The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.

This protocol follows the LUMC and AMC guidelines of human research ethics committee.
1. Surgical Procedure and Tissue Collection
Note: Although various techniques for surgical excision of Dupuytren&#8217;s contracture exist, the current gold standard is the partial fasciectomy 15. Most patients are treated in the day-surgery clinic.
<ol>
	<li>Perform surgery under general or regional anesthesia according to patient&#39;s and anaesthesiologist&#39;s preferences and to patient&#8217;s co-morbidities. These considerations are beyond the scope of this paper. Prior to incision, touch the skin with a sharp object to verify that anesthesia is satisfactory. Use a tourniquet to facilitate surgical visualization in a bloodless field.</li>
	<li>After sterile prepping and draping, mark out the anticipated incision with a pen. Incise the skin with a scalpel under loupe magnification either longitudinally or using zigzag incisions to prevent additional contractures and also to allow for sufficient exposure.</li>
	<li>Using scissor dissection, free the skin and subcutaneous layer from the underlying contracted fascia palmaris. Identify and protect the neurovascular bundle of the finger because the diseased cord can displace it toward the midline. Once the fibrotic tissue (nodule and/or cord) is outlined, excise it sharply and regionally (subtotal) using a scalpel. Perform meticulous hemostasis under tourniquet control at the end of the procedure to prevent hematoma formation.</li>
	<li>To close the skin, use additional Z-plasties to obtain partial lengthening of the skin.</li>
	<li>For these studies use only the nodule part of the fibrotic cord, the most active and cellular part of the disease. Have the surgeon perform macroscopic identification. The isolated nodules in the palm of the hand are often precursors or a cord but are hardly ever resected, as they usually do not cause symptoms. The ones we resected were the nodules that were part of a cord of the contracted finger. Identify these nodules by the hard thick part of the cords in the handpalm (Figure 1A).</li>
	<li>Once the surgeon removes the tissue, immediately transfer it using sterile forceps to a 50 ml tube containing DMEM medium supplemented with 10% FCS and 1% (P/S). Keep the tissue for a maximum of 2 hr in this medium on a box of wet ice (4 &#176;C) (e.g., transportation of the tissue from the operation room to the cell culture facility).</li>
	<li>Keep the samples on wet ice (4 &#176;C) while preparing for the next step. Preparation of the materials and cell culture chamber requires 10-20 min.</li>
</ol>
2. Preparation of Instruments, Culture Medium and Culture Inserts
Note: All procedures are performed at RT&#160;unless specifically stated.
<ol>
	<li>Prepare 500 ml of DMEM supplemented with 10% FCS and 1% (P/S) and filter sterilize. Prewarm the medium at 37 &#176;C.</li>
	<li>Autoclave forceps, a pair of curved-bladed Mayo scissors (150-170 mm in length) and a pair of Metzenbaum scissors and store in a sterile container.</li>
	<li>Under laminar flow, open a sterile 12-well culture plate. Place one culture insert (0.45 &#181;m pore size, 12 mm diameter) in each well of the 12-well plate (Figure 1B, lower panel).</li>
	<li>Fill the 12-well plate with 600 &#181;l of prewarmed (37 &#176;C) culture media by pipetting on the well and not directly on the membrane of the insert. Place the plate into the incubator (37 &#176;C, 5% CO2) (Figure 1B, upper panel).
	<ol>
		<li>Alternatively, use a 24-well plate and add 300 &#181;l of medium per well. The volume of medium per well is optimal when a meniscus layer is formed between the liquid and the lower part of the membrane insert. 
		Note: the medium at the bottom of the well diffuses through the nitrocellulose membrane forming the meniscus layer as depicted in schemes of Figure 1B (lower panel).</li>
	</ol>
	</li>
</ol>
3. Tissue Preparation and Ex Vivo Tissue Culture Setup
Note: All procedures are performed at RT unless specifically stated.
<ol>
	<li>Transfer the nodule part of the cord from the 50 ml tube on a 100 mm Petri dish and add 10 ml of prewarmed (37 &#176;C) medium. Ensure that the tissue is in contact with the liquid during the entire procedure.
	<ol>
		<li>Remove the perinodular fat layer using the Metzenbaum scissors. Discard the fat tissue. With the use of forceps and the curved-bladed scissors, cut transversally the tissue into smaller pieces (maximum thickness of 200 &#181;m).</li>
	</ol>
	</li>
	<li>Transfer the 12-well plate from the incubator into the laminar hood. Check that the membrane of the insert has turned transparent (wet) and therefore is in contact with the medium.</li>
	<li>Using forceps carefully lift a tissue part by touching the outer layer (do not apply tension on the tissue). Place one tissue part onto the membrane of the cell culture insert so that the tissue remains in the air-medium interphase. Culture a maximum of two tissue parts on the same insert/ well.</li>
	<li>Ensure that the tissue is placed flat on the membrane, in longitudinal contact with the medium such that the tissue neither floats off the membrane nor is fully covered by medium. Avoid air bubbles. 
	Note: At this stage, it is possible to add growth factors or inhibitors of interest to the media; for instance, test compounds (A, B, C, D, E) in replicates and/or in concentration curve. Include at least 2 control (untreated) pieces of tissue under normal growth conditions to ensure that the experiment has been set up properly. Incubate tissues for 3-7 days in a tissue culture incubator.</li>
	<li>Infect the tissue with either lentiviral or adenoviral construct if overexpression or knock down experiments of a gene-of-interest need to be performed (Figure 2). Transfer a tissue part from the 12-well plate into a 35 mm dish using forceps.
	<ol>
		<li>Use a maximum volume of 10 &#181;l of high titer virus.</li>
		<li>Perform the injection under a dissection microscope with an insulin syringe loaded with 50 &#181;l PBS containing 10 &#181;l of the selected virus. Place the needle perpendicular to the center of the tissue.</li>
		<li>Slowly inject the content of the syringe. Do not retract the needle directly. When puncturing the tissue, do not let the needle go through the other side but maintain the needle within the tissue itself (around the center of the tissue).</li>
		<li>Transfer the tissue back into the 12-well plate, close the culture plate and place it into the incubator (37 &#176;C, 5% CO2).</li>
	</ol>
	</li>
</ol>
4. Whole-mount Immunofluorescence Staining and 3D Reconstruction
Note: All procedures are performed at RT unless specifically stated. The protocol for whole mount immunostaining and imaging described below is an adaptation from previously reported methods used for other tissues 16-19. Buffers and materials are described in Table 1 and Materials List.
<ol>
	<li>Transfer tissues from the culture plate to 2 ml microcentrifuge tubes containing PBS. Wash 2x for 5 min in PBS solution on a rotating platform. Fix tissues in 4% buffered PFA (1.5 ml solution per tissue in 2 ml microcentrifuge tubes), O/N at 4 &#176;C on a rotating platform.</li>
	<li>Remove PFA and wash 3x in PBS for 5 min each.</li>
	<li>Dehydrate tissues by immersion in a 25% methanol solution for 2 hr at RT on a rotating platform. Increase the methanol percentage gradually (50%, 75%, 100%) with the tissues immersed at each solution for 2 hr at RT on a rotating platform.</li>
	<li>Transfer tissues in DMSO/methanol solution (permeabilization step) for 3 weeks at 4 &#176;C, as described previously 18. 
	Note: At this stage, tissues can be stored long term in 100% methanol at -20 &#176;C.</li>
	<li>Proceed with staining procedure 16, 17; gradually rehydrate tissues (75%-50%-25%-0% methanol series). Perform each rehydration step for 2 hr at RT or O/N at 4 &#176;C, under constant agitation.</li>
	<li>Incubate samples with primary antibodies in PBS containing 1% bovine serum albumin (BSA) and 20% DMSO O/N at 4 &#176;C. Use antibodies at the following ratios using the commercial stocks in PBS: anti-&#945; smooth muscle actin (1:500, mouse), anti-collagen type I (1:500, goat), anti-collagen type III (1:500, goat).</li>
	<li>Wash extensively in PBS for 48 hr at 4 &#176;C (change PBS solution 3 to 4 times).</li>
	<li>Dilute appropriate secondary antibodies (Alexa 555 anti-mouse, Alexa 488 anti-goat) at 1:250 dilution in PBS containing 1% (BSA) and 20% DMSO. Incubate O/N at 4 &#176;C.</li>
	<li>Wash extensively in PBS for 48 hr at 4 &#176;C and incubate with nuclear counterstain, TO-PRO-3 diluted 1:500 in PBS at the final washing step. 
	Note: TO-PRO-3 is a far-red emitting DNA dye (with a He-Ne 633 nm laser line) and is combined for simultaneous imaging with other channels; in this case collagen type I / collagen type III (488 nm), &#945; smooth muscle actin (555 nm), TO-PRO-3 (647 nm).</li>
	<li>Transfer the tissues to a methanol series (25%-50%-75%) to slowly dehydrate the tissue; perform each dehydration step for 2 hr at RT or O/N at 4 &#176;C under constant agitation.
	<ol>
		<li>Transfer tissues in polypropylene tubes. Clear tissues in methylsalicylate (handle under chemical hood) 18 or as an alternative use BABB solution 19. Incubate at RT under constant agitation until the tissues become transparent. Mount between a slide and a coverslip sealed with hard set mounting medium (Figure 1C, upper panel).</li>
	</ol>
	</li>
	<li>Image samples on an inverted confocal microscope equipped with high NA 63X and/or 100X oil immersion objectives (Figure 1C).
	<ol>
		<li>Clean the lens with cleaning solution provided by the microscope manufacturer.</li>
		<li>Apply one drop of the oil on the objective lens. Place the slide with the specimen on the microscope stage and focus on the sample.</li>
	</ol>
	</li>
	<li>Set the configurations on the microscope for simultaneous imaging of three-channel fluorescence with laser lines 488 nm, 514 nm and 633 nm.</li>
	<li>Acquire single XY images or multiple focal plane images (Z stack) (Figure 3A). Use increased width of Z stack for performing 3D reconstruction. To obtain a high-resolution image use low scanning speed, number of average scans (&#8805; 3) and acquire 16-bit images of 1,024 x 1,024 pixel resolution.</li>
	<li>Analyze the Z stack acquired images: first rename and save image file (xyz). Using the software program of the confocal microscope, configure a maximum intensity projection of the Z stack images into a 3D reconstructed image.
	<ol>
		<li>Select the xyz file, in the &#8220;Process Tools&#8221;, click under &#8220;Visualisation&#8221; and &#8220;3D Projection&#8221;. Enter &#8220;Maximum&#8221; in the method of projection (or &#8220;Average&#8221; if the fluorescent signal is saturated). For a single projection do not change the X, Y, and Z planes, and apply the projection tool (Figure 3B, r1).</li>
	</ol>
	</li>
	<li>Use the Z stack images to create a 3D projection with animation (confocal software) for viewing purposes. Select the xyz file, in the Process Tools, click under &#8220;Visualisation&#8221; and &#8220;3D Projection&#8221;.
	<ol>
		<li>Click on &#8220;Create Movie&#8221;. Enter the Start Rotation angle in degrees (e.g., 0&#176; as starting point of the movie, Y axis) and click on &#8220;Set Start&#8221;. Enter the End Rotation angle in degrees (Y axis) as end point of the movie (e.g., 180&#176; or 360&#176; for a complete rotation) and click on &#8220;Set End&#8221;.</li>
		<li>Select &#8220;Maximum&#8221; as a method of projection (or &#8220;Average&#8221; if the fluorescent signal is saturated). Enter the &#8221;Number of Frames&#8221; for the rotation of the 3D reconstruction (e.g.,&#160;20 rotations or higher to reduce the speed of rotation). Click on &#8220;Apply&#8221;. Export the movie as visual file (e.g., &#8220;avi&#8221;) (Supplementary&#160;Movie 1) or export as &#8220;tiff&#8221; file which creates an image file for each rotation angle (Figure 3B; rotation r4, r10, r18).</li>
	</ol>
	</li>
</ol>
5. Combined Second Harmonic Generation (Collagen) and Two-photon Excited Fluorescence (Elastin) Imaging on Ex Vivo Tissue during Culture
Note: All procedures are performed at RT unless specifically stated.
<ol>
	<li>Remove transwell plates from incubator and place a single (unfixed) tissue part in a 35 mm cell culture plate containing 500 &#181;l of medium.</li>
	<li>Position the tissue so that the long axis is flat on the plate. Keep tissue wet at all times however, not floating.</li>
	<li>Place water-immersed objective of the multiphoton microscope directly in contact with the tissue (Figure 1C).</li>
	<li>Obtain images with an excitation wavelength of 800 nm and collect emitted light between 371-425 nm (SHG, collagen) and 474-532 nm (autofluorescence, elastin) (Figure 3C). Perform two-photon microscopy in an upright confocal microscope equipped with a femtosecond laser using a 20X/1.0 NA water-immersion objective. Process confocal stacks with manufacturer&#8217;s software. 
	Note: Excitation with a near infrared laser of collagen molecules creates high contrast imaging of native collagen structures in different depths.</li>
</ol>

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The authors have filed a Patent Application for Dupuytren&#8217;s disease: 3D organ culture. # GB1307200. Commercial license and service contracts are available.

The most critical steps of culturing ex vivo human connective tissue are the immediate use of the tissue after surgical removal to ensure viability; the tissue should remain in medium or saline solution at all times; maintain a sterile culture; transverse sections of tissue should have maximum 200 &#181;m thickness; set up of the ex vivo culture system is optimal when tissue is in contact with the medium but not fully submerged. Medium should be added only on the outside chamber of the transwell in a sm...

Dupuytren&#39;s disease (DD), a benign fibroproliferative disease causes permanent flexion of the fingers due to the formation of nodules and cords in the palm of the hand. Although the disease spread is particularly high among Caucasians of Northern Europe, the underlying genetic etiology of the disease remains unknown 1. The main characteristic of DD is the excess production of extracellular matrix (ECM) proteins (e.g., collagen), which form a tough fibrous tissue occupying the space between the tendons and skin of the palm of the hand and fingers, permanently disrupting the fine movements of the hand 2, 3. The recurrence of the disease suggests underlying genetic alterations as a cause of fibrosis 1, 4. An effective treatment could be to target directly the uncontrollable fibrotic mechanisms at the cellular and molecular level.
Our recent work on fibrosis has led us to the development of a novel 3D culture system that allows short-term culture of human fibrotic tissue with the potential of drug testing. This system has helped to overcome the limiting approach of 2D fibroblast cultures and to define a role for the partial down regulation, achieved by exon skipping, of TGF&#946; pathway activation in mediating fibrosis 5.
We have developed a method to culture ex vivo human resection specimens from DD patients to study the interaction between myofibroblasts and the surrounding ECM 5, 6. The study of DD connective tissue fibrosis as well as other fibrotic diseases relies on histopathological analysis of the excised surgical specimens, isolation of fibroblasts from the tissue and establishment of primary cultures or cell sorting procedures. These approaches are quite static since they do not permit exogenous manipulation of the disease properties or therapeutic intervention by the experimenter. In addition, primary cell cultures tend to adapt to the culture conditions and their gene expression properties differ essentially from the in vivo situation upon every passage, even during early passages (among passage 3 and 6) 7, 8. We have managed to maintain the waste surgical material in ex vivo culture conditions for a time period that allows study of the patient-specific characteristics and screening of anti-fibrotic or anti-inflammatory drug compounds.
The system is based on a nitrocellulose membrane that permits contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue upon attachment, as previously observed when culturing DD fibroblasts as well as other cell types 9. No collagen gel or other ECM protein substrate is required, since the DD tissue itself produces large amounts of these proteins. This is advantageous for the maintenance of native ECM microenvironment and turnover since matrix substrates are important regulator of tissue architecture and function 10, 11. For instance, ECM proteins such as fibronectin, laminin and collagen, may influence front-rear polarity of fibroblasts as similarly shown for apical-basal polarity in epithelial cells 12, 13. Polarized cells have asymmetrical distribution of extracellular molecules which determines cell migration and gene expression, e.g., &#945;1&#946;1 integrin accessibility on the membrane affects cell adhesion to type I collagen14. Since a primary goal of this 3D model was to preserve the native microenvironment, no artificial ECM matrix substrate was used.
In brief: resection specimens are equally cut in a sterile environment and placed on nitrocellular membranes. If treatment administrated via injection is required the tissues are injected after they have been placed on the membrane. If treatment does not require to be administrated via injection then the compound is added to the culture media (Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), with 1% fetal calf serum (FCS), 1% penicillin-streptomycin (P/S)). The cultures are maintained for a maximum of ten days after which the tissue is fixed in 4% paraformaldehyde (PFA), processed through 30% sucrose solution, embedded in O.C.T. compound and stored at -80 &#176;C, as previously described 5.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Invitrogen</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td></td>
			<td>high glucose, heat inactivated</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Millicell</td>
			<td>PIHA01250</td>
			<td>0.45um pore size, 12mm diameter</td>
		</tr>
		<tr>
			<td>anti-collagen type I</td>
			<td>Southern Biotech</td>
			<td>1310-08</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- &#945; smooth muscle actin</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-collagen type III</td>
			<td>Southern Biotech</td>
			<td>1330-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor555 Donkey Anti-Mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>methylsalicylate</td>
			<td>Sigma</td>
			<td>M6752</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek OCT</td>
			<td>Sakura</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsccope glass coverslips</td>
			<td>Menzel-Glaser</td>
			<td>BB024060A1</td>
			<td>24x60mm</td>
		</tr>
		<tr>
			<td>Microscope SuperFrost slides</td>
			<td>Menzel-Glaser</td>
			<td>AA00000102E</td>
			<td>76x26mm</td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 II confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Argon-488, 514 nm and HeNe-633 nm laser lines</td>
		</tr>
		<tr>
			<td>Zeiss 710 NLO upright confocal microscope</td>
			<td>Jena, Germany</td>
			<td></td>
			<td>equipped with femtosecond Spectra - Physics Deep See MP laser (Santa Clara, United States) using a Plan -Apochromat 20x/1.0 NA 
			water-immersion objective.</td>
		</tr>
	</tbody>
</table>

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.

The method of ex vivo, 3D culture of connective tissue is an easy and robust set up system to understand the relation between ECM and other cell components constituting the DD tissue and potentially other types of fibrosis as well. Moreover this system allows a reliable method to test the effects of compounds on different cell types and their effects on the fibrotic load 20.
The steps from collection of surgical waste material derived from Dupuytren&#8217;s fibrosis, the as...

Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

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.

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 ...

human-dupuytren-s-ex-vivo-culture-for-study-myofibroblasts

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10.3K Views.  Leiden University Medical Center. Dupuytren’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.

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

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.

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

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 ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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.

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

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

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.