eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Generation of HIS mice
<ol>
	<li>Mouse husbandry of BALB/c -Rag2null Il2R&#947;Cnull Sirp&#945;NOD (BRGS) mice 
	NOTE: This strain is extremely immunodeficient, with no T, B, or natural killer (NK) cells. Therefore, rigorous measures must be used to prevent opportunistic infections. Maintain the colony on a diet containing trimethoprim and sulfadiazine on an alternating 2-week schedule with a normal diet. Maintain in the highest level of precaution housing room possible (e.g., a barrier shower-in facility with limited access).
	<ol>
		<li>Maintain colonies of BALB/c -Rag2null Il2R&#947;Cnull Sirp&#945;NOD (BRGS) and BALB/c Rag2null Il2R&#947;Cnull SirpaBalb/c (BRG) homozygous mice as breeders.</li>
		<li>Breed BRGSN/N&#215;BRGB/B to generate BRGSB/N pups, to be used as recipients of human stem cells. In this colony, BRGSB/N are healthier than BRGSN/N, and engraft at equivalent levels (more than BRG).</li>
	</ol>
	</li>
	<li>CD34+ human stem cell isolation from umbilical cord blood (CB) 
	NOTE: No antibiotics are used for this procedure. Therefore, good sterile technique is imperative.
	<ol>
		<li>Place a 50 mL conical tube rack, as well as 3x 15 mL and ~10x 50 mL conical tubes in a sterilized biosafety cabinet (BSC). Spray a blood collection bag with 70% ethanol and let it dry in the BSC.</li>
		<li>Calculate the number of 50 mL conical tubes required for CB density gradient isolation = CB volume/15, rounded up and to an even tube number. Calculate blood volume per tube = CB volume/number of tubes. Pour blood carefully from the CB bag into each conical tube; this is a maximum of 15 mL per tube. Use an automatic pipettor and a 25 mL serological pipette to mix the blood 1:1 with sterile PBS by pipetting up and down.</li>
		<li>Use an automatic pipettor on low speed and a 10 mL serological pipette to slowly underlay the blood with room temperature (RT) 1.077 g/mL density gradient solution (see Table of Materials) without disturbing the interface. Keep the pipette tip from touching the tube bottom. Repeat for all tubes. Then centrifuge for 30 min at 850 x g, with no braking, at RT to ensure maintenance of the density gradient.</li>
		<li>Visualize the cellular buffy coat on top of the 1.077 g/mL density gradient as a cloudy white layer. Remove and discard the plasma layer down to about 10 mL above the buffy coat using a 25 mL serological pipette and an automatic pipettor.</li>
		<li>Collect the buffy coat with a sterile transfer or serological pipette. Use the pipette like a spatula to scrape the cells off the side of the conical tube while releasing the bulb (or pipetting slowly) to draw the cells up. Combine the buffy coats from two 50 mL conical tubes into one new 50 mL conical tube.</li>
		<li>Wash the cells by pouring 45 mL of sterile Hanks&#39; balanced salt solution (HBSS) containing 2% fetal bovine serum (FBS) into each conical tube. Centrifuge for 11 min at 360 x g at RT.</li>
		<li>Aspirate the wash media down to the pellet in all tubes. Use a 10 mL serological pipette and an automatic pipettor to resuspend the first pellet in 10 mL of HBSS containing 2% FBS. Resuspend each pellet in the same 10 mL of HBSS, and rinse each tube with an additional 10 mL of HBSS to collect all the cells into a single tube.</li>
		<li>Pour 45 mL of sterile HBSS containing 2% FBS in the conical tube. Centrifuge for 10 min at 360 x g, at 4 &#176;C.</li>
		<li>Aspirate the wash buffer down to the cell pellet and resuspend the pellet in 20 mL of magnetic cell separator buffer (see Table of Materials). Remove a small aliquot to count the cells with a hemacytometer at a 1:20 dilution in methylene blue. Add the number of blue and white cells. Centrifuge at 360 x g, for 10 min at 4 &#176;C. 
		NOTE: This protocol uses magnetic bead technology (see Table of Materials). The protocol can be modified for use with any cell-separation technology, with sufficient purity and yield of the CD34+ stem cells.</li>
		<li>Aspirate the supernatant and resuspend the CD34+ cell pellet isolated from cord blood in 300 &#181;L of magnetic cell separator buffer per 1 x 108 cells. Add 100 &#181;L of Fc receptor (FcR) blocking reagent first, and then 100 &#181;L of CD34+ magnetic beads per 1 x 108 cells. Incubate at 4 &#176;C for 30 min (no ice).</li>
		<li>Add 5 mL of magnetic cell separator buffer per 1 x 108 cells and spin at 360 x g for 10 min at 4 &#176;C. Repeat the wash step and resuspend the pellet in 500 &#181;L of magnetic cell separator buffer per 1 x 108 cells in a 15 mL conical tube labeled &#34;unfractionated&#34;.</li>
		<li>Label two more 15 mL conical tubes &#34;CD34-&#34; and &#34;CD34+&#34;. Place the three 15 mL conical tubes (unfractionated, CD34-, and CD34+) to slots A1, B1, and C1, respectively, on a cooling rack (see Table of Materials). Separate the cells using the two-column positive selection program on an automatic magnetic cell separator (see Table of Materials) in a BSC, according to the manufacturer&#39;s instrument instructions.</li>
	</ol>
	</li>
	<li>Expanding and freezing CD34+ human stem cells
	<ol>
		<li>Aliquot 10 &#181;L of the recovered CD34+ cell suspension (2 mL) on a hemocytometer slide and count the cells under 10x magnification. Calculate the total number of CD34+ cells by multiplying the cell count by 2 x 104. Divide the total CD34+ cell number by 250,000 to calculate the number of vials to freeze (50,000 cells per mouse pup prior to in vitro expansion).</li>
		<li>Prepare CB medium Iscove&#39;s 10% fetal calf serum (FCS) (plus 1 mL extra for filtering loss), supplemented with 40 ng/mL stem cell factor, 20 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L), and 10 ng/mL interleukin-6 (IL-6), and pass through a 0.22 &#181;m filter. Resuspend the CD34+ cells at 100,000 per mL of CB medium, and incubate at 37 &#176;C. On day 3, add an equivalent volume of CB medium without cytokines to the flask containing the cells and the CB medium with cytokines. 
		NOTE: Addition of these cytokines to the CB medium promotes survival and expansion of the CD34+ cells while preventing differentiation.</li>
		<li>Harvest the expanded CD34+ cells on day 5. Pipette the cell suspension up and down and collect in a 50 mL conical tube. Add enough CB medium to cover the bottom of the flask. Using a cell scraper, scrape the entire bottom of the flask. Collect all the media into the same 50 mL tube, and centrifuge at 360 x g for 11 min.</li>
		<li>Resuspend the cells in 2 mL of CB medium. Save the final drop from the pipette into a 96-well plate for counting. Dilute the cells 1:1 in trypan blue and add 10 &#181;L to the hemacytometer, then count and average cells from four quadrants. Calculate the total number of CD34+ cells by multiplying the cell count by 4 x 104, and record the viability.</li>
		<li>Make n+1 mL of freezing medium, where n is the number of freezing vials calculated in step 1.3.1. Prepare freezing medium by adding 10% (v/v) dimethyl sulfoxide (DMSO) to FBS and keep on ice. Label the cryovials with CB#, CD34+ d5, and date.</li>
		<li>Spin down the CD34+ cells at 360 x g for 10 min at 4 &#176;C. Aspirate the medium down to the pellet and resuspend the cell pellet in freezing medium. Aliquot 1 mL of cell suspension to each vial and divide any remainder evenly between the vials. Add the vials to an isopropanol cell freezer chilled to 4 &#176;C, place at -80 &#176;C, and transfer to liquid nitrogen to store for &#62;90 days.</li>
	</ol>
	</li>
	<li>Irradiation of mouse pups
	<ol>
		<li>Collect the BRGSB/N pups, 1-3 days after birth, into an autoclaved plastic box with padding. Add a small amount of bedding with the pups. Label the box with the cage number and the number of pups.</li>
		<li>Set up the irradiator (see Table of Materials) for a dose of 300 rad. Set the box of pups into the irradiator and expose them to 300 rad. Take the pups back to their cage, place them in a pile, and cover with bedding.</li>
	</ol>
	</li>
	<li>Pup injections and CD34+ cell preparation
	<ol>
		<li>Begin CD34+ cell preparation ~3 h post irradiation. Warm 10 mL of CB media in a 50 mL conical tube. Perform all steps in a sterile BSC.</li>
		<li>Retrieve one vial of in vitro expanded and frozen CD34+ cells for every four to six pups to inject. Rapidly thaw at 55 &#176;C, until just a small amount of ice is visible, and add the cells to the warmed CB medium (the vial should still be cool to the touch.) Use 1 mL of medium to rinse each vial and spin the cells at 360 x g for 12 min at 4 &#176;C. 
		&#8203;NOTE: Rapid thawing at 55 &#176;C was found to yield better cell viability (90%-95%) than thawing at 37 &#176;C.</li>
		<li>Aspirate the medium carefully. Resuspend the (small) pellet in 2 mL of CB media, gently mix, and add ~30 &#181;L of the cell suspension to a single well in a counting plate. Dilute 1:1 in trypan blue, then add 10 &#181;L to a hemocytometer and count and average the cells from four quadrants.</li>
		<li>Calculate the total number of CD34+ cells by multiplying the cell count by 4 x 104 and then record the viability. Spin at 360 x g for 12 min at 4 &#176;C.</li>
		<li>Aspirate the medium carefully and resuspend the cell pellet in 100 &#181;L of sterile PBS per n+1 pups to inject, resulting in 250,000-450,000 CD34+ cells per mouse. Place the conical tube on ice in a transport container and travel to the vivarium for pup injection.</li>
		<li>Bring a heat lamp, diapers, 1 mL syringe, 18 G needle, 30 G needle, and the CD34+ cell preparation in sterile containers to the vivarium BSC. Place a sterile diaper ~2 ft underneath the heat lamp. Retrieve the cage with the litter to be injected and place it in the BSC.</li>
		<li>Assemble the syringe with an 18 G beveled needle. Matching the angle of the conical tube with the bevel of the needle, gently mix and draw up the cell suspension. Place the pups on the diaper to warm (watch for overheating). Remove air from the syringe and replace the 18 G needle with the 30 G needle, and then carefully push the syringe until the cell suspension is just at the needle tip. 
		NOTE: Alternatively, an insulin syringe can be used.</li>
		<li>Take one pup at a time to the edge of the diaper away from the heat lamp. Immobilize the pup on its side under the thumb and forefinger, allowing for a clear view of the face. Notice the vein across the cheek under where the ear will be. Insert the needle shallowly into the vein (IV) nearest to the eye, and slowly inject 50 &#181;L of cells.</li>
		<li>Check if a bubble is forming from a subcutaneous injection. If so, insert the needle deeper and proceed to inject cells. A small drop of blood/hematoma will be visible when successful.</li>
		<li>With the pup still immobilized, perform an intrahepatic injection (IH) with another 50 &#181;L of cells (100 &#181;L total per pup of IV+IH). The liver can be visualized as a dark spot between the white milk band and the thoracic cage. Place the injected pup on a diaper further away from the un-injected pups and heat. 
		NOTE: IV injection results in better long-term chimerism than IH injection alone, but IV injections are not always successful. Therefore, splitting the injection between IV and IH ensures engraftment into a larger percentage of mice.</li>
		<li>Repeat both facial vein and IH injections for all pups. Clean off any blood, return pups to the nest in their cage, and cover with bedding.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe w/needles</td>
			<td>McKesson</td>
			<td>1031815</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Grenier Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Grenier Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi</td>
			<td>130-092-545</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP1600100</td>
			<td></td>
		</tr>
		<tr>
			<td>Chill 15 Rack</td>
			<td>Miltenyi</td>
			<td>130-092-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryonic Stemcell FCS</td>
			<td>Gibco</td>
			<td>10439001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes; 1.5 mL volume</td>
			<td>Grenier Bio-One</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Benchmark</td>
			<td>100-106 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Hypaque</td>
			<td>GE Healthcare</td>
			<td>45001752</td>
			<td></td>
		</tr>
		<tr>
			<td>glass pipettes</td>
			<td>DWK Life Sciences</td>
			<td>63A53</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/ Ca &#38; Mg</td>
			<td>Sigma</td>
			<td>55037C</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiRad 350</td>
			<td>Precision X-Ray</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr>
			<td>PRISM</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu FLT3L</td>
			<td>R&#38;D systems</td>
			<td>308-FK-005/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu IL6</td>
			<td>R&#38;D systems</td>
			<td>206-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu SCF</td>
			<td>R&#38;D systems</td>
			<td>255SC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes 25 mL</td>
			<td>Fisher Scientific</td>
			<td>1367811</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile molecular water</td>
			<td>Sigma</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeti Cell Analyzer</td>
			<td>Bio-Rad</td>
			<td>12004279</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating cord blood hematopoietic stem cells to generate human immune system mice

Source: Lanis, J. M., et al. <a href="https://app.jove.com/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

Begin with human umbilical cord blood cells, including CD34+ hematopoietic stem cells, or HSCs.
Add antibodies to block cellular Fc receptors, preventing non-specific binding of HSC-specific magnetic beads.
Introduce the antibody-coated magnetic beads targeting CD34+ HSCs. Centrifuge and discard the supernatant.
Resuspend the cells in a buffer. Pass them through a magnetic separator to retain bead-bound HSCs; unbound cells flow through.
Remove the magnetic field to collect the HSCs.
Centrifuge and resuspend the cells in a growth medium. Plate the cells and incubate.
The medium contains growth factors and cytokines that promote HSC survival and expansion.
Harvest the cells. Add a freezing medium, transfer to a cryovial, and freeze.
To generate a human immune system, or HIS, mouse model, thaw the HSCs. Add them to a pre-warmed medium.
Centrifuge and resuspend the cells in a buffer.
The cells are ready for injection into an immunodeficient mouse pup for HIS model generation.

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JoVE Encyclopedia of Experiments

Immunology

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91 Views. Fonte: Lanis, J. M., et al. Test di immunoterapie contro il cancro in un modello murino umanizzato portatore di tumori umani. J. Vis. Exp. (2022) Questo video dimostra una tecnica per generare topi del sistema immunitario umano (HIS). Le cellule staminali ematopoietiche (HSC) CD34+ sono isolate dalle cellule del buffy coat del sangue del cordone ombelicale umano utilizzando perle magnetiche rivestite di anticorpi. Le HSC CD34+ vengono espanse in un terreno di crescita arricchito con citochine e...

Video: Isolamento delle cellule staminali ematopoietiche del sangue del cordone ombelicale per generare topi del sistema immunitario umano - Concept

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The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

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We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

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The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

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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.

Mitrai NOG umanizzati per l'esposizione all'HIV intravaginale e il trattamento dell'infezione da HIV

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Immunologia e infezioni

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

Trattamento antiretrovirale combinato orale in topi umanizzati infetti da HIV-1

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Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

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Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice