Name: Ex Vivo Culture of Patient Tissue and Examination of Gene Delivery (Video) | JoVE Journal
Uploaded: 2010-12-20T18:00:00.000+00:00
Duration: 5 min
Description: Learn ex vivo culture of patient tissue slices for gene delivery studies using IVIS bioluminescence imaging. This method enables accurate analysis of gene expression in human cancer tissues.

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5 minutes

This work was funded through the Cork South Infirmary Victoria University Hospital Breast fund, the Irish Cancer Society (CRI07TAN) and the Cork Cancer Research Centre. Replication incompetent recombinant Adenovirus 5 particles under the transcriptional control of the CMV promoter was a kind gift from Prof. Andrew Baker, University of Glasgow.

Preparation of Media and Reagents
<ol>
 <li>Antibiotic Solution 
 Phosphate buffered saline (PBS) with 200 U/ml penicillin, 200 μg/ml streptomycin, 5 μg/ml fungizone</li>
 <li>Collection and Treatment medium 
 Dulbecco's modified eagle medium (DMEM) with 200 U/ml penicillin, 200 μg/ml streptomycin, 5 μg/ml fungizone</li>
 <li>Culture medium 
 Collection medium containing 10% heat-inactivated fetal bovine serum</li>
</ol>
I. Tissue collection and storage
Approval for patient tissue collection was obtained from the Clinical Research Ethics Committee of the Cork Teaching Hospitals and informed consent was obtained from the patients the day before surgery. Liver tissue was obtained from patients undergoing partial hepatectomy for malignant disease.
<ol>
 <li> Fresh patient tissue is obtained at the time of surgical resection.</li>
 <li> Tissue is stored in collection media at 4°C (Tissue may be stored refrigerated for up to 12 h without significant loss of viability, however immediate processing is recommended).</li>
</ol>
II. Tissue slice preparation and culture
Slicing was performed using a vibratome (Leica, Germany). The tissue slicing system was used according to the manufacturer's instructions. Tissue slice preparation was performed under a sterile hood using instruments cleaned with 70% 2-propanol. Slice thickness is set at 2000 microns and cut using a reciprocating blade at 22-26 rpm.
<ol>
 <li> Tissue is washed in antibiotic solution three times.</li>
 <li> Tissue is attached to mounting stage using non-toxic adhesive (Dermabond).</li>
 <li> Slice thickness is set and slicing is achieved at 22-26 rpm.</li>
 <li> Slices are maintained in culture medium in 6-well culture dishes (one slice per well) at 37°C with 5% CO2 in a humidified environment.</li>
</ol>
III. Gene delivery to tissue slices
<ol>
 <li> Slices are pre-incubated at 37°C for 2 h prior to treatment.</li>
 <li> Culture medium is replaced with treatment medium.</li>
 <li> Slices are directly injected with viral vector (25 μl of viral particles Ad5.CMVluc [1x109]). Alternatively, particles may be added to the medium, for passive infusion.</li>
 <li> After two hours of incubation, serum is added to medium.</li>
</ol>
IV. Analysis of gene expression by bioluminescent analysis
<ol>
 <li> Slices are injected with 100 μl of luciferin substrate (3 mg/ml).</li>
 <li> 6-well culture dish is placed on stage and incubated for 10 min.</li>
 <li> Slices are imaged for 5 min at high sensitivity (Figure 1).</li>
</ol>
V. Representative Results
<img src="/files/ftp_upload/2378/2378fig1.jpg" alt="Bioluminescence assay, photon detection, 4-well plate, experimental result, emission analysis, chart." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Bioluminescent imaging of patient liver slices using 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/pubmed?term=((Precision-cut+liver+slices+as+a+new+model+to+study+toxicity-induced+hepatic+stellate+cell+activation+in+a+physiologic+milieu%5BTitle%5D)+AND+%22Toxicol+Sci%22%5BJournal%5D)">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/pubmed?term=((Short-term+primary+culture+of+epithelial+cells+derived+from+human+breast+tumours%5BTitle%5D)+AND+%22Br+J+Cancer%22%5BJournal%5D)">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/pubmed?term=((A+novel+ex+vivo+model+system+for+evaluation+of+conditionally+replicative+adenoviruses+therapeutic+efficacy+and+toxicity%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Non-invasive+in+vivo+optical+imaging+of+the+lacZ+and+luc+gene+expression+in+mice%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice.</a> Gene Ther. 14, 1587-1593 (2007).</li>
</ol>

No conflicts of interest declared.

We describe an ex vivo patient tissue culture method and bioluminescent detection system for the assessment of gene delivery within non-fixed human tissue. The method offers a simple and reproducible way of culturing tissue slices. It has significant potential as it allows the study of gene delivery into intact human tissue, the analysis of gene delivery into a variety of human tissue including malignant tissue and can provide important information concerning the effects of the high degree of variability between...

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Firefly Luciferin</td><td>Biosynth International, Inc</td><td></td><td></td></tr><tr><td>Dermabond</td><td>Johnson &amp;amp; Johnson</td><td></td><td></td></tr><tr><td>Vibrotome</td><td>Leica Microsystems</td><td>VT 1000 A</td><td></td></tr></tbody></table>

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

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

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 is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

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 ...

Hello everyone. My name is Simon Regenron. I'm one of the surgical registrars here in the Core Cancer Research Center in Ireland.The technique I'm showing you today involves the use of intact patient tissue for the study of gene delivery. Gene therapy offers exciting therapeutic approaches for the treatment of cancer. However, have recent clinical trials have failed to replicate results found in animal models.The main reason, I believe, to be the variation in tissue physiology between animal and man tumor heterogeneity and differences in tumor phenotype. X vivo patient tissue model I'm demonstrating is the most stringent preclinical model available today. This model involves slicing patient tissue into thin slices and maintaining these slices in vitro.Many gene delivery methods may be tested using this model, the viral vectors and DNA constructs I'm using express a firefly luciferase under the transcription control of the CMV promoter, allowing quantification of gene expression by bioluminescent Imaging. Okay, let's go start this. The materials that are needed for this model are fresh human tissue, vibram tissue slicing system, physical gene delivery methods to be Assessed.An ultrasound machine, six plate tissue culture dish 27 gauge incident needle non-toxic glue gene delivery vector, which may be viral and non-viral media Containing serum and culture media. The block of tissue is attached to the slicing stage using derma bond ad heath. The material, the tissue is submerged in PBS during slicing to aid with slicing tissue Is submerged in ice and refrigerated for 30 minutes.The required slice thickness in this case two millimeters is set and slices are cut At 20 RPM slices are cultured in a six well dish after two hours of Incubation at 37 degrees Celsius. Viral vectors are injected directly Into the slices. Slices are incubated for 24 hours in a 37 degrees incubator.The next day, slices are analyzed for gene expression, but by a luminescent imaging.

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Learn ex vivo culture of patient tissue slices for gene delivery studies using IVIS bioluminescence imaging. This method enables accurate analysis of gene expression in human cancer tissues.

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

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