eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice housing and maintenance
<ol>
	<li>Purchase 6-8-week-old NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ).</li>
	<li>House the mice under SPF conditions with air exchange, prefilters, and high-efficiency particulate air (HEPA) filters (0.22 &#956;m) in a room with controlled temperature, humidity, and pressure.
	<ol>
		<li>House and maintain the mice in autoclaved individual microisolator cages in a rack system capable of managing air exchange with prefilters and HEPA filters (0.22 &#956;m).</li>
	</ol>
	</li>
	<li>Perform all procedures and manipulations of the mice in a Class II Type A2 biological safety fume hood that has been pre-treated with 70% ethanol. Before working in the animal room, shower and change into clean scrubs, Tyvek suit, boot covers, hair cap, face mask, and gloves. Wear a surgery gown over the Tyvek suit during surgical procedures.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Ultraviolet (UV) light decontamination is also preferred prior to procedures.
	<ol>
		<li>Autoclave all instruments and reagents if possible, and then disinfect them prior to transferring them to the fume hood.</li>
		<li>During procedures, remove the animals&#39;&#160;individually ventilated cages from the rack and disinfect them prior to transferring them to the fume hood.</li>
	</ol>
	</li>
	<li>Feed the mice an irradiated diet and provide autoclaved water. Provide irradiated food and supplement with additional irradiated food as needed. Change autoclaved water weekly or as needed.&#160;&#160;&#160; 
	NOTE: Autoclaved acidified water can also be used. The irradiated diet provided had a shelf-life of 6 months after manufacture.</li>
</ol>
2. Generation of humanized bone-marrow, liver, and thymus (BLT) mice
NOTE: The generation of hu-BLT mice has been described previously.
<ol>
	<li>On the day of surgery, give the mice whole-body irradiation at a dose of 12 cGy/g of body weight.</li>
	<li>Prepare the mice for surgery.
	<ol>
		<li>Give each irradiated mouse a mixture of Ketamine at the working concentration of 100 mg/kg and Xylazine at the concentration of 12 mg/kg, which ranged from 130-170 &#956;L&#160;per mouse based on body weight by intraperitoneal (IP) injection for anesthesia. To prepare 3 ml of the Ketamine and Xylazine mixture, add 0.27 mL of Ketamine at the stock concentration of 100 mg/mL and 0.03 mL of Xylazine at the concentration of 100 mg/mL to 2.7 mL of sterile saline and mix well.&#160;Disinfect the skin with 70% isopropanol prior to injection.</li>
		<li>Give each irradiated mouse Buprenorphine 1 mg/kg of body weight (half-live 72 h, SR-LAB) by subcutaneous injection for long lasting pain management.</li>
		<li>Give each irradiated mouse 100 &#956;L (858 &#956;g) of Cefazolin by IP injection for preoperative antibiotic prophylaxis.</li>
		<li>Shave the hair of the mice using electric hair clippers around the left lateral and medial side of the mouse at the later surgical site used to expose the left kidney.</li>
		<li>Verify proper level of anesthesia by pedal reflex (firm toe pinch).</li>
		<li>Give isoflurane gas at 3-5% if additional anesthesia is needed at any point during surgery.</li>
		<li>Apply ophthalmic ointment to both eyes to prevent corneal desiccation. Apply ear tag if needed.</li>
	</ol>
	</li>
	<li>Perform surgery to implant the liver and thymus tissues into the left kidney capsule.
	<ol>
		<li>Disinfect the skin at the surgical site by applying iodine scrub starting from the center of the surgical site and moving towards the outside in a circular manner. Repeat this process with 70% isopropanol and a third time with iodine.</li>
		<li>Using forceps, first load one human fetal liver tissue fragment into a trocar. Then using forceps, load one human fetal thymus tissue fragment into the trocar. Then using forceps, load another human fetal liver tissue fragment into the trocar. Tissues should be cut to 1-1.6 mm3&#160;to fit the inner dimensions of the trocar.</li>
		<li>Use forceps to lift the skin and use scissors to make a small cut longitudinally. Extend the cut to 1.5-2 cm in the left side of the mouse.</li>
		<li>Use forceps to lift the muscle layer and use scissors to make a small cut longitudinally. Extend the cut as needed to expose the kidney.</li>
		<li>Expose the kidney by gently grasping the fatty tissue surrounding the kidney. Do not directly touch the kidney parenchyma.</li>
		<li>Make a 1-2 mm incision at the posterior end of the kidney capsule using a scalpel.</li>
		<li>Slowly insert the preloaded trocar through the incision parallel to the long axis of the kidney and release the tissues between the kidney capsule and kidney.</li>
		<li>Carefully return the kidney and bowel to their normal position. Use absorbable 5/0 P-3 (P-13) sutures with 13 mm 3/8 circle needle to close the muscle layer and surgical staples to close the skin.</li>
	</ol>
	</li>
	<li>After surgery, put the mouse into a clean autoclaved microisolator cage for recovery.
	<ol>
		<li>To minimize heat loss during post-surgical recovery, put the cage containing the mice on a heating pad that is connected to a water pump that warms and circulates the water.</li>
		<li>Monitor the mice until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Within 6 h of surgery completion, via the tail-vein, inject CD34+ hematopoietic stem cells isolated from human fetal liver tissue.
	<ol>
		<li>Warm up the mice with a heat-lamp. Disinfect the tail with 70% isopropanol and then inject 1.5 to 5 &#215; 105&#160;stem cells/200 &#956;L into the tail vein.</li>
		<li>Stop any bleeding from the injection and return mice to the microisolator cage and the microisolator cage to the cage rack.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: Post-surgery mice are typically housed together, five per microisolator cage, during and after recovery. However, mice are only housed together if they all received surgery on the same day.</li>
	</ol>
	</li>
	<li>Check the mice daily. Carefully monitor the surgical staples and replace them as needed. Closely monitor the mice for any sign of infection or discomfort. Supply autoclaved food on the floor of the microisolator cage for a few days post-surgery.</li>
	<li>Remove the surgical staples 7-10 days after surgery. Give isoflurane gas at 3-5% to anesthetize the mice. Carefully remove the staples and then apply antibiotic and pain-relieving ointment onto the site.</li>
	<li>Allow 9-12 weeks for reconstitution of human immune cells, then collect peripheral blood from the medial saphenous vein from each of the humanized mice.
	<ol>
		<li>Restrain conscious mice using an appropriately sized plastic cone restraint with an opening near the head of the mouse for breathing and an opening near the back of the mouse to isolate one leg. Put the mice into the restraint cone head first and then gently pull one leg through the leg opening.</li>
		<li>Spray the medial side of the isolated leg with 70% isopropanol, then spread antibiotic and pain-relieving ointment onto the same site.&#160; 
		NOTE: The ointment helps to reveal the location of the vein without the need for hair removal and also assists in blood droplet formation.</li>
		<li>Using a 25-gauge needle at a 90&#176; angle, puncture the vein and collect 50-100 &#956;L of blood using an ethylenediaminetetraacetic acid (EDTA) coated blood collection tube. Stop the bleeding by applying pressure to the site with sterile gauze. Once the bleeding has stopped, return the mice to their cage. 
		NOTE: The maximum blood volumes collected are typically 50 &#956;L per week or 100 &#956;L every two weeks.</li>
		<li>Use the collected peripheral blood to test the level of human immune cell reconstitution using flow cytometry with antibodies for hCD45, mCD45, hCD3, hCD4, hCD8, hCD19.</li>
	</ol>
	</li>
</ol>
3. Antibiotic treatment
<ol>
	<li>Prior to antibiotic treatment, collect pre-treatment fecal samples. Move the mice to a new autoclaved microisolator cage.</li>
	<li>Prepare a fresh cocktail of broad-spectrum antibiotics daily.
	<ol>
		<li>Prepare 250 mL of water containing freshly prepared Metronidazole (1 g/L), Neomycin (1 g/L), Vancomycin (0.5 g/L), and Ampicillin (1 g/L) for each microisolator cage of mice.&#160;&#160; 
		NOTE: Use autoclaved or sterile water for the antibiotic supplemented drinking water.</li>
		<li>Add 9.2 g of grape sugar sweetened drink mix to 250 mL of antibiotic supplemented water.&#160; 
		NOTE: The use of grape sugar sweetened drink mix masks the bitter taste of the antibiotics and helps prevent dehydration in the mice.</li>
		<li>Change the antibiotic and grape sugar sweetened drink mix supplemented water and place the mice in a new autoclaved cage daily.&#160; 
		NOTE: Mice are coprophagic and changing the cages daily prevents the mice from re-inoculating themselves with gut bacteria.</li>
		<li>For maximal engraftment of the human-like gut microbiome, provide antibiotics for 14 days. During antibiotic treatment, monitor the mice for weight loss and dehydration. Weight loss is expected during the first 3-4 days and plateaus after that for the duration of the treatment. If dehydration occurs, give the mice saline or Ringers Solution via intraperitoneal injection. 
		NOTE: Other gut microbiome humanization protocols call for oral gavage of antibiotics. While effective at depleting the murine gut bacteria, we found that the less invasive method of adding antibiotics to the drinking water put less stress on our humanized mice and led to better outcomes.</li>
	</ol>
	</li>
</ol>
4. Donor samples and fecal transplant
<ol>
	<li>Prepare human donor fecal samples.
	<ol>
		<li>Use properly prepared sources of fecal microbiota transplant (FMT) material for fecal transplant into the humanized mice.</li>
		<li>Thaw FMT preparations and aliquot them under anaerobic conditions in an anaerobic chamber.</li>
		<li>If desired, at this step mix equal parts of the fecal samples together to create an unbiased &#34;human&#34;&#160;sample.</li>
		<li>Keep freezing and thawing of the FMT material to a minimum, if aliquoting or mixing of samples is not needed, only thaw immediately before the procedure.</li>
	</ol>
	</li>
	<li>Human fecal transplant
	<ol>
		<li>After completion of 14 days of antibiotic treatment, change the drinking water to autoclaved water and move the mice into a new autoclaved cage. Stop daily cage changes and implement a once every 1-2 weeks cage changing schedule.</li>
		<li>Give two fecal transplants at 24 and 48 hours post-cessation of antibiotics.</li>
		<li>24 h after antibiotics treatment, thaw the needed amount of FMT material and give each mouse 200 &#956;L of FMT material via oral gavage. Repeat the procedure again at 48 h post antibiotics.</li>
		<li>Spread any remaining or leftover thawed FMT material on the fur of the humanized mice or onto the cage bedding.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal Feeding Needles 18G</td>
			<td>Cadence Science</td>
			<td>9928B</td>
			<td></td>
		</tr>
		<tr>
			<td>Clidox-s Activator</td>
			<td>Pharmacal Research Laboratories</td>
			<td>95120F</td>
			<td></td>
		</tr>
		<tr>
			<td>Clidox-s Base</td>
			<td>Pharmacal Research Laboratories</td>
			<td>96125F</td>
			<td></td>
		</tr>
		<tr>
			<td>DGM 108 cage rack</td>
			<td>Techniplast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flat Brown Grocery Bag 3-5/8&#34;D x 6&#34;W x 11-1/16&#34;L</td>
			<td>Grainger</td>
			<td>12R063</td>
			<td></td>
		</tr>
		<tr>
			<td>FMT Upper Delivery Microbiota Preparations</td>
			<td>OpenBiome</td>
			<td>FMP30</td>
			<td></td>
		</tr>
		<tr>
			<td>Grape Kool-Aid</td>
			<td>Kraft Foods Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hCD19-PE/Cy5</td>
			<td>Biolegend</td>
			<td>302209</td>
			<td></td>
		</tr>
		<tr>
			<td>hCD3-PE</td>
			<td>Biolegend</td>
			<td>300408</td>
			<td></td>
		</tr>
		<tr>
			<td>hCD4-Alexa 700</td>
			<td>Biolegend</td>
			<td>300526</td>
			<td></td>
		</tr>
		<tr>
			<td>hCD45-FITC</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>hCD8-APC/Cy7</td>
			<td>Biolegend</td>
			<td>301016</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactate Buffered Ringer&#39;s Solution</td>
			<td>Boston BioProducts Inc</td>
			<td>PY-906-500</td>
			<td></td>
		</tr>
		<tr>
			<td>mCD45-APC</td>
			<td>Biolegend</td>
			<td>103111</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette 100 K3E</td>
			<td>Microvette</td>
			<td>20.1278.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin First Aid Antibiotic/Pain Relieving Ointment</td>
			<td>Neosporin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide 25 G Needle</td>
			<td>BD</td>
			<td>305127</td>
			<td></td>
		</tr>
		<tr>
			<td>RS200 X-ray irradiator</td>
			<td>RAD Source Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sealsafe Plus GM500 microisolator cages</td>
			<td>Techniplast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Non-woven Gauze</td>
			<td>Fisherbrand</td>
			<td>22-028-558</td>
			<td></td>
		</tr>
		<tr>
			<td>Teklad global 16% protein irradiated mouse chow</td>
			<td>Teklad</td>
			<td>2916</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a double humanized blt (bone-marrow, liver, thymus) mouse model

Source: Daharsh, L. et al., <a href="https://app.jove.com/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mice ...

Begin with an anesthetized, immunodeficient mouse that lacks immune cells.
Treat the mouse with a sub-lethal dose of X-rays to eliminate existing stem cells from the bone marrow.
Dissect the mouse and deliver fragments of human fetal liver and thymus tissue between the kidney and its capsule.
Close the incision.
The implanted liver and thymus tissue grow, forming organoids, which are cell aggregates resembling an organ.
After recovery, inject human hematopoietic stem cells or HSCs into the tail vein.
Injected HSCs migrate to the thymus organoid and bone marrow, differentiating into various human immune cells.
Provide water supplemented with antibiotics to the mouse to kill existing gut microbes.
Using an oral tube, introduce human fecal microbes directly into the mouse&#39;s gut to promote microbial colonization.
This results in the development of a double humanized BLT-mouse model featuring a human immune system and gut microbes.

generating-a-double-humanized-blt-bone-marrow-liver-thymus-mouse-model

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

77 Views. Fonte: Daharsh, L. et al., Un modello di topi BLT doppiamente umanizzati con un microbioma intestinale stabile simile a quello umano e un sistema immunitario umano.J. Vis. Exp. (2019) Questo video dimostra il metodo per generare un topo BLT (midollo, fegato e timo) osseo umanizzato doppio con un sistema immunitario umano funzionale e un microbioma intestinale stabile simile a quello umano innestato. 

Video: Generazione di un modello murino BLT (midollo osseo, fegato, timo) doppiamente umanizzato - Concept

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Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

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Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Generazione di un umanizzato mouse Fegato Utilizzando epatici umani cellule staminali

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

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Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

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Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Trascrizione

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model