Pre-operatorio <ol><li> Receptor de ratón y células de donantes </li><li> Plataforma de funcionamiento con un hilo de sutura y la cinta </li><li> 1cc jeringa </li><li> 23G aguja </li><li> Anestesia: avertina 25 mg / ml (alternativamente ketamina 9 mg / ml y xylazin 1 mg / ml) </li><li> Cortapelos con cuchilla # 40 </li><li> Guantes estériles </li><li> Torundas con alcohol </li><li> Yodo </li><li> Esterilizar los instrumentos quirúrgicos </li><li> Sutura hilo </li><li> Catéter de 22G </li><li> 200 ul pipeta (estéril) </li><li> Esterilizar pipetas </li><li> Ungüento antibiótico pegamento </li><li> Sharpie contenedor </li></ol> Operatorio Técnicas de asepsia se utilizan en este procedimiento. <ol><li> Pesar del ratón. </li><li> Inyección intraperitoneal de anestésico (250 mg / kg avertina o, alternativamente, la ketamina 90 mg / kg y xilacina 10 mg / kg) en dosis suficientes para permitir la respiración espontánea, sino que aportan quirúrgica-la concentración de la anestesia. Para la cirugía para comenzar la pizca dedo fue utilizado como un indicador de un dolor profundo, sin respuesta del animal anestesiado. </li><li> Coloque el ratón con los incisivos escondido en hilo de sutura y la cola para estabilizar la cinta. </li><li> Broche para el cabello con una cuchilla # 40 clipper. </li><li> Cambie los guantes (continuar con guantes estériles). </li><li> Desinfectar con alcohol dos veces antes de la incisión de la piel. </li><li> Esterilizar la zona con un yodóforo. </li><li> Utilizando instrumentos estériles hacer una pequeña incisión (&lt;1 cm) en paralelo a la tráquea. </li><li> Cuidadosamente tejido separado del cartílago. </li><li> Un empate se inserta debajo de la tráquea. </li><li> Levante suavemente hacia arriba y atar insertar la aguja en la tráquea expuestos. </li><li> Se retira la aguja e introduzca el tubo sin problemas, sin embargo, rápidamente, hasta que se sienta una ligera resistencia. </li><li> Pipeta de células (hasta 50 microlitros de volumen) directamente en el catéter. </li><li> Inmediatamente retire el tubo y la corbata, mientras que la liberación del ratón de aparatos de maniobra. </li><li> Si es necesario aplique un lado del ratón en posición vertical (vertical), aferrándose las patas anteriores y se mueven suavemente. </li><li> Cerrar la herida con una sutura o pegamento con antibióticos. </li></ol> Post-operatorio <ol><li> Mantenga caliente del ratón. </li><li> La buprenorfina (0,05 mg / kg) se debe administrar como analgesia postoperatoria cada 12 horas durante las primeras 24 horas después del procedimiento. </li></ol><img src="/files/ftp_upload/old/163_2008_7_15_4/163_figure-1.png" alt="Figura 1" width="587" height="531" />

<table><tbody><tr><td>23 G needle</td><td></td><td></td><td></td></tr><tr><td>1 cc syringe</td><td></td><td></td><td></td></tr><tr><td>22 G catheter</td><td></td><td></td><td></td></tr><tr><td>suture thread</td><td></td><td></td><td></td></tr><tr><td>antibiotic glue ointment</td><td></td><td></td><td></td></tr><tr><td>Anesthetic</td><td></td><td></td><td>Avertin 25 mg/mL. Alternatively ketamine 9 mg/mL and xylazin 1 mg/ml.</td></tr></tbody></table>

tracheotomy: a method for transplantation of stem cells to the lung

La enfermedad pulmonar es la principal causa de muerte y de probabilidades de convertirse en una epidemia dado el aumento de la contaminación y en todo el mundo fumando. Los avances en la terapia de células madre puede aliviar muchos de los síntomas asociados con la enfermedad de los pulmones y provocar la reparación alveolar en los adultos. Al mismo tiempo que la búsqueda permanente de las células madre de aplicación para el tratamiento humano, la entrega precisa y yendo (a la localización de la enfermedad) deben estar tranquilos para una terapia exitosa. Aquí, puedo informar de que las células madre de manera segura puede ser inculcado a través de la tráquea, la apertura de una ruta sin escalas a los pulmones. Este método consiste en una incisión en la piel, la inserción de una cánula de caudal en ya lo largo de la luz traqueal, y la inyección de una mezcla madre de vehículos de células en las vías respiratorias de los pulmones. Una amplia gama de soluciones de medios de comunicación y los estabilizadores pueden ser inculcados a través de traqueotomía, lo que resulta en la capacidad de ofrecer una gama más amplia de tipos de células. Con epitelio alveolar confinar a estas células a la luz, y la expansión de los pulmones durante la inhalación de presión negativa también puede ayudar en la integración de células madre. Entrega de la tráquea de las células madre, con una absorción rápida y la capacidad de manejar una amplia gama de tratamientos, podrían acelerar el desarrollo de terapias basadas en células, abriendo nuevas vías para el tratamiento de la enfermedad pulmonar.

Watch this Scientific Journal Video about Tracheotomy: A Method for Transplantation of Stem Cells to the Lung at JoVE.com

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Avances significativos en la identificación de células madre están continuamente haciendo. Para traducir estos descubrimientos, sin embargo, nuevos métodos para la entrega de celulares deben ser concebido. Aquí me informan de que las vías aéreas proporcionen una ruta segura para el trasplante de células madre a los pulmones.

Traqueotomía: Un método para el trasplante de células madre para el pulmón

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

tracheotomy-a-method-for-transplantation-of-stem-cells-to-the-lung

Universidad de Cartagena UDEC

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JoVE Journal

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Video: Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

Trasplante ortotópico de células de Adenocarcinoma de pulmón Syngeneic para estudiar la expresión de PD-L1

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Investigación

Investigación sobre el cáncer

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

El desarrollo de bronquiolitis obliterante en un modelo murino de trasplante de pulmón ortotópico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Medicina

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

El trasplante heterotópico traqueal y ortotópico en ratones utilizados como modelos para estudiar el desarrollo de la enfermedad de las vías respiratorias obliterante

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Biología

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Rata heterotópico abdominal Corazón / Trasplante de un solo pulmón en una configuración de volumen cargado

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Siembra e implantación de un injerto traqueal tejido-dirigida biosintético en un modelo murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Bioingeniería

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

Tejidos pulmonares diseñados preparados a partir de rodajas pulmonares descelularizadas

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Instilación intratraqueal de células madre en ratas neonatales a término

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Un método mínimamente invasivo para la instilación intratraqueal de medicamentos en roedores neonatales para tratar la enfermedad pulmonar

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Exploración de la enfermedad obliterante de las vías respiratorias en los trasplantes de pulmón

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions.
In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. 
Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months.
In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

Autor destacado: Investigación de los factores clave de la bronquiolitis obliterante después del trasplante de pulmón

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

Trasplante de tráquea intrapulmonar murina: un modelo para investigar la enfermedad obliterante de las vías respiratorias después de un trasplante de pulmón

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...

Traqueotomía: Un método para el trasplante de células madre para el pulmón

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Trasplante de tráquea intrapulmonar murina: un modelo para investigar la enfermedad obliterante de las vías respiratorias después de un trasplante de pulmón