Name: application of hemostatic devices in laparoscopic hepatectomy
Uploaded: 2022-04-19T13:00:00
Description: Watch this Scientific Journal Video about Application of Hemostatic Devices in Laparoscopic Hepatectomy at JoVE.com

This work was supported by grants from the Science and Technology Project of Guangzhou City (202102010090).

The operation is routine and received ethics approval. The approvals are as follows: Approval for rapid review by the Clinical Research and Application Ethics Committee of the Second Affiliated Hospital of Guangzhou Medical University: The scientific research ethics review project &#34;Application of simple and easy Hemostatic Devices in Laparoscopic Hepatectomy&#34; (acceptance No.: 2022-hg-ks-02) was approved by the clinical research and application ethics committee of the Second Affiliated Hospital of Guangzhou Medica...

<ol>
	<li>Hess, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+patterns+in+adenocarcinoma.">Metastatic patterns in adenocarcinoma.</a> Cancer. 106 (7), 1624-1633 (2006).</li><li>van de Velde, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EURECCA+colorectal:+multidisciplinary+management:+European+consensus+conference+colon+&amp;+rectum.">EURECCA colorectal: multidisciplinary management: European consensus conference colon &amp; rectum.</a> European Journal of Cancer. 50 (1), 1-34 (2014).</li><li>Adams, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+for+hepatic+resection+of+colorectal+liver+metastases:+expert+consensus+statement[J].">Selection for hepatic resection of colorectal liver metastases: expert consensus statement[J].</a> HPB. 15 (2), 91-103 (2013).</li><li>Reich, H., McGlynn, F., DeCaprio, J., Budin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+excision+of+benign+liver+lesions.">Laparoscopic excision of benign liver lesions.</a> Obstetrics and Gynecology. 78 (5), 956-958 (1991).</li><li>Ciria, R., Cherqui, D., Geller, D. A., Briceno, J., Wakabayashi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+short-term+benefits+of+laparoscopic+liver+resection:+9000+cases+and+climbing.">Comparative short-term benefits of laparoscopic liver resection: 9000 cases and climbing.</a> Annals of Surgery. 263 (4), 761-777 (2016).</li><li>Makuuchi, M., Hasegawa, H., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonically+guided+subsegmentectomy.">Ultrasonically guided subsegmentectomy.</a> Surgery, Gynecology, and Obstetrics. 161 (4), 346-350 (1985).</li><li>Agrawal, S., Belghiti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncologic+resection+for+malignant+tumors+of+the+liver.">Oncologic resection for malignant tumors of the liver.</a> Annals of Surgery. 253 (4), 656-665 (2011).</li><li>Wakabayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+repeat+liver+resection+after+open+liver+resection:+A+comparative+study+from+a+single-centre.">Laparoscopic repeat liver resection after open liver resection: A comparative study from a single-centre.</a> Journal of Minimal Access Surgery. 16 (1), 59-65 (2020).</li><li>Berardi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenchymal+sparing+anatomical+liver+resections+with+full+laparoscopic+approach:+description+of+technique+and+short-term+results.">Parenchymal sparing anatomical liver resections with full laparoscopic approach: description of technique and short-term results.</a> Annals of Surgery. 273 (4), 785-791 (2021).</li><li>Cho, J. Y., Han, H. S., Yoon, Y. S., Shin, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+laparoscopic+liver+resection+for+tumors+located+in+the+posterosuperior+segments+of+the+liver,+with+a+special+reference+to+overcoming+current+limitations+on+tumor+location.">Feasibility of laparoscopic liver resection for tumors located in the posterosuperior segments of the liver, with a special reference to overcoming current limitations on tumor location.</a> Surgery. 144 (1), 32-38 (2008).</li><li>Ome, Y., Honda, G., Doi, M., Muto, J., Seyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+anatomic+liver+resection+of+segment+8+using+intrahepatic+Glissonean+approach.">Laparoscopic anatomic liver resection of segment 8 using intrahepatic Glissonean approach.</a> Journal of the American College of Surgeons. 230 (3), 13-20 (2020).</li><li>Ryu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perioperative+and+oncological+outcomes+of+laparoscopic+anatomical+hepatectomy+for+hepatocellular+carcinoma+introduced+gradually+in+a+single+center.">Perioperative and oncological outcomes of laparoscopic anatomical hepatectomy for hepatocellular carcinoma introduced gradually in a single center.</a> Surgical Endoscopy. 32 (2), 790-798 (2018).</li><li>Makabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+occlusion+of+hepatic+artery+and+risk+of+carbon+dioxide+gas+embolism+during+laparoscopic+hepatectomy+in+a+pig+model.">Efficacy of occlusion of hepatic artery and risk of carbon dioxide gas embolism during laparoscopic hepatectomy in a pig model.</a> Journal of Hepato-Biliary-Pancreatic Sciences. 21 (8), 592-598 (2014).</li><li>Miyagi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+laparoscopic+hepatectomy+combined+with+a+pure+laparoscopic+pringle+maneuver+in+patients+with+severe+cirrhosis.">Pure laparoscopic hepatectomy combined with a pure laparoscopic pringle maneuver in patients with severe cirrhosis.</a> Case Reports in Gastroenterology. 9 (1), 101-105 (2015).</li></ol>

Hepatectomy is one of the most important treatment methods for patients with primary liver cancer and secondary liver cancer. In 1991, Reich et al. applied laparoscopy to remove benign tumors of the liver and completed the world&#39;s first laparoscopic hepatectomy4. After years of rapid development and promotion, laparoscopic hepatectomy has become one of the mainstream methods to treat liver malignant tumors and has been widely carried out in recent years. With the advantages of less intraoperat...

Hepatocellular carcinoma is one of the most common malignant tumors of the digestive system. According to the different primary sites, it can be divided into primary hepatocellular carcinoma and secondary liver cancer. Cells can metastasize from primary tumors from organs outside the liver into the liver in various ways, leading to carcinoma in the liver. It has been reported that more than 50% of metastatic cells in the liver come from colorectal cancer, while others are from breast, pancreas, lung, and stomach tumors, etc1. In recent years, many treatments for secondary liver cancer are available, including systematic chemotherapy, interventional therapy, molecular targeted therapy, surgery, etc2. However, radical resection is still the most effective treatment because it can completely remove the cancer3.
With the rapid development of laparoscopic technology, laparoscopic anatomical hepatectomy is gradually recognized by surgeons but is still not widely performed, especially in primary medical institutions. One of the reasons is the requirement for high-grade hemostatic devices. These are required to reduce the risks of bleeding and bile leakage during the operation process. Here, we present a set of simple and easy hemostatic equipment, including a single lumen catheter, harmonic scalpel, and monopole electrocoagulation, for performing laparoscopic hepatectomy. To do this, first the porta hepatis is occluded intermittently by using a single lumen catheter. Liver parenchyma tissue is then resected by using the harmonic scalpel. Bleeding spots are coagulated by monopole electrocoagulation point-to-point. This simple and easy hemostatic equipment uses a single lumen catheter to perform the pringle maneuver and utilizes the hemostasis of the harmonic scalpel and monopole electrocoagulation. The equipment can be found easily in hospitals, thereby providing added ease for performing laparoscopy or training. Thus, these simple and easy hemostatic devices are suitable for conducting procedures in primary medical institutions.
In this study, the patient was a 67-year-old male diagnosed with a moderately differentiated adenocarcinoma of the sigmoid colon metastasized in the liver. Radical resection of the sigmoid colon cancer was performed in January 2021. The pathological results were moderately differentiated adenocarcinoma with the TNM stage of pT4aN2aM1. FOLFOX chemotherapy was given four times after the operation. Following this, the patient&#39;s body condition was deemed suitable to perform laparoscopic anatomical hepatectomy to completely remove the lesion of liver metastases. The Child-Pugh grade of liver function was Grade A. For the liver reserve function test, R15 in the ICG clearance test was 1.6% (&#60;10%). CT showed a 57 mm x 68 mm x 76 mm tumor across the dorsal part of S5 and ventral part of S6 of the liver; the three-dimensional reconstruction model is shown in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HARMONIC ACE Ultrasonic Surgical Devices</td>
			<td>Ethicon Endo-Surgery,LLC</td>
			<td>HAR36</td>
			<td></td>
		</tr>
		<tr>
			<td>Hem-o-lock</td>
			<td>Teleflex Medical</td>
			<td>544233</td>
			<td></td>
		</tr>
		<tr>
			<td>Monopole Electrocoagulation</td>
			<td>KANGJI MEDICAL</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>single lumen ureter</td>
			<td>WELL LEAD MEDICAL CO.,LTD.</td>
			<td>12F</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of hemostatic devices in laparoscopic hepatectomy

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. Despite its non-invasive aspect, bleeding and bile leakage occur in liver parenchyma tissue resection during the operation or in the post-operation period, indicating the requirement for high-grade hemostatic devices, such as ultrasonic surgical aspiration, bipolar electrocoagulation, etc. The lack of availability of these high-grade hemostatic devices prevents laparoscopic hepatectomy from becoming a generalized procedure in basic medical organizations. In view of the situation mentioned above, a suite of simple and easy hemostatic devices is developed in this protocol, which includes a harmonic scalpel, monopole electrocoagulation, and a single lumen catheter, to innovatively perform liver parenchyma tissue resection. First of all, the porta hepatis or hepatic pedicle is occluded intermittently by a single lumen catheter, followed by clamping for 15 min and releasing for 5 min. Subsequently, using the harmonic scalpel, clamping and crushing of the liver are done to cut off the hepatic parenchyma tissue and to reveal the intrahepatic arteries, veins, and bile ducts. Lastly, the bleeding spots are coagulated by using monopole electrocoagulation at each spot. Intrahepatic pipeline structures are then visible by using these methods, which could stop bleeding easily, reduce the incidence rate of bile leakage, and improve the safety and feasibility of laparoscopic hepatectomy. Therefore, the simple and easy hemostatic devices shown here are suitable for conducting procedures in primary medical institutions.

The operation was completed within 2.5 h and the intraoperative bleeding volume was 100 mL without the blood transfusion. There were no short-term complications, and the patient was discharged from the hospital on the 8th day after the operation. The carcinoembryonic antigen (CEA) level post operation decreased to 110.64 &#181;g/L from the pre-operation level of 1058.69 &#181;g/L (Figure 12). Two months after the operation, the patient returned to the hospital to continue chemothe...

Watch this Scientific Journal Video about Application of Hemostatic Devices in Laparoscopic Hepatectomy at JoVE.com

Application of Hemostatic Devices in Laparoscopic Hepatectomy

High-grade hemostatic devices are essential for laparoscopic hepatectomy. However, these devices are not generalized in basic medical organizations. Therefore, a suite of simple and easy hemostatic devices is shown in this article, which can make the laparoscopic hepatectomy easier to perform.

Laparoscopic hepatectomy is considered a conventional method for treating benign and malignant liver diseases because it is a minimally invasive method. ...

The main advantage of this technique is that the hemostatic device is simple and easy to obtain and can be carried out in basic hospitals. This set of hemostatic equipment can be further applied to laparoscopic hepatectomy and other systems. It is highly recommended to have a certain basis of laparoscopic surgery, especially skilled use of a harmonic scalpel to apply this set of hemostatic devices in laparoscopic hepatectomy.Begin by applying direct puncture to establish pneumoperitoneum in laparoscopy. Place a 10 millimeter trocar in each observation hole in the subumbilical area, right mid-clavicular line under the costal margin and ventral midline. Then place a five millimeter trocar in the right anterior auxiliary line and the ventral midline.Assemble the hemostatic devices starting from the harmonic scalpel, monopole electrocoagulation, and single lumen catheter to be innovatively used in liver parenchyma tissue resection. To perform a Pringle maneuver, occlude the first porta hepatis intermittently by using a single lumen catheter. Clamp the lumen catheter with a hemostatic clip for 15 minutes followed by releasing it for five minutes using the harmonic scalpel to cut off the clip and take the clip out to restore blood supply to the first porta hepatis.Separate the hepatic pedicles supplying the tumor. Resect and divide them one by one with a harmonic scalpel. Observe the ischemia line on the liver surface, which becomes visible after the resection of the hepatic pedicles.Use the hook to mark it. Resect the liver parenchyma tissue using the harmonic scalpel while clamping and crushing the liver gradually. Observe the internal hepatic artery, vein, and bile duct.Resect and divide these pipeline structures. Once bleeding appears, coagulate the bleeding spots on the liver dissection surface immediately using point to point monopole electrocoagulation. Use the right posterior hepatic pedicle as a signpost for intrahepatic parenchyma tissue resection.Reserve the main posterior hepatic pedicle and dissect the branch of pedicles supplying the tumor. To complete the operation, wash the liver surface so one can see if there are still bleeding spots. Stop the bleeding on the liver dissection surface and remove the tumor specimen from the abdominal cavity.The operation was completed within 2.5 hours. The intraoperative bleeding volume was 100 milliliters without the blood transfusion and no short-term complications were observed. The carcinoembryonic antigen level post-operation decreased to 110.64 micrograms per liter from the pre-operation level of 1058.69 micrograms per liter.The chemotherapy treatment was resumed two months after the operation, according to the tumor pathology results. The postoperative CT examination was compared with the preoperative CT and no signs of tumor regrowth were observed. The most important step in the surgical process is the use of the harmonic scalpel.A good amount of skill is required to avoid damage to vital structures.

application-of-hemostatic-devices-in-laparoscopic-hepatectomy

Research

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Cancer Research

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Laparoscopic Pancreatoduodenectomy With Modified Blumgart Pancreaticojejunostomy

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal pancreatectomy, laparoscopic pancreatoduodenectomy (LPD) has not yet obtained wide acceptance, probably due to technical challenges, especially regarding the pancreatic anastomosis.
The study describes and demonstrates all steps of LPD, including the modified Blumgart pancreaticojejunostomy. Indications for LPD are all pancreatic and peri-ampullary tumors without vascular involvement. Relative contra-indications are body mass index &#62;35 kg/m2, chronic pancreatitis, mid-cholangiocarcinomas and large duodenal cancers.
The patient is in French position, 6 trocars are placed, and dissection is performed using an (articulating) sealing device. A modified Blumgart end-to-side pancreaticojejunostomy is performed with 4 large needles (3/0) barbed trans-pancreatic sutures and 4 to 6 duct-to-mucosa sutures using 5/0 absorbable multifilament combined with a 12 cm, 6 or 8 Fr internal stent using 3D laparoscopy. Two surgical drains are placed alongside the pancreaticojejunostomy.
The described technique for LPD including a modified Blumgart pancreatico-jejunostomy is well standardized, and its merits are currently studied in the randomized controlled multicenter trial. This complex operation should be performed at high-volume centers where surgeons have extensive experience in both open pancreatic surgery and advanced laparoscopic gastro-intestinal surgery.

Laparoscopic pancreatoduodenctomy (LPD) may offer advantages over open pancreatoduodenectomy, including early postoperative mobilization, less delayed gastric emptying and a shorter hospital stay. However, LPD is technically challenging and not well-standardized, especially regarding the pancreatic anastomosis. We describe a standardized technique for the pancreatic anastomosis during LPD: modified Blumgart pancreaticojejunostomy.

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal ...

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Application of the En Bloc Concept Combined with Anatomic Resection in Laparoscopic Hepatectomy

Laparoscopic hepatectomy has been reported in many studies, and it is the mainstream method of liver resection. In some particular cases, such as when there are tumors adjacent to the cystic bed, surgeons cannot palpate the surgical margins through the laparoscopic approach, which leads to uncertainty about R0 resection. Conventionally, the gallbladder is resected first, and the hepatic lobes or segments are resected second. However, tumor tissues can be disseminated in the above cases. To address this issue, based on the recognition of the porta hepatis and intrahepatic anatomy, we propose a unique approach to hepatectomy combined with gallbladder resection by en bloc anatomic resection in situ. Firstly, after dissecting the cystic duct, without cutting the gallbladder primarily, the porta hepatis is pre-occluded by the single lumen ureter; secondly, the left hepatic pedicle is made free by the gap of the Laennec membrane and Hilar plate; thirdly, the assistant is asked to drag the fundus of the gallbladder, and the liver parenchyma tissue is resected using a harmonic scalpel along the ischemia line on the liver surface and intraoperative ultrasound. The whole middle hepatic vein (MHV) and its tributaries appear completely; lastly, the left hepatic vein (LHV) is disconnected, and the specimen is taken out from the abdominal cavity. The tumor, gallbladder, and other surrounding tissues are resected en bloc, which meets the tumor-free criterion, and a wide incisal margin and R0 resection are achieved. Therefore, the laparoscopic hepatectomy with the combination of the en bloc concept and anatomic resection is a safe, effective, and radical method with low postoperative recurrence and metastasis.

Numerous studies have demonstrated the advantages of anatomic resection. Nonetheless, whether anatomic resection can increase R0 resection rates remains controversial. Consequently, the present study describes an innovative procedure involving the en bloc concept combined with anatomic resection in laparoscopic hepatectomy, which can reduce postoperative recurrence and metastasis.

Laparoscopic hepatectomy has been reported in many studies, and it is the mainstream method of liver resection. In some particular cases, such as when ...