Diese Arbeit wurde durch die Cork South Infirmary Victoria University Hospital Breast Fonds, die Irish Cancer Society (CRI07TAN) und Cork Krebsforschungszentrum finanziert. Replication inkompetent rekombinanten Adenovirus 5 Partikel unter der transkriptionellen Kontrolle des CMV-Promotors war eine Art Geschenk von Prof. Andrew Baker, University of Glasgow.

Vorbereitung der Medien und Reagenzien <ol><li> Antibiotika-Lösung Phosphat-gepufferte Kochsalzlösung (PBS) mit 200 U / ml Penicillin, 200 ug / ml Streptomycin, 5 ug / ml Fungizone </li><li> Sammlung und Behandlung Medium Dulbecco modifiziertem Eagle Medium (DMEM) mit 200 U / ml Penicillin, 200 ug / ml Streptomycin, 5 ug / ml Fungizone </li><li> Nährboden Sammlung Medium mit 10% Hitze-inaktiviertem fetalem Rinderserum </li></ol> I. Tissue Datenerhebung und-speicherung Zulassung für Patienten Gewebe Sammlung wurde von der Clinical Research Ethics Committee der Cork Teaching Hospitals erhalten und Einwilligung wurde von den Patienten am Tag vor der Operation erhalten. Lebergewebe wurde von Patienten, bei denen eine partielle Hepatektomie für bösartige Erkrankung erhalten. <ol><li> Frisches Gewebe des Patienten zum Zeitpunkt der chirurgischen Resektion erhalten. </li><li> Das Gewebe wird in der Sammlung Medien Lagerung bei 4 ° C (Tissue gespeichert werden können gekühlt bis zu 12 h ohne nennenswerten Verlust der Lebensfähigkeit, aber sofortige Verarbeitung wird empfohlen). </li></ol> II. Gewebeschnitt Vorbereitung und Kultur Slicing wurde mit einem Vibratom (Leica, Deutschland). Das Gewebe schneiden System wurde nach den Anweisungen des Herstellers verwendet. Gewebeschnitt Präparat wurde unter einer sterilen Haube mit Instrumenten mit 70% 2-Propanol gereinigt durchgeführt. Schichtdicke ist bei 2000 Mikrometer eingestellt und schneiden mit einer Hin-Klinge bei 22-26 Umdrehungen pro Minute. <ol><li> Das Gewebe wird in Antibiotika-Lösung dreimal gewaschen. </li><li> Das Gewebe wird zur Montage der Bühne mit ungiftigen Kleber (Dermabond) befestigt. </li><li> Schichtdicke eingestellt ist und das Schneiden ist bei 22-26 Umdrehungen pro Minute erreicht. </li><li> Scheiben sind in Kulturmedium in 6-well Kulturschalen (eine Scheibe pro Well) bei 37 ° C mit 5% CO 2 in eine feuchte Umgebung. </li></ol> III. Gene Lieferung an Gewebeschnitten <ol><li> Scheiben werden bei 37 vorinkubiert ° C für 2 h vor der Behandlung. </li><li> Kulturmedium wird mit der Behandlung Medium ersetzt. </li><li> Slices sind direkt mit viralen Vektor (25 ul von Viruspartikeln Ad5.CMVluc [1x10 9]). Injiziert Alternativ können Partikel, die zugesetzt werden, für passive Infusion. </li><li> Nach zwei Stunden Inkubation wird Serum zugesetzt. </li></ol> IV. Die Analyse der Genexpression durch Biolumineszenz-Analyse <ol><li> Scheiben sind mit 100 ul Luciferin-Substrat (3 mg / ml) injiziert. </li><li> 6-well Kulturschale auf der Bühne platziert und für 10 min inkubiert. </li><li> Scheiben sind für 5 min bei hoher Empfindlichkeit (Abbildung 1) abgebildet. </li></ol> V. repräsentative Ergebnisse <img src="/files/ftp_upload/2378/2378fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Bioluminescent Bildgebung von Patienten mit Leber-Scheiben IVIS Detection System.

<ol>
	<li>van de Bovenkamp, M., Groothuis, G. M., Draaisma, A. L., Merema, M. T., Bezuijen, J. I., van Gils, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+liver+slices+as+a+new+model+to+study+toxicity-induced+hepatic+stellate+cell+activation+in+a+physiologic+milieu.">Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu.</a> Toxicol Sci. 85, 632-638 (2005).</li><li>Speirs, V., Green, A. R., Walton, D. S., Kerin, M. J., Fox, J. N., Carleton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+primary+culture+of+epithelial+cells+derived+from+human+breast+tumours.">Short-term primary culture of epithelial cells derived from human breast tumours.</a> Br J Cancer. 78, 1421-1429 (1998).</li><li>Kirby, T. O., Rivera, A., Rein, D., Wang, M., Ulasov, I., Breidenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ex+vivo+model+system+for+evaluation+of+conditionally+replicative+adenoviruses+therapeutic+efficacy+and+toxicity.">A novel ex vivo model system for evaluation of conditionally replicative adenoviruses therapeutic efficacy and toxicity.</a> Clin Cancer Res. 10, 8697-8703 (2004).</li><li>Josserand, V., Texier-Nogues, I., Huber, P., Favrot, M. C., Coll, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+optical+imaging+of+the+lacZ+and+luc+gene+expression+in+mice.">Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice.</a> Gene Ther. 14, 1587-1593 (2007).</li></ol>

Wir beschreiben eine ex vivo Patienten Gewebekultur-Methode und Biolumineszenz Detection System für die Beurteilung der Gen-Lieferung innerhalb von nicht fixierten menschlichen Gewebe. Das Verfahren bietet eine einfache und reproduzierbare Weise der Kultivierung Gewebeschnitten. Es hat ein erhebliches Potenzial, wie es das Studium der Gentransfer in intakte menschliche Gewebe, die Analyse der Gene Delivery ermöglicht in einer Vielzahl von menschlichem Gewebe einschließlich bösartiger Gewebe und können wich...

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ex vivo culture of patient tissue &amp; examination of gene delivery

Dieses Video beschreibt die Verwendung von Gewebe des Patienten als ex vivo-Modell für die Untersuchung der Gene Delivery. Frisches Gewebe des Patienten zum Zeitpunkt der Operation gewonnen wird in Scheiben geschnitten und in Kultur gehalten. Die ex vivo-Modell-System ermöglicht die physische Lieferung von Genen in intakte Gewebe des Patienten und die Genexpression durch Biolumineszenz-Bildgebung mit dem IVIS Detection System analysiert. Die Biolumineszenz Detection System zeigt eine schnelle und genaue Quantifizierung der Genexpression in einzelne Segmente, ohne dass Gewebe zu opfern. Diese Scheibe Gewebekultur-System kann in einer Vielzahl von Gewebetypen einschließlich der normalen und malignen Gewebe verwendet werden und ermöglicht es uns, die Auswirkungen der heterogenen Natur der intakten Gewebes und der hohe Grad an Variabilität zwischen den einzelnen Patienten zu untersuchen. Dieses Modell System könnte in bestimmten Situationen als Alternative zu Tierversuchen und als ergänzende präklinische Modus vor dem Eintritt in klinische Studie verwendet werden.

Watch this Scientific Journal Video about Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery at JoVE.com

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

Dieser Artikel beschreibt die Kultur der Patientensicherheit Gewebeschnitten für den Gentransfer-Studien und die anschließende Analyse der Genexpression mittels IVIS Biolumineszenz-Bildgebung.

Ex vivo-Kultur der Patient Tissue &amp; Prüfung von Gentransfer

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

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Research

JoVE Journal

Medicine

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

11.1K Aufrufe.  University College Cork. Dieser Artikel beschreibt die Kultur der Patientensicherheit Gewebeschnitten für den Gentransfer-Studien und die anschließende Analyse der Genexpression mittels IVIS Biolumineszenz-Bildgebung.

Serie: Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex-vivo-Infektion von lebendem Gewebe mit onkolytischen Viren

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Forschung

Medizin

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Ein orthotopen Blasenkrebs-Modell für Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Menschen Dupuytren&#39;sche<em&gt; Ex vivo</em&gt; Kultur für das Studium der Myofibroblasten und extrazellulären Matrix-Interaktionen

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex-vivo-Live-Imaging von Lungenmetastasen und deren Mikromilieu

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Die Erzeugung von Microtumors Mit 3D Human Biogel Kultursystem und Patienten stammGlioblastomZellen für Kinomic Profilieren and Drug Response-Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Muttermilch fördert Wachstum der Enteroids: ein Ex-Vivo -Modell der Zellproliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Ein kleines Tier Modell der Ex Vivo Normothermic Leber Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adeno-assoziierten Virus-vermittelte Lieferung von CRISPR für kardiale gen Bearbeitung bei Mäusen

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Lentivirale Vektor-vermittelte Gentherapie von Hepatozyten Ex Vivo für autologe Transplantation bei Schweinen

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Methods Article

Ex vivo-Kultur der Patient Tissue & Prüfung von Gentransfer