fibrosis) were specifically analyzed. RESULTS: We report data from eighteen reports, 13 prospective non-randomized trials, 5 retrospective trials, and two meta-analyses assessing diagnostic accuracy, with one analysis also assessing costs, duration of the examination, and anatomical mapping. Overall, LUS was shown to provide highly sensitive mapping of the extra-pancreatic biliary anatomy in 92%-100% of patients, with more difficulty encountered in delineation of the intra-pancreatic segment of the biliary tract (73.8%-98%). Identification of vascular and biliary variations has been documented in two studies. Although inflammatory disease hampered accuracy, LUS was still advantageous vs IOC in patients with obscured anatomy. LUS can be performed before any dissection and repeated at will to guide the surgeon especially when hilar mapping is difficult due to fibrosis and inflammation. In two studies LUS prevented conversion in 91% of patients with difficult scenarios. Considering CBDS detection, LUS sensitivity and specificity were 76%-100% and 96.2%-100%, respectively. LUS allowed the diagnosis/treatment of incidental findings of adjacent organs. No valuable data for BDI prevention or detection could be retrieved, even if no BDI was documented in the reports analyzed. Literature analysis proved LUS as a safe, quick, non-irradiating, cost-effective technique, which is comparatively well known although largely under-utilized, probably due to the perception of a difficult learning curve. CONCLUSION: We highlight the advantages and limitations of laparoscopic ultrasound during cholecystectomy, and underline its value in difficult scenarios when the anatomy is obscured.