The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.

All procedures involving animals were carried out in accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The protocol for this study was approved by the Royal North Shore Hospital Animal Care and Ethics Committee. Female Fischer 344 rats (F344, 150-200 g) were maintained at the Kearns Facility, Kolling Institute under standard conditions (12 hr light/dark cycles and free access to food and water).
Note: A flow chart for all experimental procedures is presented in Figure 1.
1. Preparation of Cells for Implantation
<ol>
	<li>Culture the rat mesothelioma II-45 cell line (also known as IL-45; derived by peritoneal introduction of crocidolite asbestos) in RPMI 1640 (RPMI) media supplemented with 10% fetal bovine serum (FBS) and grow in standard conditions (37 &#176;C humidified incubator with 5% CO2). Maintain by passaging and sub-culturing at approximately 1:50 twice a week in a 75 cm2 flask.</li>
	<li>Prepare reagents for cell culture and warm aliquots at 37 &#176;C. Required reagents include serum free RPMI media (SFM), RPMI with 10% FBS, phosphate buffered saline (PBS) and 0.5% trypsin-EDTA.</li>
	<li>Culture cells for implantation to approximately 70-80% confluence. This ensures they are in the linear growth phase.</li>
	<li>Harvest cells by discarding the media, washing once with 5 ml&#160;of sterile PBS and then adding 3 ml&#160;of 0.5% trypsin-EDTA.</li>
	<li>Return flasks to the incubator for approximately 5 min until all cells become non-adherent.</li>
	<li>Once cells are non-adherent, add 3 ml&#160;of RPMI with 10% FBS to inactivate the trypsin. Collect and centrifuge cells at 300 x g for 3 min.</li>
	<li>Wash the cell pellet in 10 ml&#160;of SFM and centrifuge again at 300 x g for 3 min.</li>
	<li>Wash cell pellet again with 10 ml&#160;of SFM and centrifuge as above.</li>
	<li>Resuspend the cells in 10 ml&#160;of SFM and perform a cell count using a hemocytometer or similar instrumentation.</li>
	<li>Dilute cells so that 100 &#181;l&#160;contains the amount of cells to be implanted. 
	Note: Tumor growth has been demonstrated at a dose as low as 100 cells in 100 &#181;l but a standard dose is 500,000&#160;cells in 100 &#181;l.</li>
	<li>Prepare sufficient cells in media for the number of rats to be implanted (i.e., 100 &#181;l/rat), plus at least 0.5 ml&#160;extra to compensate for losses from priming and the dead volume of the needle.</li>
	<li>Prepare sufficient SFM (without cells) to be implanted into control rats (i.e.,&#160;100 &#181;l/rat), plus at least 0.5 ml&#160;extra. 
	Note: The cells and SFM are now ready for implantation. They should be kept at 37&#160;oC and implanted within 2 hr of harvesting to maintain viability.</li>
</ol>
2. In vivo&#160;Implantation of Cells
<ol>
	<li>Place the F344 rat (&#62; 13 weeks of age) into the induction chamber and anesthetize using 1.4% isoflurane inhalation (or the method in use in the facility). Once the rat appears to be asleep move it from the chamber to a nose cone (with 1.4% isoflurane flowing), place it on its back with chest facing up (ventral view). This allows the internal organs to settle away from the chest cavity. Check reflexes according to institutional protocols to ensure the rat is fully anesthetized.</li>
	<li>Shave the right regio costalis (chest) area to remove the fur.</li>
	<li>Clean the shaven area with 80% v/v ethanol.</li>
	<li>&#160;Identify the injection site: on the right side, find the 2nd gland starting cranial. The injection site is 0.5 cm proximal to this, in between the 3rd and 4th rib from the caudal end of the rib cage.&#160;(Figure 2A).</li>
	<li>Gently mix the II-45 cells to resuspend. Slowly draw the cell suspension (or SFM for control rats) into a 1 ml&#160;syringe without a needle attached. If a needle is attached for the drawing up of cells there is the potential for cells to grow along the needle injection line. Attach a 23G x 1&#188; needle. Prime the needle and remove any air bubbles.</li>
	<li>Once the syringe and needle are primed, place a 20 mm long and 5 mm diameter spacer over the needle shaft. This is used to prevent the needle from penetrating too deeply into the pleural cavity during the injection. Approximately 5 mm-12 mm of exposed needle is sufficient for penetration through the ribs without damaging any organs.</li>
	<li>Slowly insert the needle between the ribs, draw back on the syringe to ensure a blood vessel has not been punctured (no blood should appear in the syringe) then inject 100 &#956;l&#160;cells or SFM.&#160;(Figure 2B).</li>
	<li>Remove the needle and gently roll the rat from side to side to spread cells in the chest cavity.</li>
	<li>Place the rat into a cage and check for recovery. The rat should be awake within 1 min&#160;and starting to move around.</li>
	<li>Repeat for each rat using a new needle. Reusing the same needle will result in cell growth along the injection line of the needle.</li>
	<li>Monitor the well-being of the animals daily.</li>
	<li>Euthanize animals at ethically defined endpoints as governed by the institutional animal ethics committee. The ethical endpoints for the rats in these experiments were weight loss of greater than 10% or labored breathing.</li>
</ol>
3. Tail Vein Blood Collection
<ol>
	<li>If blood is to be collected immediately post-cell implantation, keep the rat anesthetized. If sampling blood at another time point, anesthetize the rat using 1.4% isoflurane inhalation. Check reflexes according to institutional protocols to ensure the rat is fully anaesthetized.</li>
	<li>Place the rat onto its side and locate a lateral tail vein.</li>
	<li>Sterilize the tail with 80% ethanol and label a 0.5 ml&#160;EDTA collection tube.</li>
	<li>To collect blood, always start at the caudal end of the tail (approximately one third of the way along). This allows further attempts closer to the cranial end of the tail in case the first attempt is unsuccessful. Never resample caudally as this can cause a blood clot.</li>
	<li>Position a 23G x 1&#188; needle parallel to the lateral vein and slide it into the vein at a shallow angle so it penetrates approximately 10 mm (Figure 3A).</li>
	<li>Note: If the vein has been successfully punctured blood will be visible in the attachment end of the needle (Figure 3B).</li>
	<li>A drop of blood will form on the tail at the site of the needle puncture. Collect this blood using a pipette and transfer into the labelled 0.5 ml (or smaller) EDTA collection tube. For the immune cell assay 25 &#956;l is sufficient. Apply gauze with pressure to puncture site until bleeding stops.</li>
	<li>Flick the blood tube to mix the blood and EDTA to prevent clotting. Keep the time between blood collection and mixing with the EDTA as short as possible to prevent clotting.</li>
	<li>When collecting blood from multiple rats store EDTA-blood samples in a rack at RT until analysis. Process blood within 2 hr&#160;of collection.</li>
</ol>
4. Sample Preparation for Immune Cell Profiling Using the Bead-based Method
Note: This single platform method relies on using commercially available absolute counting tubes that have a known number of beads for each sample. These tubes contain lyophilized pellets that dissolve during sample preparation, releasing the beads. The beads are fluorescently labelled and by gating on the bead population, absolute counts can be calculated.
<ol>
	<li>Ensure the EDTA whole blood sample is well mixed by placing it on a slow rotary mixer for several min. Label one absolute counting tube for each sample. A pellet containing the beads should be visible underneath the metal bead holder at the bottom of the tube.</li>
	<li>Transfer 25 &#181;l&#160;of EDTA whole blood into a labelled absolute counting tube. The bead pellet will dissolve upon addition of the blood.</li>
	<li>To each tube add 20 &#181;l&#160;of anti-rat T/B/Natural Killer (NK) cell cocktail, 10 &#181;l&#160;of anti-rat CD8a PE, 10 &#181;l&#160;of anti-rat CD4 (domain 1) FITC and 10 &#181;l of anti-rat CD45 PE/Cy7 (Figure 4A). Fluorophores are defined in Table 1.</li>
	<li>Centrifuge the tube briefly (300 x g) to ensure the antibodies and cells are in the bottom of the tube and not stuck to the side of the tube. Vortex to mix and incubate for 15 min&#160;at RT.</li>
	<li>To lyse red blood cells add 400 &#181;l&#160;of 10 mM Tris, 0.15 M ammonium chloride buffer (pH 7.5) and vortex to mix. Lysis is complete when the sample appears translucent and not cloudy (Figures 4B and C). Failure to lyse the sample completely will lead to increased background and falsely elevated counts when analyzing by flow cytometry.</li>
</ol>
5. Flow Cytometric Processing of Samples
Note: Perform on a 4 color flow cytometer.
<ol>
	<li>Open the software in acquisition mode and a new template with 8 plots as depicted in Figure 5.</li>
	<li>Adjust instrument settings to those listed in Table 1 and set up gate R1 (FITC [FL-1] versus APC [FL-4]), Figure 5Ai) to count the fluorescent beads. The other gates are not as important at this acquisition stage but will be required for analysis. The absolute counting beads used in this protocol contain fluorescent dyes and can be detected in any channel although are weakest in the blue channel.</li>
	<li>Using a prepared control blood sample, vortex and then load onto the cytometer and run at a low speed (12 &#181;l/min) on setup mode so data acquisition gates can be adjusted.</li>
	<li>Set the acquisition to collect 10,000 events in the R1 bead gate.</li>
	<li>Set up a folder to record data and set file number and label sample file in acquisition menu.</li>
	<li>Load the sample to be analyzed onto the cytometer and set the flow rate to medium (35 &#181;l/min). Run each sample at the same flow rate. The flow rate may need to be varied to low (12 &#181;l/min) or high (60 &#181;l/min), but medium is generally appropriate. At this rate it takes approximately 90 to 120 sec&#160;to acquire 10,000 bead events for each sample.</li>
	<li>Once the sample is loaded watch the scatter plots to make sure events are appearing in the R1 bead gate. Initially there can be some instability in the sample pressure causing drift in the scatter plots. Wait for this to stabilize.</li>
	<li>Once stabilized, click on acquire and allow sample to run. Once the cytometer has finished acquiring 10,000 bead events in R1 the cytometer will stop acquiring and save all data.</li>
	<li>Remove the sample and discard flow tube. The cytometer is now ready for the next sample. Run all samples and then proceed to analysis mode.</li>
</ol>
6. Immune Cell Analysis
Note: Gating strategies and Boolean algebra are used to define each cell population. Boolean algebra is a logic based analysis method that allows for multiple operations in a single definition. The analysis software of the flow cytometer (e.g., BD CELLQuest) allows for the use of Boolean algebra. The equations are used to actively account for the significant negative reactivity that assists in defining the cell to more specifically identify each cell population. &#8216;Regions&#8217; are used to define a &#8216;gate&#8217;. Regions define a 2 dimensional space whereas gates can be composed of numerous regions connected by algebraic operators (+, *,-, defined in Table 2).
<ol>
	<li>Switch the software to analysis mode. An analysis template should be generated to match Figure 5 with the plots and gates shown.</li>
	<li>Analyze each individual file (i.e., each individual sample) separately. Set up gates R1 through to R9 and then set up the algorithms for each cell type as defined in Table 2 (also shown in Figure 5) .</li>
	<li>Use the cell statistics counter to calculate individual cell populations defined by gates and algorithms (Table 2 and Figure 5). The algorithms will adjust cell numbers automatically in the cell statistics counter.</li>
	<li>Calculate cell subsets using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/53019/53019eq1.jpg" /> 
	Note: Number of cell events counted (e.g., CD4 T cell events) is enumerated using the above equation to give cell number per &#181;l of blood. Examples are shown in Figure 5.</li>
</ol>

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The authors have nothing to disclose.

This paper details a method for the generation of a rat syngeneic orthotopic model of pleural mesothelioma and a simple method for monitoring disease progression through longitudinal blood sampling.
The II-45 model was developed by exposing Fischer 344 rats to asbestos fibers13. Although this exposure represents the true dynamics of host-asbestos-immune system interactions for mesothelioma pathogenesis, it has a long lag time (taking years to generate) and can be dangerous for the r...

Malignant mesothelioma (MM) is an aggressive malignancy which arises from transformed cells in the membrane (mesothelium) that lines the lung and abdominal cavities, heart and internal reproductive organs, and is the most common primary tumor of the lung cavity or pleura1,2. Exposure to asbestos fibres accounts for 80% of all MM, and while bans on asbestos use were introduced decades ago in most western countries, its widespread use in the community has left a deadly legacy. The World Health Organization has estimated that 107,000 people worldwide die each year from asbestos related diseases, with mortality rates continuing to increase. A new non-occupational incidence wave is also emerging and there is little understanding of when, and at what level this will peak3.
The majority of people with MM are diagnosed late when systemic chemotherapy represents one of the only viable options4. The most effective chemotherapy and current &#8216;standard of care&#8217; (pemetrexed together with cisplatin5) was identified over 10 years ago. However failure of this treatment is inevitable and there are no proven second line options, leaving patients with a grim prognosis and median survival of only 12 months2. Therefore, there is an urgent unmet need for more effective treatments. Despite the examination of a number of novel therapies in clinical trials none has resulted in changes in practice. This is due in part to the low (5%) transference of pre-clinical results, generally performed in xenograft mouse models, to the clinical setting6-8. Such models do not faithfully recapitulate the complex aspects of the tumor microenvironment occurring in non-physiological locations, frequently in the absence of a functioning immune system9.
Syngeneic orthotopic models create a significantly more realistic tumor environment than the commonly used subcutaneous xenograft models as the tumors occur in the correct physiological location with an intact immune system10,11. The larger size of the rat enhances its use as a rodent disease model, especially in drug studies where serial blood draws are required to assess treatment response and toxicity12. Furthermore, in models in which monitoring disease progression is difficult due to the location of the tumors (such as in the pleural cavity), the ability to monitor disease progression using factors found in the circulation is extremely attractive. The generation of a syngeneic orthotopic model of pleural mesothelioma using immune-competent rats is described. In addition, an easy and relatively non-invasive method for monitoring pleural disease progression by measuring circulating immune cells is also described.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EDTA Collection tube (0.5ml)</td>
			<td>Greiner Bio One GmbH</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat T/B/NK Cell cocktail</td>
			<td>BD Pharmingen</td>
			<td>558509</td>
			<td>anti-Rat CD3&#160; APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78)</td>
		</tr>
		<tr>
			<td>anti-RAT CD8a PE&#160;</td>
			<td>Biolegend</td>
			<td>200608</td>
			<td>(IgG1vClone G28)</td>
		</tr>
		<tr>
			<td>anti-Rat CD4 FITC (Domain 1)&#160;</td>
			<td>Biolegend</td>
			<td>203406</td>
			<td>(IgG1 Clone OX-38)</td>
		</tr>
		<tr>
			<td>anti-Rat CD45 PE/Cy7&#160;</td>
			<td>Biolegend</td>
			<td>202214</td>
			<td>(IgG1 Clone OX-1)</td>
		</tr>
		<tr>
			<td>TruCount&#8482; Tubes</td>
			<td>Becton Dickinson</td>
			<td>340334</td>
			<td>Box of 50 absolute counting tubes</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>foetal bovine serum (FBS)</td>
			<td>Scientifix</td>
			<td>FBS500-S (lot# 010101-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago AB</td>
			<td>09-9400-100</td>
			<td></td>
		</tr>
		<tr>
			<td>23Gx1&#188; Needle</td>
			<td>Becton Dickinson</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fischer 344 Rat</td>
			<td>Animal Resources Centre, Perth Australia</td>
			<td>F344</td>
			<td></td>
		</tr>
		<tr>
			<td>I.S.O (Isoflurane USP)</td>
			<td>Veterinary Companys Australia (VCA)&#160;</td>
			<td>B7058</td>
			<td></td>
		</tr>
		<tr>
			<td>II-45 Rat Mesothelioma line</td>
			<td>Zurich University</td>
			<td></td>
			<td>Note: The cell line was given as a gift and is not commercially available at the ATCC</td>
		</tr>
		<tr>
			<td>FACSCalibur&#8482; 4 colour</td>
			<td>Becton Dickinson</td>
			<td>342975</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCL</td>
			<td>SIGMA</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>SIGMA</td>
			<td>9718</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthetic Machine (The stinger)</td>
			<td>Advanced Anaesthesia specialists</td>
			<td>#00449</td>
			<td></td>
		</tr>
	</tbody>
</table>

orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The method used in this paper for the generation of an orthotopic model of pleural mesothelioma using II-45 cells resulted in animals succumbing to mesothelioma in a reproducible and rapid timeframe, with no rats dying due to the implantation method. Titration of the number of cells implanted determined that 1x 103 cells was the minimum number required for a fully penetrant model (100% engraftment). The different number of cells implanted in the rats changed the time course of the disease without appearing to ...

Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

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9.1K Views.  Royal North Shore Hospital, University of Sydney. The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 µl blood sample is also described.

Video: Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...