Funding for this project is provided by the Clayton Foundation for Research and the Retina Research Foundation.

1.	Preparation of retinoblastoma tumorspheres
<ol>
	<li>Obtain retinoblastoma tumor sample under an IRB-approved protocol.</li> 
 <li>Prepare fresh defined stem cell media (Neurobasal-A media, 1X B-27 supplement minus vitamin A, 1X non-essential amino acid solution, 1X L-glutamine, 20 ng/mL EGF, 10 ng/mL bFGF).</li>
<li>Mechanically disaggregate the tumor tissue with a scalpel using a cross-cutting technique in a sterile, tissue culture-treated 6-well plate. Immediately add 100 &mu;L of defined stem cell media to the tissue and gently spread the minced tissue using the scalpel. Add 5 mL of defined stem cell media to the well.</li>
<li>Incubate at 37&deg;C, 5% CO2 in a humidified incubator and inspect daily. Some tumorspheres should arise from the disrupted tumor tissue almost immediately, with increasing numbers over the course of 2-4 days.</li>
<li>Weekly, centrifuge tumorspheres at 300xg for 5 minutes, resuspend in freshly prepared defined stem cell media, and pipet up and down 10 times to disrupt larger, centrally necrotic spheres and allow new, healthy spheres to form.</li>
</ol>
2.	Preparation of tumor cells for injection
<ol>
	<li>If injecting cultured tumorspheres, gently pipet spheres into a 15-mL conical tube. Gently pipet up and down with a serological pipet 10-15 times to disrupt the spheres.</li> 
<li>Count the cells to be injected using a hemacytometer, and aliquot at least 50,000 cells per injection into a new tube. Prepare for as many injections as possible based on the number of cells available.</li>
<li>Centrifuge at 300xg for 5 minutes. Carefully aspirate the supernatant and resuspend in 5 mL PBS. Repeat centrifugation and aspiration, and resuspend in 10 &mu;L PBS per injection.</li> 
</ol>
3.	Preparation of animals
<ol>
<li>Using an insulin syringe, administer an intraperitoneal injection of rodent anesthesia cocktail (ketamine 37.6 mg/mL, xylazine 1.92 mg/mL, acepromazine 0.38 mg/mL) at 1 &mu;L per gram body weight (gbw). Wait 5 minutes for the animal to become sedated. Check for heartbeat and place the animal on a heated pad. Maintain animals on a heated pad at all times until recovery.</li>
<li>After sedation, administer 1 drop of proparacaine HCl to the right eye. Replace animal on heated pad and wait 1 minute.</li>
<li>Administer 1 drop of phenylephrine HCl to the right eye. Replace the animal on the heated pad and wait 5 minutes. If dilation of the pupil has not occurred, re-administer 1 drop and wait another 5 minutes.</li>
<li>Monitor animals for signs of movement or twitching, which may occur at &gt;30 minutes post-sedation. At first signs of movement, administer 1 &mu;L/gbw of ketamine HCl (diluted to 10 mg/mL in PBS) and wait 5 minutes.</li>
</ol>
4.	Injection of tumor cells
<ol>
	<li>Place the anesthetized animal under the microscope on its side, with the right eye facing up and with the head resting on gauze and centered to obtain a red reflex of the right eye fundus (pupil should be dilated at this time) when observed through the microscope.</li>
<li>Prop up the right globe by gently pushing down simultaneously with two fingers on the eyelids and hold this position steadily for the remainder of the procedure.</li> 
<li>Using a sterile 30-gauge needle attached to a Luer-lock syringe, pierce the globe laterally through the conjunctiva and sclera adjacent to the corneal limbus in the area of the pars plana (Figure 1) and just through the choroid into the vitreous cavity. Remove the needle from the opening.</li>
<li>Sterilize the Hamilton needle with an alcohol swab. Draw the cell suspension into the Hamilton syringe and insert the needle into the opening until the needle is visualized through the microscope behind the lens in the vitreous near the retina. With the help of a second person while holding the needle steadily in this position, depress the plunger slowly. Remove the needle. If necessary, use a cotton swab to absorb any fluid from the opening.</li>
<li>Gently release the pressure from the eyelids to close the mouse eyelids.</li> 
<li>Administer 1 drop of hypromellose to each eye and return animal to heated pad for recovery.</li>
</ol>
5.	Monitoring of injected mice and harvesting of tumors
<ol>
	<li>Daily, examine the injected eye for leukocoria (white papillary reflex) and/or periocular distention that usually becomes apparent 2-4 weeks post-injection.</li> 
<li>Once tumor growth is detected, euthanize animal according to institutional guidelines.</li>
<li>If the eye and tumor are covered by the eyelids, use the scalpel to make two incisions orthogonal to the palpebral fissure and lift the skin flaps.</li> 
<li>Under visualization using the stereoscopic microscope, make a circumferential incision at the limbus, removing the cornea and the lens, and carefully dissect the bulk of the tumor mass from the other tissues using tweezers. Place the tumor in sterile RPMI-1640 media.</li> 
<li>Use tweezers to slowly pull the open globe from the socket. To ensure an attached optic nerve so tumor invasion can be visualized, pull the globe until the nerve is exposed and use scissors to cut the nerve as long as possible. Place in 10% formalin for regular processing for histological examination.</li>
</ol>
6. Representative Results:
Retinoblastoma tumorspheres will begin to appear from the disaggregated tissue almost immediately as they are liberated from the tumor mass. Within 2-4 days, more tumorspheres will begin to form and will increase in size. The spheres tend to be regular and exhibit a well-defined, secondary membrane surrounding the aggregate (Figure 2).
The animal typically presents with leukocoria within 4 weeks post-injection (Figure 3b), followed by enlargement of the globe and distention of the surrounding tissues 5-8 weeks after injection as the tumor grows (Figure 3c). 
<img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Figure 1"/> Figure 1. Cross-sectional diagram of the mouse eye highlighting features referenced in the protocol.
<img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Figure 2"/> Figure 2. Culture of human retinoblastoma cells in vitro imaged at a) 4x, and b) 10x objective magnification. Retinoblastoma primary tumor cells produce tumorspheres with a regular spheroid shape and a crisp outer membrane. Scale bars represent a) 500 &mu;m, and b) 200 &mu;m.
<img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Figure 3"/> Figure 3. Eye of Rag2-/- mouse showing a) normal features, b) leukocoria indicative of a tumor mass in vitreous cavity, and c) a large tumor mass filling the globe with associated periocular distension, intraocular hemorrhage.

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Baylor College of Medicine IACUC Committee.

The technique described herein facilitates the propagation retinoblastoma tumors in their intraocular, intravitreal milieu. The intraocular injection technique has been used in the past to create tumors from retinoblastoma-derived cell lines1 as well as to deliver viral vectors for intraocular gene therapy2,3. This technique has now been successfully utilized to produce human retinoblastoma tumors by direct injection of cells from the primary tumor and injection of tumorspheres as well as serial propagation of xenograft tumors. Visible evidence of tumor formation (usually leukocoria) is usually first noted within 4 weeks, after which intraocular hemorrhage and distention of the globe and/or tissues surrounding the orbit develop within 5-8 weeks. A minority of injected mice injure the injected eye, leading to permanent closure of the eyelids. In these cases, distention is the only sign of tumor formation.
Establishment of murine xenograft tumors has not been successful with all human retinoblastoma tumors, though propagation of established xenograft tumors is highly successful. This observation suggests that certain characteristics of the primary tumor, such as invasiveness and level of differentiation or some other unknown factor, may be influencing the ability of these tumors to persist in the murine ocular environment.
The amount of tissue that can be acquired from a human retinoblastoma tumor is quite small, and there are significant limitations on the ability to culture human retinoblastoma cells in vitro such as limited life span, loss of solid tumor histology and changes in cellular phenotype. This protocol provides a relatively simple way to propagate the human tumor and establish a murine model of the human disease. This allows further in vitro and in vivo experimentation of the biology of retinoblastoma and longer maintenance of the tumor outside of the patient.

<table border="1">
	<tbody>
 <tr>
 	<th>Name</th>
 <th>Company</th>
 <th>Catalog #</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Phenylephrine HCl 2.5%</td>
 <td>Bausch &amp; Lomb</td>
 <td>053-11</td>
 <td>Ophthalmic solution</td>
 </tr>
 <tr>
 	<td>30-ga Needle</td>
 <td>BD</td>
 <td>305128</td>
 <td>Regular bevel</td>
 </tr>
 <tr>
 	<td>10-mL Luer-Lock Syringe</td>
 <td>BD</td>
 <td>309604</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>3/10-cc Insulin Syringe</td>
 <td>BD</td>
 <td>328431</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Alcohol swabs</td>
 <td>BD</td>
 <td>326895</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>6-Well Plate, Tissue Culture-Treated</td>
 <td>BD</td>
 <td>353046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Proparacaine HCl 0.5%</td>
 <td>Butler AHS</td>
 <td>017239</td>
 <td>Ophthalmic solution</td>
 </tr>
 <tr>
 	<td>Ketamine HCl 100mg/mL</td>
 <td>Fort Dodge</td>
 <td>4402A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>10-&mu;L Hamilton Syringe</td>
 <td>Hamilton Company</td>
 <td>7648-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>32-ga Hamilton Needle</td>
 <td>Hamilton Company</td>
 <td>7803-04</td>
 <td>Custom length - 0.5"</td>
 </tr>
 <tr>
 	<td>Neurobasal-A Media</td>
 <td>Invitrogen</td>
 <td>10888-022</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>B-27 Supplement Minus Vitamin A, 50X</td>
 <td>Invitrogen</td>
 <td>12587-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RPMI-1640 Media</td>
 <td>Mediatech</td>
 <td>10-040-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Non-essential Amino Acid Solution, 100X</td>
 <td>Mediatech</td>
 <td>25-025-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>L-Glutamine, 100X</td>
 <td>Mediatech</td>
 <td>25-005-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Disposable #11 Scalpel</td>
 <td>Miltex</td>
 <td>4-411</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Rodent Anesthesia Combo</td>
 <td>n/a</td>
 <td>n/a</td>
 <td>In-house pharmacy formulation 
(ketamine 37.6 mg/mL, xylazine 1.92 mg/mL, acepromazine 0.38 mg/mL)
</td>
 </tr>
 <tr>
 	<td>Recombinant Human Epidermal Growth Factor (EGF)</td>
 <td>STEMCELL Technologies</td>
 <td>02633</td>
 <td>Reconstitute at 10 &mu;g/mL stock solution</td>
 </tr>
 <tr>
 	<td>Recombinant Human Basic Fibroblast Growth Factor (bFGF)</td>
 <td>STEMCELL Technologies</td>
 <td>02634</td>
 <td>Reconstitute at 10 &mu;g/mL stock solution</td>
 </tr>
 <tr>
 	<td>OMS-75 Operation Microscope</td>
 <td>Topcon Medical Systems</td>
 <td>OMS-75</td>
 <td>This model has been discontinued</td>
 </tr>
 <tr>
 	<td>10% Formalin</td>
 <td>VWR</td>
 <td>95042-908</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

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 ...

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11.7K ビュー.  Baylor College of Medicine. メソッドは、マウスにヒトの網膜芽細胞腫の腫瘍を伝播するために記述されています。腫瘍細胞は、直接免疫欠損マウスの眼に注入されています。二次腫瘍が正常に直接ヒト腫瘍および培養tumorspheresから採取した細胞の両方を使用して確立されている。

ビデオ: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.