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.

Variability in the Processing of Fresh Osteochondral Allografts

The indications for fresh osteochondral allograft continue to increase. As a result, variations in graft processing and preservation methods have emerged. An understanding of these techniques is important when evaluating the optimal protocol for processing fresh osteochondral allografts prior to surgical implantation. The aim of this study is to review the literature and understand various tissue processing protocols of four leading tissue banks in the United States. Donor procurement, serological and microbiological testing, and storage procedures were compared among companies of interest. Similarities between the major tissue banks include donor screening, aseptic processing, and testing for microorganisms. Variability exists between these companies with relation to choice of storage media, antibiotic usage, storage temperature, and graft expiration dates. Potential exists for increased chondrocyte viability and lengthened time-to-expiration of the graft through a protocol of delicate tissue handling, proper choice of storage medium, adding hormones and growth factors like insulin growth factor-1 (IGF-1) to serum-free nutrient media, and storing these grafts closer to physiologic temperatures.

EP.TU.40Tendon Repair Training: An Alternative Approach

Aims The COVID-19 pandemic has seen reduced training opportunities for surgical trainees. Tendon repair is an important surgical skill for all core surgical trainees. It is particularly essential for those training within Plastic and Orthopaedic surgery. Tendon repair simulation often involves the use of expensive materials or animal tissues, posing ethical predicament. Here we aimed to devise a simple and reproducible method for tendon repair simulation. Methods Our tendon simulation model is an inexpensive and easily set up arrangement consisting of edible strawberry laces. Results The tendon simulator gives reasonable replication of a tendon, with the laces depicting an inner and outer core of a severed tendon. In addition the materials are easy to acquire, handle, dispose of and are free from ethical limitations. Conclusions Surgical trainees will be able to practice varying tendon repair techniques on this model, applying basic surgical principles such as instrument and tissue handling and develop their expertise.

Discussions on hypospadius repair

Surgery for Hypospadius is fraught with complications. The very fact that there are more than a hundred techniques described for this problem is evidence enough to confirm that no single technique is the perfect answer to our efforts. The presenting author is a student of Dr.H.S.Asopa and an advocate of the perpucial island flap technique, though not dogmatic in its propagation. He has also had the opportunity to train with Mr. Aviar Bracka and uses different techniques judiciously. This presentation is about the details no author stresses upon for Hypospadius and surgery. Pre and post operative pictures are not the only teaching aids. Ten Commandments for Hypospadius surgeons. These are in themselves self explanatory for the students of Hypospadius as well as the senior specialist. We have been achieving consistant results even though we have not struck to any one technique. We have been using the modified Asopa-I technique (for almost half the cases) and Bracka technique as the two main methods for surgery. This step by step on pitfalls in hypospadius surgery is not to highlight surgical techniques but to discuss about steps which have their own importance to produce acceptable results.In spite of so many methods to perform surgery for patients of hypospadius, some general principles on tissue handling, suture management, prevention of edema, general method of dissection, dressing techniques etc go a long way in giving consistant results whatever the method used for the repair.

Gingival Biotype: A Secret for Esthetic Success

Gingival biotype, in the recent years, has gained substantial interest as one of the important pillars for esthetic success. This review provides recommendations to be considered prior to performing any dental procedure to attain best clinical results. Several methods for measuring biotype have been suggested. Gingiva is often subjected to various insults during routine dental procedures. Hence, understanding the gingival biotype can provide insights into precautions rendered necessary during tissue handling to avoid undesirable treatment outcomes.

Creation of a High Fidelity, Cost Effective, Real World Surgical Simulation for Surgical Education

Background How do surgical residents learn to operate? What is a surgical plane? How does one learn to see and dissect the plane? How do surgical residents learn tissue handling and suturing (sewing)? One method to learn and practice performing surgery is through the use of simulation training. Surgical training models include laparoscopic box trainers (a plastic box with holes for instruments) with synthetic materials inside to simulate tissues, or computer-based virtual reality simulation for laparoscopic, endoscopic, and robotic techniques. These methods, however, do not use real tissues. They lack the haptic and kinesthetic feedback of real tissue. These simulations fail to recreate the fidelity of soft tissues, do not foster the ability to accurately see surgical planes, do not accurately mimic the act of dissecting surgical planes, do not allow for complex surgical procedures, and do not provide accurate experience to learn tissue handling and suturing. Despite their poor performance, these plastic and virtual trainers are extremely costly to purchase, maintain, and keep up to date - with prices starting at $700 for basic plastic training boxes to thousands of dollars for virtual simulation. Also, there are additional costs of maintenance and software curriculum. Despite the cost of software, virtual simulators do not include a simulation for every surgery. Our aim was to create a life-like surgical simulation as close to real world as possible that allows trainees to learn how to see and dissect surgical planes, learn how soft tissues move, and learn the dynamics of soft tissue manipulation. We created a laparoscopic simulator using porcine tissues for gallbladder removal, acid reflux surgery, and surgery to treat swallowing difficulties (cholecystectomy, Nissen fundoplication, and Heller myotomy, respectively). Second year general surgery residents were able to practice these procedures on real tissues, enabling them to learn the steps of each procedure, increase manual dexterity, improve use of laparoscopic equipment, all while maintaining life-like haptic, soft-tissue feedback and enabling them to develop the ability to see real surgical planes. Methods The abdomen was recreated by purchasing intact porcine liver, gallbladder, (Cholecystectomy simulation) and intact esophagus, stomach, and diaphragm (Nissen and Heller simulation) from a packing supplier. Each organ system was placed into a laparoscopic trainer box with the ability to re-create laparoscopic ports. Surgical residents were then able to perform the procedures using real laparoscopic instruments, laparoscopic camera/video imaging, and real-time electrocautery. The simulation included all critical steps of each procedure such as obtaining the critical view of safety and removing the gallbladder from the liver bed (cholecystectomy), wrapping the stomach around the esophagus and laparoscopic suturing (Nissen fundoplication), and dissecting the muscular portion of the esophageal wall (Heller myotomy). Because these porcine tissues were readily available, several stations were set-up to teach multiple residents during each session (10-12 residents / session). Discussion Surgeons develop haptic perception of soft tissues by cutaneous or tactile feedback and kinesthetic feedback (Okamura, 2009). Kinesthetic feedback is the force and pressure transmitted by the soft tissues along the shaft of the laparoscopic instruments (Okamura, 2009). This soft tissue simulation re-creates the ability to experience what soft tissue feedback feels like, outside a normal operative environment. Real tissue learning allows trainees to learn how to see surgical planes, learn how soft tissues feel and move, develop proficiency in surgical dissection, and learn how to suture laparoscopically. This is the only model that recreates the movement of soft tissues and visualization of dissection planes outside the operative environment. Because this model utilizes the laparoscopic instruments used in the operating room, residents also develop familiarity with laparoscopic instruments, thus, flattening another learning curve. A literature review found that this is the only real tissue simulation being performed for foregut procedures used specifically for resident training. By building a realistic, anatomical model with inherent accurate soft tissue surgical planes, surgical trainees can have a more realistic surgical experience and develop skills in a safe, low pressure environment without sacrificing the hepatic learning and surgical visualization that is critical to performing safe laparoscopic surgery. All residents that participated in the stimulation reported positive feedback and felt that is contributed to their surgical education.

Design of Soft Fingers for a Surgical Robotic Hand with Hybrid Structure

Utilization of robot manipulators started to be preferred in many medical applications due to the rapid technological developments occurred in the last decade. Thanks to the studies and applications in the related literature, leaving the usage of classical industrial robot manipulator structures, new designs with respect to the application constraints have been focused on. In light of this, current study focuses on the design of soft fingers that will allow the usage of a robotic hand with hybrid structure on soft tissue handling that requires high precision and compliance. Throughout the study various prototype trials were carried out and their suitability for the system was discussed.

Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA’s compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.