Use of fluorescence imaging and indocyanine green during laparoscopic cholecystectomy: Results of an international Delphi survey.

BACKGROUND: Published empirical data have increasingly suggested that using near-infrared fluorescence cholangiography during laparoscopic cholecystectomy markedly increases biliary anatomy visualization. The technology is rapidly evolving, and different equipment and doses may be used. We aimed to identify areas of consensus and nonconsensus in the use of incisionless near-infrared fluorescent cholangiography during laparoscopic cholecystectomy. METHODS: A 2-round Delphi survey was conducted among 28 international experts in minimally invasive surgery and near-infrared fluorescent cholangiography in 2020, during which respondents voted on 62 statements on patient preparation and contraindications (n = 12); on indocyanine green administration (n = 14); on potential advantages and uses of near-infrared fluorescent cholangiography (n = 18); comparing near-infrared fluorescent cholangiography with intraoperative x-ray cholangiography (n = 7); and on potential disadvantages of and required training for near-infrared fluorescent cholangiography (n = 11). RESULTS: Expert consensus strongly supports near-infrared fluorescent cholangiography superiority over white light for the visualization of biliary structures and reduction of laparoscopic cholecystectomy risks. It also offers other advantages like enhancing anatomic visualization in obese patients and those with moderate to severe inflammation. Regarding indocyanine green administration, consensus was reached that dosing should be on a milligrams/kilogram basis, rather than as an absolute dose, and that doses >0.05 mg/kg are necessary. Although there is no consensus on the optimum preoperative timing of indocyanine green injections, the majority of participants consider it important to administer indocyanine green at least 45 minutes before the procedure to decrease the light intensity of the liver. CONCLUSION: Near-infrared fluorescent cholangiography experts strongly agree on its effectiveness and safety during laparoscopic cholecystectomy and that it should be used routinely, but further research is necessary to establish optimum timing and doses for indocyanine green.

3D scaffold-free microlivers with drug metabolic function generated by lineage-reprogrammed hepatocytes from human fibroblasts.

Generating microliver tissues to recapitulate hepatic function is of increasing importance in tissue engineering and drug screening. But the limited availability of primary hepatocytes and the marked loss of phenotype hinders their application. Human induced hepatocytes (hiHeps) generated by direct reprogramming can address the shortage of primary hepatocytes to make personalized drug prediction possible. Here, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Transducing human fetal and adult fibroblasts with low vector dosage generated human induced hepatocyte-like cells (hiHeps) displaying characteristics of mature hepatocytes and capable of drug metabolism. To mimic the physiologic liver microenvironment and improve hepatocyte function, we prepared 3D scaffold-free microliver spheroids using hiHeps and human liver nonparenchymal cells through self-assembly without exogenous scaffolds. We then introduced the microliver spheroids into a two-organ microfluidic system to examine interactions between hepatocytes and tumor cells. The hiHeps-derived spheroids metabolized the prodrug capecitabine into the active metabolite 5-fluorouracil and induced toxicity in downstream tumor spheroids. Our results demonstrate that hiHeps can be used to make microliver spheroids and combined with a microfluidic system for drug evaluation. Our work could make it possible to use patient-specific hepatocyte-like cells to predict drug efficacy and side effects in various organs from the same patient.

Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform.

Current drug development techniques are expensive and inefficient, partially due to the use of preclinical models that do not accurately recapitulate in vivo drug efficacy and cytotoxicity. To address this challenge, we report on an integrated, in vitro multi-organoid system that enables parallel assessment of drug efficiency and toxicity on multiple 3D tissue organoids. Built in a low-cost, adhesive film-based microfluidic device, these miniaturized structures require less than 200 µL fluid volume and are amenable to both matrix-based 3D cell culture and spheroid aggregate integration, each supported with an in situ photocrosslinkable hyaluronic acid hydrogel. Here, we demonstrate this technology first with a three-organoid device consisting of liver, cardiac, and lung constructs. We show that these multiple tissue types can be kept in common circulation with high viability for 21 days and validate the platform by investigating liver metabolism of the prodrug capecitabine into 5-fluorouracil (5-FU) and observing downstream toxicity in lung and cardiac organoids. Then we expand the integrated system to accommodate six humanized constructs, including liver, cardiac, lung, endothelium, brain, and testes organoids. Following a 14-day incubation in common media, we demonstrate multi-tissue interactions by metabolizing the alkylating prodrug ifosfamide in the liver organoid to produce chloroacetaldehyde and induce downstream neurotoxicity. Our results establish an expandable, multi-organoid body-on-a-chip system that can be fabricated easily and used for the accurate characterization of drug interactions in vitro. STATEMENT OF SIGNIFICANCE: The use of 3-dimensional (3D) in vitro models in drug development has advanced over the past decade. However, with several exceptions, the majority of research studies using 3D in vitro models, such as organoids, employ single tissue types, in isolated environments with no "communication" between different tissues. This is a significant limiting factor because in the human body there is significant signaling between different cells, tissues, and organs. Here we employ a low-cost, adhesive film-based microfluidic device approach, paired with a versatile extracellular matrix-derived hyaluronic acid hydrogel to support integrated systems of 3 and 6 3D organoid and cell constructs. Moreover, we demonstrate an integrated response to drugs, in which downstream toxicity is dependent on the presence of liver organoids.

Immersion Bioprinting of Tumor Organoids in Multi-Well Plates for Increasing Chemotherapy Screening Throughput.

The current drug development pipeline takes approximately fifteen years and $2.6 billion to get a new drug to market. Typically, drugs are tested on two-dimensional (2D) cell cultures and animal models to estimate their efficacy before reaching human trials. However, these models are often not representative of the human body. The 2D culture changes the morphology and physiology of cells, and animal models often have a vastly different anatomy and physiology than humans. The use of bioengineered human cell-based organoids may increase the probability of success during human trials by providing human-specific preclinical data. They could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high-throughput screening (e.g., 96- and 384-well plates). Bioprinting can be used to scale up deposition of such organoids and tissue constructs. Unfortunately, it has been challenging to 3D print hydrogel bioinks into small-sized wells due to well-bioink interactions that can result in bioinks spreading out and wetting the well surface instead of maintaining a spherical form. Here, we demonstrate an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. A hydrogel bioink comprised of hyaluronic acid and collagen is bioprinted into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form. This method was validated using several cancerous cell lines, and then applied to patient-derived glioblastoma (GBM) and sarcoma biospecimens for drug screening.

Design of an Adhesive Film-Based Microfluidic Device for Alginate Hydrogel-Based Cell Encapsulation.

To support the increasing translational use of transplanted cells, there is a need for high-throughput cell encapsulation technologies. Microfluidics is a particularly promising candidate technology to address this need, but conventional polydimethylsiloxane devices have encountered challenges that have limited their utility, including clogging, leaking, material swelling, high cost, and limited scalability. Here, we use a rapid prototyping approach incorporating patterned adhesive thin films to develop a reusable microfluidic device that can produce alginate hydrogel microbeads with high-throughput potential for microencapsulation applications. We show that beads formed in our device have high sphericity and monodispersity. We use the system to demonstrate effective cell encapsulation of mesenchymal stem cells and show that they can be maintained in culture for at least 28 days with no measurable reduction in viability. Our approach is highly scalable and will support diverse translational applications of microencapsulated cells.

Model of Patient-Specific Immune-Enhanced Organoids for Immunotherapy Screening: Feasibility Study.

INTRODUCTION: We hypothesized that engineering a combined lymph node/melanoma organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. METHODS: Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. RESULTS: Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor organoids between September 2017 and June 2018. Successful establishment of viable organoid sets was 90% (9/10), although organoid yield varied with biospecimen size. Average time from organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. CONCLUSION: Development of 3D mixed immune-enhanced tumor/node organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.

Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Normal and Malignant Hemopoietic Cell-Niche Interactions.

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC-niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)-on-a-chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell-cell/cell-matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue-engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions-homing and retention-of circulating normal and malignant HSPC with distinct niches.

Appendiceal Cancer Patient-Specific Tumor Organoid Model for Predicting Chemotherapy Efficacy Prior to Initiation of Treatment: A Feasibility Study.

INTRODUCTION: We have hypothesized that biofabrication of appendiceal tumor organoids allows for a more personalized clinical approach and facilitates research in a rare disease. METHODS: Appendiceal cancer specimens obtained during cytoreduction with hyperthermic intraperitoneal chemotherapy procedures (CRS/HIPEC) were dissociated and incorporated into an extracellular matrix-based hydrogel system as three-dimensional (3D), patient-specific tumor organoids. Cells were not sorted, preserving tumor heterogeneity, including stroma and immune cell components. Following establishment of organoid sets, chemotherapy drugs were screened in parallel. Live/dead staining and quantitative metabolism assays recorded which chemotherapies were most effective in killing cancer cells for a specific patient. Maintenance of cancer phenotypes were confirmed by using immunohistochemistry. RESULTS: Biospecimens from 12 patients were applied for organoid development between November 2016 and May 2018. Successful establishment rate of viable organoid sets was 75% (9/12). Average time from organoid development to chemotherapy testing was 7 days. These tumors included three high-grade appendiceal (HGA) and nine low-grade appendiceal (LGA) primaries obtained from sites of peritoneal metastasis. All tumor organoids were tested with chemotherapeutic agents exhibited responses that were either similar to the patient response or within the variability of the expected clinical response. More specifically, HGA tumor organoids derived from different patients demonstrated variable chemotherapy tumor-killing responses, whereas LGA organoids tested with the same regimens showed no response to chemotherapy. One LGA set of organoids was immune-enhanced with cells from a patient-matched lymph node to demonstrate feasibility of a symbiotic 3D reconstruction of a patient matched tumor and immune system component. CONCLUSIONS: Development of 3D appendiceal tumor organoids is feasible even in low cellularity LGA tumors, allowing for individual patient tumors to remain viable for research and personalized drug screening.

A multi-site metastasis-on-a-chip microphysiological system for assessing metastatic preference of cancer cells.

Metastatic disease remains one of the primary reasons for cancer-related deaths, yet the majority of in vitro cancer models focus on the primary tumor sites. Here, we describe a metastasis-on-a-chip device that houses multiple bioengineered three-dimensional (3D) organoids, established by a 3D photopatterning technique employing extracellular matrix-derived hydrogel biomaterials. Specifically, cancer cells begin in colorectal cancer (CRC) organoid, which resides in a single microfluidic chamber connected to multiple downstream chambers in which liver, lung, and endothelial constructs are housed. Under recirculating fluid flow, tumor cells grow in the primary site, eventually enter circulation, and can be tracked via fluorescent imaging. Importantly, we describe that in the current version of this platform, HCT116 CRC cells preferentially home to the liver and lung constructs; the corresponding organs of which CRC metastases arise the most in human patients. We believe that in subsequent studies this platform can be implemented to better understand the mechanisms underlying metastasis, perhaps resulting in the identification of targets for intervention.

A cost-effective fluorescence mini-microscope for biomedical applications.

We have designed and fabricated a miniature microscope from off-the-shelf components and a webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters, such as cell/tissue viability (e.g. live/dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60×, achieves a resolution as high as <2 µm, and possesses a long working distance of 4.5 mm (at a magnification of 8×). The mini-microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including, but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread application in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required.

Atención de las emergencias y urgencias médico-quirúrgicas en un hospital oncológico / Emergencies and urgent medical-surgical conditions attended at a comprehensive cancer center

La incidencia global de las emergencias y urgencias médicoquirúrgicas en pacientes con cáncer ha sido descrita esporádicamente. El objetivo del estudio fue identificar los principales síntomas y diagnósticos de los pacientes que acudieron al Servicio de Urgencias del Hospital de Oncología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social. El diseño fue observacional y retrospectivo. La información fue obtenida del registro de la consulta diaria del Servicio de Admisión Continua. En un periodo de seis meses fueron atendidos 4,937 pacientes. Los cuadros clínicos evaluados como emergencias correspondieron a 3.7 %, como condiciones médicas urgentes 52.5 % y como condiciones no urgentes, 43.7 %. Los síntomas más frecuentes motivo de las consultas de emergencia o urgencias en los pacientes con cáncer fueron dolor grave en 69.5 % y deshidratación con desequilibrio hidroelectrolítico en 11.4 %. Los principales síntomas fueron provocados por el tumor primario o su diseminación metastásica, en 89 %. Los tumores malignos sólidos más frecuentes fueron los carcinomas mamario, de colon/recto, cervicouterino, broncogénico y gástrico. Las principales emergencias registradas en los pacientes con cáncer en este estudio fueron choque séptico y neutropenia severa (20 %), choque hipovolémico por sangrado en diversos sitios (16.5 %) y disnea agudizada por neumonía o derrame pleural (12 %). En aproximadamente 80 % de quienes son tratados paso a paso de manera racional, el dolor por cáncer pudo ser controlado sólo con analgésicos. La analgesia no efectiva se asoció frecuentemente con prescripción inadecuada o ingesta insuficiente de analgésicos opioides. Los servicios de urgencias establecidos funcionalmente en los hospitales monográficos de cáncer ofrecen la mejor oportunidad de tratamiento a los pacientes con cáncer con condiciones emergentes o urgentes.

The global incidence of emergencies and urgent medical?surgical conditions in cancer patients has not been well described. The aim of the study was to identify the main symptoms and diagnoses in patients seen for consultation at the Urgent Care Service in a Mexican Comprehensive Cancer Center. This was a retrospective observational study. The information was obtained from the Continuous Admission Service daily consultation records at the Oncology Hospital, National Medical Center 21st Century, Institute of Social Security, Mexico City. During a 6-month period, 4937 patients were seen for consultation. True oncologic emergencies were 3.7%, urgencies 52.5% and non-urgent were 43.7%. Most common symptoms for emergency and urgency patient consultations were severe pain (69.5%) and dehydration with electrolyte imbalance (11.4%). Prevalent symptoms were associated with the primary tumor or metastatic dissemination (89% cases). The most frequent baseline diseases were breast, colorectal, cervical, lung and stomach carcinomas. Defined oncologic emergencies in this series were septic shock and severe neutropenia (20%), hypovolemic shock due to severe bleeding (16.5%), and severe dyspnea due to pneumonia or pleural efusion (12%). Data evaluating the use of analgesic drug therapy for cancer pain alone indicate that 80% of patients report adequate analgesia. Analgesia failures were associated with an insufficient prescription or with inadequate consumption of opioid analgesics. The Urgent Care Center at a Comprehensive Cancer Center offers the best opportunity for diagnosis and treatment of emergencies and urgent care conditions in cancer patients.

[Emergencies and urgent medical-surgical conditions attended at a comprehensive cancer center]. / Atencián de las emergencias y urgencias médico-quirúrgicas en un hospital oncológico.

The global incidence of emergencies and urgent medical?surgical conditions in cancer patients has not been well described. The aim of the study was to identify the main symptoms and diagnoses in patients seen for consultation at the Urgent Care Service in a Mexican Comprehensive Cancer Center. This was a retrospective observational study. The information was obtained from the Continuous Admission Service daily consultation records at the Oncology Hospital, National Medical Center "21st Century," Institute of Social Security, Mexico City. During a 6-month period, 4937 patients were seen for consultation. True oncologic emergencies were 3.7%, urgencies 52.5% and non-urgent were 43.7%. Most common symptoms for emergency and urgency patient consultations were severe pain (69.5%) and dehydration with electrolyte imbalance (11.4%). Prevalent symptoms were associated with the primary tumor or metastatic dissemination (89% cases). The most frequent baseline diseases were breast, colorectal, cervical, lung and stomach carcinomas. Defined oncologic emergencies in this series were septic shock and severe neutropenia (20%), hypovolemic shock due to severe bleeding (16.5%), and severe dyspnea due to pneumonia or pleural efusion (12%). Data evaluating the use of analgesic drug therapy for cancer pain alone indicate that 80% of patients report adequate analgesia. Analgesia failures were associated with an insufficient prescription or with inadequate consumption of opioid analgesics. The Urgent Care Center at a Comprehensive Cancer Center offers the best opportunity for diagnosis and treatment of emergencies and urgent care conditions in cancer patients.

Programa de calidad de las Historias Clínicas del Hospital Zonal Virgen del Carmen de Zárate / Quality of Medical Record Program of the Hospital Zonal Virgen del Carmen

La medicina actual nos exige pericia, diligencia, respeto de los derechos del paciente y un registro adecuado y representativo de las circunstancias del servicio asistencial. El Foro de Medicina Legal de la Ciudad de Bs. As. y sus integrantes con participación de colegas y la institución hospitalaria citada, producen el Programa de Calidad y mejora en Historia Clínica. La técnica aplicada y los resultados alcanzados se describen en el presente trabajo con el objeto de poner en manos de otras entidades de salud las herramientas y experiencia adquirida por los autores en esta experiencia piloto.

La atención médica y la ley de defensa del consumidor / Medical care and the consumer defense law

Concluído el siglo y el milenio, a lo largo de su historia, la humanidad ha luchado por mejorar su calidad de vida. Dos aspectos centrales de dichos conceptos son el cuidado de su salud y la refirmación de sus derechos como integrantes de una sociedad. Hoy esta sociedad adquiere una complejidad que, por momentos, nos sorprende e incluso nos abruma a partir de los descubrimientos que superan nuestra capacidad de análisis y adaptación; valgan como ejemplo: el genoma humano y la clonación. Dicha tecnología, el aumento de la población, la inversión de la pirámide de crecimiento, son aspectos de la complejidad que exige elaborar normas protectoras del individuo y la sociedad. En esta presentación, analizaremos dos principios que representan las intenciones de la humanidad referidas a la salud y sus derechos como individuos.

Introducción a la calidad de atención médica / Introduction to medical service quality

El ejercicio de la profesión médica y la atención de la salud adquiere una inusitada complejidad en el transcurso de las tres últimas décadas. El avance incesante de la tecnología y el conocimiento; el nuevo rol del paciente a través de la reafirmación de sus derechos y el ejercicio de los mismos; las demandas de mala praxis; las leyes y normatizaciones gubernamentales; como así también una nueva concepción de atención en salud, asimilada a una empresa de servicios, plantea la necesidad de difundir en la población médica nociones básicas de estos temas. Esta propuesta debe permitirle al médico, interpretar el curso de los acontecimientos en la evolución de la atención de la salud, y comprometerlo a participar activamente en diseños de los nuevos modelos en calidad de atención médica.