Name: tracheotomy: a method for transplantation of stem cells to the lung
Uploaded: 2007-02-25T00:00:00.000+00:00
Duration: 3 min 45 s
Description: Watch this Scientific Journal Video about Tracheotomy: A Method for Transplantation of Stem Cells to the Lung at JoVE.com

Pre-operative <ol><li> Empfänger Maus und Spenderzellen </li><li> Operating-Plattform mit Klebeband und Faden </li><li> 1cc Spritze </li><li> 23G Nadel </li><li> Betäubungsmittel: Avertin 25 mg / ml (alternativ Ketamin 9 mg / ml und xylazin 1 mg / ml) </li><li> Haarschneider mit # 40 Klinge </li><li> Sterile Handschuhe </li><li> Alkoholtupfer </li><li> Jod </li><li> Sterilisiert chirurgische Instrumente </li><li> Faden </li><li> 22G-Katheter </li><li> 200 ul Pipette (sterile) </li><li> Sterilisierte Pipettenspitzen </li><li> Antibiotika-Salbe Kleber </li><li> Sharpie Container </li></ol> Operativ Aseptische Techniken werden in diesem Verfahren verwendet. <ol><li> Wiegen Sie mit der Maus. </li><li> Intraperitoneale Injektion von Anästhetika (250 mg / kg Avertin oder alternativ Ketamin 90 mg / kg und Xylazin 10 mg / kg) bei Dosierungen ausreichen, um Spontanatmung zu ermöglichen, bieten aber chirurgisch-Konzentration Anästhesie. Für die Operation beginnen die Zehe Prise als Indikator für die tiefen Schmerz ohne Antwort aus dem narkotisierten Tier verwendet wurde. </li><li> Lay-Maus mit Schneidezähnen in Faden und Klebeband Schwanz zur Stabilisierung versteckt. </li><li> Clip Haare mit einem # 40 clipper Klinge. </li><li> Wechseln Sie die Handschuhe (weiter mit sterilen Handschuhen). </li><li> Desinfizieren Sie zweimal mit Alkohol vor dem Hautschnitt. </li><li> Sterilisieren Bereich mit einem Iodophor. </li><li> Mit sterilen Instrumenten einen kleinen Einschnitt (&lt;1 cm) parallel zur Luftröhre. </li><li> Sorgfältig trennen Gewebe aus Knorpel. </li><li> Eine Krawatte ist unterhalb der Luftröhre eingeführt. </li><li> Vorsichtig heben nach oben Krawatte und legen Nadel in exponierten Luftröhre. </li><li> Entfernen Sie die Nadel und stecken Sie den Schlauch problemlos, aber schnell, bis das Gefühl einen leichten Widerstand. </li><li> Pipette Zellen (bis zu 50 Mikroliter Volumen) direkt in den Katheter. </li><li> Unverzüglich entfernen Rohr und Krawatte, während der Befreiung der Maus aus dem operativen Apparat. </li><li> Bei Bedarf sanft halten Maus aufrecht (vertikal) durch Festhalten seiner vorderen Pfoten und bewegen sanft. </li><li> Schließen Sie die Wunde mit einer Naht oder Antibiotika Leim. </li></ol> Post-operative <ol><li> Halten Sie mit der Maus warm. </li><li> Buprenorphin (0,05 mg / kg) als postoperative Analgesie gegeben werden alle 12 Stunden für die ersten 24 Stunden nach diesem Verfahren. </li></ol><img src="/files/ftp_upload/old/163_2008_7_15_4/163_figure-1.png" alt="Abbildung 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

Lung Krankheit ist eine der häufigsten Todesursachen und wahrscheinlich eine Epidemie gegeben steigt in der Umweltverschmutzung und Rauchen weltweit geworden. Advances in Stammzell-Therapie kann zu lindern viele der Symptome von Lungenkrankheiten und induzieren alveolären Reparatur bei Erwachsenen. Zeitgleich mit der andauernden Suche nach Stammzellen gelten für menschliche Behandlung muss präzise Lieferung und Homing (an den Ort der Erkrankung) für eine erfolgreiche Therapie beruhigt sein. Hier berichte ich, dass Stammzellen sicher über die Luftröhre Eröffnung eines Non-Stop-Route in die Lunge instilliert werden. Bei dieser Methode wird ein Hautschnitt, caudal Einführen einer Kanüle in und entlang der Tracheallumen und Injektion eines Stammzell-Fahrzeug Mischung in Atemwegen der Lunge. Eine breite Palette von Medien-Lösungen und Stabilisatoren können via Tracheotomie eingeflößt werden, was in der Fähigkeit, ein breiteres Spektrum von Zelltypen zu liefern. Mit Alveolarepithel Beschränkung dieser Zellen in das Lumen, kann Lungenkrebs Expansion und Unterdruck beim Einatmen auch in Stammzellen Integration zu unterstützen. Tracheal Lieferung von Stammzellen, mit einer schnellen Aufnahme und die Fähigkeit, eine große Auswahl an Behandlungen Griff, konnte Beschleunigung der Entwicklung von Zell-basierte Therapien, eröffnet neue Wege für die Behandlung von Lungenkrankheiten.

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

Bedeutende Durchbrüche in der Stammzellenforschung Identifikation werden ständig gemacht. Zu übersetzen, diese Entdeckungen, müssen jedoch neue Methoden für die zelluläre Lieferung erarbeitet werden. Hier habe ich berichten, dass die Atemwege eine sichere Route für Stammzelltransplantation in der Lunge liefern.

Tracheotomie: Eine Methode zur Transplantation von Stammzellen in die Lunge

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

Research

JoVE Journal

Biology

11.7K Aufrufe.  Harvard Medical School. Aseptic techniques are used th

Serie: 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.

Orthotopen Transplantation von Phänomen Lungenzellen Adenokarzinom, PD-L1 Ausdruck zu studieren

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

Forschung

Krebsforschung

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.

Entwicklung einer Bronchiolitis obliterans in einem Mausmodell der orthotopen Lungentransplantation

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

Medizin

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.

Heterotope und orthotopen Tracheal Transplantation in Mäuse als Modelle benutzt, um die Entwicklung von obliterans Airway Disease Study

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

Biologie

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.

Aussaat und Implantation von einem biosynthetischen Tissue-Engineering trachealen Transplantat in einem Mausmodell

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

Biotechnik

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolierung von Basalzellen und Submuköse Gland Duct Zellen aus Maus Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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.

Intratracheale Instillation von Stammzellen bei Term Neonatal Ratten

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

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Transplantation einer biotechnologisch hergestellten Lunge unter Verwendung von dezellularisierten Mauslungen und primären humanen Endothelzellen

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

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.

Eine minimal-invasive Methode zur intratrachealen Instillation von Medikamenten bei neonatalen Nagetieren zur Behandlung von Lungenerkrankungen

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 ...

Erforschung der obliterativen Atemwegserkrankung bei Lungentransplantationen

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 im Rampenlicht: Untersuchung der Schlüsselfaktoren der obliterativen Bronchiolitis nach Lungentransplantation

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.

Intrapulmonale Trachealtransplantation bei Mäusen: Ein Modell zur Untersuchung obliterativer Atemwegserkrankungen nach Lungentransplantation

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

Tracheotomie: Eine Methode zur Transplantation von Stammzellen in die Lunge

Transkript

Zusammenfassung

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Heterotope und orthotopen Tracheal Transplantation in Mäuse als Modelle benutzt, um die Entwicklung von obliterans Airway Disease Study

Aussaat und Implantation von einem biosynthetischen Tissue-Engineering trachealen Transplantat in einem Mausmodell

Isolierung von Basalzellen und Submuköse Gland Duct Zellen aus Maus Trachea

Intratracheale Instillation von Stammzellen bei Term Neonatal Ratten

Transplantation einer biotechnologisch hergestellten Lunge unter Verwendung von dezellularisierten Mauslungen und primären humanen Endothelzellen

Eine minimal-invasive Methode zur intratrachealen Instillation von Medikamenten bei neonatalen Nagetieren zur Behandlung von Lungenerkrankungen

Intrapulmonale Trachealtransplantation bei Mäusen: Ein Modell zur Untersuchung obliterativer Atemwegserkrankungen nach Lungentransplantation

Intrapulmonale Trachealtransplantation bei Mäusen: Ein Modell zur Untersuchung obliterativer Atemwegserkrankungen nach Lungentransplantation